CN116539901A - Application of differential metabolite as diagnostic marker and kit - Google Patents
Application of differential metabolite as diagnostic marker and kit Download PDFInfo
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- CN116539901A CN116539901A CN202310822021.0A CN202310822021A CN116539901A CN 116539901 A CN116539901 A CN 116539901A CN 202310822021 A CN202310822021 A CN 202310822021A CN 116539901 A CN116539901 A CN 116539901A
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- equal
- metabolite
- differential
- differential metabolite
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Links
- 239000002207 metabolite Substances 0.000 title claims abstract description 91
- 239000003550 marker Substances 0.000 title claims abstract description 24
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 claims abstract description 17
- WBWWGRHZICKQGZ-HZAMXZRMSA-N taurocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 WBWWGRHZICKQGZ-HZAMXZRMSA-N 0.000 claims abstract description 16
- DMASLKHVQRHNES-UPOGUZCLSA-N (3R)-beta,beta-caroten-3-ol Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C DMASLKHVQRHNES-UPOGUZCLSA-N 0.000 claims abstract description 14
- NBZANZVJRKXVBH-ITUXNECMSA-N all-trans-alpha-cryptoxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CCCC2(C)C)C NBZANZVJRKXVBH-ITUXNECMSA-N 0.000 claims abstract description 14
- DMASLKHVQRHNES-ITUXNECMSA-N beta-cryptoxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2=C(C)CCCC2(C)C DMASLKHVQRHNES-ITUXNECMSA-N 0.000 claims abstract description 14
- 235000002360 beta-cryptoxanthin Nutrition 0.000 claims abstract description 14
- 239000011774 beta-cryptoxanthin Substances 0.000 claims abstract description 14
- 229940087168 alpha tocopherol Drugs 0.000 claims abstract description 13
- 229960000984 tocofersolan Drugs 0.000 claims abstract description 13
- 239000002076 α-tocopherol Substances 0.000 claims abstract description 13
- 235000004835 α-tocopherol Nutrition 0.000 claims abstract description 13
- DWEXIFLNCXYYAA-QQHSWTODSA-N 4beta-(hydroxymethyl)-4alpha-methyl-5alpha-cholest-7-en-3beta-ol Chemical compound OC[C@@]1(C)[C@@H](O)CC[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@H](C)CCCC(C)C)CC[C@H]33)C)C3=CC[C@H]21 DWEXIFLNCXYYAA-QQHSWTODSA-N 0.000 claims abstract description 12
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 claims abstract description 12
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims abstract description 12
- 108010007979 Glycocholic Acid Proteins 0.000 claims abstract description 8
- RFDAIACWWDREDC-UHFFFAOYSA-N Na salt-Glycocholic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)C(O)C2 RFDAIACWWDREDC-UHFFFAOYSA-N 0.000 claims abstract description 8
- RFDAIACWWDREDC-FRVQLJSFSA-N glycocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 RFDAIACWWDREDC-FRVQLJSFSA-N 0.000 claims abstract description 8
- 229940099347 glycocholic acid Drugs 0.000 claims abstract description 8
- BTNMPGBKDVTSJY-UHFFFAOYSA-N keto-phenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=CC=C1 BTNMPGBKDVTSJY-UHFFFAOYSA-N 0.000 claims abstract description 8
- JCCZCUUYYHWRGX-VGMNWLOBSA-N Leucyl-Hydroxyproline Chemical compound CC(C)C[C@H](N)C(=O)N1C[C@H](O)C[C@H]1C(O)=O JCCZCUUYYHWRGX-VGMNWLOBSA-N 0.000 claims abstract description 7
- -1 hydroxypropionyl histidine Chemical compound 0.000 claims abstract description 7
- JKQUXSHVQGBODD-VOTSOKGWSA-N 1-(4-Methoxyphenyl)-2-nitroethylene Chemical group COC1=CC=C(\C=C\[N+]([O-])=O)C=C1 JKQUXSHVQGBODD-VOTSOKGWSA-N 0.000 claims abstract description 6
- WPAMZTWLKIDIOP-NQXXGFSBSA-N 2-dehydro-3-deoxy-D-galactonic acid Chemical compound OC[C@@H](O)[C@H](O)CC(=O)C(O)=O WPAMZTWLKIDIOP-NQXXGFSBSA-N 0.000 claims abstract description 6
- UGQMRVRMYYASKQ-UHTZMRCNSA-N 9-[(2r,3s,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHTZMRCNSA-N 0.000 claims abstract description 6
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims abstract description 6
- CBQJSKKFNMDLON-JTQLQIEISA-M N-acetyl-L-phenylalaninate Chemical compound CC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 CBQJSKKFNMDLON-JTQLQIEISA-M 0.000 claims abstract description 6
- 229940109239 creatinine Drugs 0.000 claims abstract description 6
- QIJRTFXNRTXDIP-YBBRRFGFSA-N (2s)-2-amino-3-sulfanylpropanoic acid;hydrate;hydrochloride Chemical compound O.Cl.SC[C@@H](N)C(O)=O QIJRTFXNRTXDIP-YBBRRFGFSA-N 0.000 claims abstract description 5
- GRXIELRCPYIEQI-UHFFFAOYSA-N 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoic acid Chemical compound OC(=O)C1=C(O)C=C(O)C=C1OC(=O)C1=CC=C(O)C(O)=C1 GRXIELRCPYIEQI-UHFFFAOYSA-N 0.000 claims abstract description 5
- LFWNPKYGVKNNAB-UHFFFAOYSA-N Trimethylaminoacetone Chemical compound CC(=O)C[N+](C)(C)C LFWNPKYGVKNNAB-UHFFFAOYSA-N 0.000 claims abstract description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims abstract description 5
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims abstract description 5
- HMMVBUVVQLUGQA-UHFFFAOYSA-N (24R)-5alpha-stigmast-3,6-dione Natural products C1C(=O)C2CC(=O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 HMMVBUVVQLUGQA-UHFFFAOYSA-N 0.000 claims abstract description 4
- HMMVBUVVQLUGQA-UMQMBAGDSA-N (5s,8s,9s,10r,13r,14s,17r)-17-[(2r,5r)-5-ethyl-6-methylheptan-2-yl]-10,13-dimethyl-2,4,5,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthrene-3,6-dione Chemical compound C([C@@H]1C(=O)C2)C(=O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]2(C)CC1 HMMVBUVVQLUGQA-UMQMBAGDSA-N 0.000 claims abstract description 4
- 239000001903 2-oxo-3-phenylpropanoic acid Substances 0.000 claims abstract description 4
- HMMVBUVVQLUGQA-CIKSSPQXSA-N Stigmastane-3,6-dione Natural products O=C1[C@H]2[C@](C)([C@@H]3[C@@H]([C@H]4[C@@](C)([C@@H]([C@@H](CC[C@H](C(C)C)CC)C)CC4)CC3)C1)CCC(=O)C2 HMMVBUVVQLUGQA-CIKSSPQXSA-N 0.000 claims abstract description 4
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 claims abstract description 4
- DEDGUGJNLNLJSR-UHFFFAOYSA-N alpha-hydroxycinnamic acid Natural products OC(=O)C(O)=CC1=CC=CC=C1 DEDGUGJNLNLJSR-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229960003732 tyramine Drugs 0.000 claims abstract description 4
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical compound [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 claims abstract description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 238000004458 analytical method Methods 0.000 claims description 18
- 208000014906 developmental dysplasia of the hip Diseases 0.000 claims description 17
- 238000012216 screening Methods 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 15
- 206010073767 Developmental hip dysplasia Diseases 0.000 claims description 14
- 208000035790 developmental dysplasia of the hip 1 Diseases 0.000 claims description 14
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 5
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- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 238000012795 verification Methods 0.000 claims description 4
- WBWWGRHZICKQGZ-UHFFFAOYSA-N Taurocholic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCCS(O)(=O)=O)C)C1(C)C(O)C2 WBWWGRHZICKQGZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000013211 curve analysis Methods 0.000 claims 1
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- 238000003908 quality control method Methods 0.000 description 31
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- AOBORMOPSGHCAX-DGHZZKTQSA-N tocofersolan Chemical compound OCCOC(=O)CCC(=O)OC1=C(C)C(C)=C2O[C@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C AOBORMOPSGHCAX-DGHZZKTQSA-N 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 206010010356 Congenital anomaly Diseases 0.000 description 2
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 2
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- XLSMFKSTNGKWQX-UHFFFAOYSA-N hydroxyacetone Chemical compound CC(=O)CO XLSMFKSTNGKWQX-UHFFFAOYSA-N 0.000 description 2
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- WUORZOPBCJKPEL-QMMMGPOBSA-N (2s)-2-(3-hydroxypropylamino)-3-(1h-imidazol-5-yl)propanoic acid Chemical compound OCCCN[C@H](C(O)=O)CC1=CNC=N1 WUORZOPBCJKPEL-QMMMGPOBSA-N 0.000 description 1
- QXCBGDZJQIWXSW-UHFFFAOYSA-N 2-oxo-3-phenylpropanoic acid Chemical compound OC(=O)C(=O)CC1=CC=CC=C1.OC(=O)C(=O)CC1=CC=CC=C1 QXCBGDZJQIWXSW-UHFFFAOYSA-N 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 101150082208 DIABLO gene Proteins 0.000 description 1
- 102100033189 Diablo IAP-binding mitochondrial protein Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- UBORTCNDUKBEOP-UHFFFAOYSA-N L-xanthosine Natural products OC1C(O)C(CO)OC1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- UBORTCNDUKBEOP-HAVMAKPUSA-N Xanthosine Natural products O[C@@H]1[C@H](O)[C@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-HAVMAKPUSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
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- 230000002776 aggregation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 229940071221 dihydroxybenzoate Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229910052602 gypsum Inorganic materials 0.000 description 1
- 239000010440 gypsum Substances 0.000 description 1
- 230000030400 head development Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 230000000399 orthopedic effect Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
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- 230000035945 sensitivity Effects 0.000 description 1
- DYDUXGMDSXJQFT-UHFFFAOYSA-N sulfuric acid [4-(2-aminoethyl)phenyl] ester Chemical compound NCCC1=CC=C(OS(O)(=O)=O)C=C1 DYDUXGMDSXJQFT-UHFFFAOYSA-N 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- UBORTCNDUKBEOP-UUOKFMHZSA-N xanthosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC(=O)NC2=O)=C2N=C1 UBORTCNDUKBEOP-UUOKFMHZSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
- G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Abstract
The invention relates to application of a differential metabolite as a diagnostic marker and a kit, and relates to the technical field of biomedicine, wherein the differential metabolite comprises one or more of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol, beta-cryptoxanthin, alpha-tocopherol, taurocholate, glycocholic acid, 2- (3, 4-dihydroxybenzoyloxy) -4,6-dihydroxybenzoate, arabinosyl hypoxanthine, leucyl hydroxyproline, hypoxanthine, stigmastane-3,6-dione, tyramine ortho-sulfate, phosphatidylcholine, 2' -dehydro-polyoinosine, 1- (4-methoxyphenyl) -2-nitroethylene, hydroxypropionyl histidine, trimethylaminoacetone, creatinine, phenylpyruvic acid, 2-keto-3-deoxy-D-gluconic acid, n-acetyl-L-phenylalanine and D-cysteine hydrochloride monohydrate.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of a differential metabolite as a diagnostic marker and a kit.
Background
Developmental dysplasia of the hip (Developmental Dysplasia of the Hip, DDH) is known as congenital dislocation of the hip (Congenital Dislocation of the Hip, CDH). The earlier the DDH treatment, the simpler the method of treatment, and the easier it is to obtain a normal or near normal hip joint. Ultrasound screening is an important means of early diagnosis. The basic physical examination should be carried out on children of 0-6 months, and ultrasonic examination is carried out on the patients with abnormal physical examination or high risk factors so as to achieve the purposes of early detection and treatment. The uk medical consultation committee (The Standing Medical Advisory Committee, SMAC) recommends that all newborns should incorporate clinical screening for DDH and emphasizes multiple examinations including the day of birth, at discharge, 6 weeks, 6-9 months and after the onset of walking. The american society for orthopedic surgery (TheAmerican Academy of Orthopaedical Surgeons, AAOS) recommends early imaging of those at high risk for DDH. The ultrasonic examination can be used for evaluating the shape, the position and the stability of the hip joint of the hip, and can effectively avoid the adverse effect of the X-ray examination on infants. Graf examination is the earliest ultrasonic examination method for measuring by using coronal section of hip joint, and is the most effective screening and diagnosis method accepted at present. Abnormality is found through standard early ultrasonic screening and is corrected in time through a physical method, so that the requirement of long-term hip joint replacement can be effectively avoided, and the long-term disability rate is reduced.
The development of the children suffering from the dysplasia of hip joint (developmental dysplasia of the hip, DDH) in China accounts for 5 per mill of population, and incomplete epidemiological investigation shows that the disease of the newborn DDH in the high-altitude area of the Tibetan area is 32.4 per mill, which is related to genetic factors, environmental influence and life habits. The treatment rationale should be early-stage for cases diagnosed with DDH, including: (1) obtaining center reset; (2) maintaining a stable reset; (3) promoting the normal growth and development of the hip joint; (4) reducing complications. For DDH infants between 0 and 6 months, the application of hip flexion abduction harnesses or braces is the primary mode of treatment. Pavlik harness has a high success rate for DDH infants less than 3 months, but a significantly lower success rate for infants older than 4 months or Graf IV. The early DDH has the best treatment effect, and if the early DDH is not timely intervened, the early DDH can seriously influence the growth and development of children, if the best treatment time is missed, hip joint pain can occur, and the disease can cause life-long disability after the development of adult severe osteoarthritis, and becomes the most common cause of hip joint replacement under 60 years.
Since DDH has the clinical feature of slightly hidden early symptoms, early diagnosis has been a difficult clinical problem. The DDH high dislocation patients can be diagnosed early through ultrasonic screening in the infant period, but the ultrasonic screening has higher requirements on professional technical capability of personnel, has misdiagnosis and missed diagnosis proportion, especially has a plurality of people in China, wide regional treatment and unbalanced economic development in each region, and only implements comprehensive ultrasonic screening in individual regions, and adopts selective ultrasonic screening in most regions. And combining the ultrasonic screening result to correct or recover the infant suffering from DDH, such as CN110464526A discloses a device for correcting infant developmental hip dysplasia; the child left thigh and right thigh supporting device comprises a first inverted V-shaped wrist support suitable for supporting the left knee wrist of the child, a second inverted V-shaped wrist support suitable for supporting the right knee wrist of the child, and a telescopic adjusting piece used for adjusting the interval between the first inverted V-shaped wrist support and the second inverted V-shaped wrist support so as to prop open the left thigh and right thigh of the child; CN205459267U discloses a developmental dysplasia gypsum mount, including the base, base one side tip intermediate position is fixed with the support, and the fixed sacrum portion tray in support body of rod upper portion, base along width direction bilateral symmetry set up a plurality of base screw, and two long limits of base slide respectively set up a slip buckle, and the carriage both ends are connected respectively on corresponding slip buckle, all offer the buckle screw on every slip buckle, cooperate through the buckle screw and realize the fixed of slip buckle with the base screw, the fixed chest tray in carriage top, chest tray is parallel with the base. These correction devices are all dependent on the results of the Graf method (hip ultrasonic examination), which is a simple device for checking hip development level, generally performed by first fixing the infant to a special examination table and performing hip ultrasonic examination of the infant by means of an ultrasonic probe guide system to obtain a standard image. The Graf method is to take three mark points (lower ilium branch edge of the acetabular bottom, acetabular labrum, section of the middle part of ilium) on a standard image, obtain three lines (base line, bone top line, cartilage top line), measure bone top angle (alpha angle) and cartilage top angle (beta angle), and then perform parting diagnosis according to the measured alpha angle and beta angle combined with the month age of the infant, while inaccurate or nonstandard images can lead to incorrect diagnosis results, cause misdiagnosis or missed diagnosis, even lead to inaccurate correction devices for people suffering from DDH, and delay optimal treatment time.
Based on the above-mentioned prior art situation, there is a technical problem in the prior art that Graf method can not accurately and timely detect and judge whether the infant has developmental hip dysplasia and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides the application of the differential metabolite as a diagnostic marker, namely the application of the differential metabolite as an in vitro diagnostic marker of developmental hip dysplasia, the differential metabolites include 4-beta-Hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol (4-beta-hydroxy-4-alpha-methyl-5-alpha-chol-7-en-3-beta-ol), beta-Cryptoxanthin (beta-Cryptoxanthin), alpha-Tocopherol (alpha-tocopheryl), taurocholate (Taurocholic acid), glycocholic acid (Glycocholic acid), 2- (3, 4-dihydroxybenzoyl-4, 6-dihydroxybenzoate (2- (3, 4-dihydroxybenzoyl) -4, 6-dihydroxybenzoate), arabinosyl Hypoxanthine (Arabinoxanthine), leucyl-Hydroxyproline (Leuc-Hydroxyproline), hypoxanthine (alpha-tocopheryl), taurocholate (taurocholate), glycocholic acid (Glycocholic acid), 2- (3, 4-dihydroxybenzoyl) -4,6-dihydroxybenzoate (2- (3, 6-dihydroxybenzoate) -4, 6-dihydroxybenzoate), arabinothioxanthine (arabinopyranol), xanthosine (xanthophylline), xanthophylline (Leucyl-Hydroxyproline), xanthophylline (2-hydroxy-hydrochloride), and (3, 6-dihydroxybenzoyl-5-dihydroxybenzoate (3-hydroxy-5-hydroxy-lactone), and 7-hydroxy-5-lactone (11-hydroxy-methyl-5-lactone), and 2-hydroxy-2-methyl-5-beta-lactone (beta-hydroxy-beta-lactone) and (beta-hydroxy-beta-alcohols) One or more of 1- (4-Methoxyphenyl) -2-nitroethylene (1- (4-Methoxyphenyl) -2-nitroethylene), hydroxypropionyl Histidine (hydroxypropyl-Histidine), trimethylaminoacetone (trimethyolamineacetone), creatinine (Creatinine), phenylpyruvic acid (phenyl pyruvic acid), 2-Keto-3-deoxy-D-gluconic acid (2-Keto-3-deoxy-D-gluconic acid), N-Acetyl-L-phenylalanine (N-Acetyl-L-phenylalanine), and D-cysteine hydrochloride monohydrate (3-sulfato-L-alaninate).
Further, the screening method of the differential metabolite comprises the following steps:
(1) Sample preparation: dividing into a case group and a control group, wherein the sample is a serum sample;
(2) Extraction and analysis of metabolites: respectively transferring samples of the case group and the control group in the step (1), and adding an extracting solution, wherein the volume ratio of the samples to the extracting solution is 1: (4-5), the extracting solution comprises methanol and acetonitrile, the extracting solution is subjected to vortex mixing for 30-60 seconds and ice water bath ultrasonic treatment for 10-20 minutes in sequence, a mixture A is obtained after standing for 1-2 hours at the temperature of minus 40 ℃, the mixture A is centrifuged for 15-30 minutes at the rotating speed of 12000 rpm at the temperature of 4 ℃, and the supernatant after centrifugation is analyzed by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry analysis technology to obtain original data;
(3) And (3) data processing: processing the original data obtained in the step (2), and analyzing the processed data to determine the structure and name of the metabolite;
(4) Verification analysis: quantifying the diagnostic performance of the metabolite in step (3) by analysis of the subject's working characteristics curve (ROC curve), and when the area under the curve (Area under the curve, AUC) of the metabolite selected is 0.7.ltoreq.AUC.ltoreq.1.0, determining that the metabolite is a differential metabolite, the differential metabolite being a diagnostic marker for determining whether the diagnosed person has developmental hip dysplasia (DDH).
Further, the case group and the control group are grouped using the Graf method (hip ultrasonography) as a diagnostic standard in the step (1).
Further, the volume ratio of methanol to acetonitrile in the extracting solution is 1:1.
further, the extracting solution also contains an isotope labeling compound used for making an internal standard.
Further, the ultra performance liquid chromatograph in the step (2) is a Vanquish (Thermo Fisher Scientific) ultra performance liquid chromatograph, wherein the detection conditions are as follows: a WatersACQUITY UPLCBEH Amide (2.1 mm ×100 mm, 1.7 μm) liquid chromatographic column is selected, the liquid chromatographic column A phase is an aqueous phase, 25 mmol/L ammonium acetate and 25 mmol/L ammonia water are contained, the B phase is acetonitrile, the temperature of a sample tray is 4 ℃, the sample injection volume is 2 μl, and the samples in the sample tray are the supernatant extracted in the step (2).
Further, in the step (2), the mass spectrum is a Orbitrap Exploris mass spectrometer, and the mass spectrometer is controlled by control software (Xcalibur, version: 4.4, thermo) to perform primary and secondary mass spectrum data acquisition, wherein specific parameters are as follows: sheath gas flow rate:50 Arb, aux gas flow rate:15 Arb, capillary temperature:320 ℃, full MS resolution:60000, MS/MS resolution: 15000,Collision energy:10/30/60 in NCE mode, spray voltage:3.8 kV (positive) or-3.4 kV (negative).
Further, the processing method in the step (3) includes that after the original data is converted into mzXML format by Proteowizard software, R program package (the kernel is XCMS software) is used for carrying out peak identification, peak extraction, peak alignment and integral processing, and then the metabolites are annotated by matching with BiotreDB (V2.1) secondary mass spectrum database.
Further, the value of the Cutoff of the algorithmic score in the processing of the raw data in the step (3) is set to 0.3, and the Cutoff value is a critical point set according to statistical analysis.
Further, the area under ROC curve (AUC) in step (4) is between 1.0 and 0.5, the closer the AUC is to 1 when AUC >0.5, the better the effect of the use of the differential metabolite as an in vitro diagnostic marker of developmental hip dysplasia; the accuracy is lower when AUC is more than 0.5 and less than 0.7, and the accuracy is higher when AUC is more than or equal to 0.7 and less than 0.9; higher accuracy is achieved when AUC is greater than or equal to 0.9, and no value is achieved when auc=0.5.
Further, the differential metabolite has an AUC of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol of 0.882 or beta-cryptoxanthin of 0.8544, or alpha-tocopherol of 0.8496, or 2 '-dehydropolyglucofuran of 0.8464, or 1- (4-methoxyphenyl) -2-nitroethylene of 8258, or 2- (3, 4-dihydroxybenzoyloxy) -4,6-dihydroxybenzoate of 8258, or arabinosyl hypoxanthine of 0.8624, or leucyl hydroxyproline of 0.8592, or hypoxanthine of 0.8592, or stigmane-3, 6-dione of 0.8544, or tyramine o-sulfate of 0.8496, or phosphatidylcholine of 0.8496, or 2' -dehydropolyglucofuran of 0.8464, or 1- (4-methoxyphenyl) -2-nitroethylene of AUC of 8224, or hydroxyacetone of N-5-D, or D-glutamic acid of the amino acid hydrochloride of 8224, or the amino acid of which is more than or equal to that of D-80, or N-acetyl-3, or D-glutamic acid of the amino acid is more than or of the amino acid is equal to or more than 3.3930.88.
Further, the AUC of the combination of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol and beta-cryptoxanthin in the differential metabolite is more than or equal to 0.91.
Further, the AUC of the combination of the alpha-tocopherol and the taurocholate in the differential metabolite is more than or equal to 0.935.
Further, the equal volume of supernatant of all samples in step (2) is mixed to prepare Quality Control (QC) samples for process quality control, wherein the process quality control steps are as follows: mixing QC sample with the extracting solution in the step (2), wherein the volume ratio of the QC sample to the extracting solution is the same as that of the sample and the extracting solution in the step (2), the extracting solution is also the same as that of the extracting solution in the step (2), vortex mixing for 30-60s and ice water bath ultrasonic for 10-20min are sequentially carried out, standing for 1-2h at-40 ℃ to obtain a mixture B, centrifuging the mixture B at the rotating speed of 12000 rpm at 4 ℃ for 15-30 min, analyzing the supernatant after centrifuging by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry analysis technology to obtain control data, the processing of the control data is the same as that of the original data in the step (3), the control data are used for determining the instrument state and evaluating the stability of the system in the experimental process of the whole screening method, namely 1 group of control data are acquired after 10-20 groups of original data are acquired, each group of control data are compared, the instrument state is determined and the stability of the system is evaluated according to an extracted ion flow chart (EIC chart), a Principal Component Analysis (PCA) score chart, a PCA-X one-dimensional distribution chart and a correlation analysis chart which are acquired after the QC sample control data are processed, in theory, QC samples are identical, but the instrument and the system have errors in the material extraction and detection analysis processes, so that the difference among QC samples is caused, and the smaller difference indicates that the stability of the whole screening method is better and the data quality is higher.
The invention also provides a kit comprising any of the aforementioned differential metabolites.
The invention has the beneficial effects that:
1. compared with the in-vitro diagnosis marker for developmental hip dysplasia, the in-vitro diagnosis marker for developmental hip dysplasia is easy to cause incorrect diagnosis results due to inaccurate or nonstandard images by using a Graf method in the prior art, so that the accuracy is greatly improved, and especially, whether the infant suffers from DDH in early stage or not can be detected, and the infant suffering from DDH can be intervened and treated in time, so that the in-vitro diagnosis marker for developmental hip dysplasia has important value;
2. the kit containing the differential metabolite provides more choices and supports for the application of the differential metabolite as an in-vitro diagnosis marker of developmental hip dysplasia and the research thereof, is easy to operate, can be used for rapid batch detection, has high detection accuracy and has wide industrial popularization value;
3. the invention provides diagnostic markers, wherein the AUC value of a single differential metabolite in the ROC curve is more than 0.8, the accuracy is certain, when two differential metabolites are combined, the AUC value of the single differential metabolite in the ROC curve is more than 0.9, and the accuracy is higher, namely, the differential metabolite is used as the diagnostic marker for in-vitro diagnosis of developmental hip dysplasia, one or more of the differential metabolites can be optionally combined, and the more the selected variety is, the more accurate the result is.
Drawings
FIG. 1 is a metabolite heat map of the case group and the control group in example 1 of the present invention;
FIG. 2 is a ROC curve of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol in the differential metabolite of example 1 of the present invention;
FIG. 3 is a ROC curve of β -cryptoxanthin in the differential metabolite of example 1 of the present invention;
FIG. 4 is a ROC curve of alpha-tocopherol in the differential metabolite of example 1 of the present invention;
FIG. 5 is a ROC curve of taurocholate in differential metabolites of example 1 of the present invention;
FIG. 6 is an EIC diagram of internal standard positive ions in QC sample of example 1 of the present invention;
FIG. 7 is an EIC chart of internal standard anions in QC sample of example 1 of the present invention;
FIG. 8 is a positive ion PCA score of QC sample of example 1 of the present invention;
FIG. 9 is a PCA score chart of QC sample negative ions of example 1 of the present invention;
FIG. 10 is a one-dimensional PCA-X distribution diagram of QC sample in positive ion mode according to example 1 of the present invention;
FIG. 11 is a one-dimensional PCA-X distribution diagram of QC sample in anion mode in example 1 of the present invention;
FIG. 12 is a correlation analysis chart of positive ion QC sample in example 1 of the present invention;
FIG. 13 is a graph showing correlation analysis of negative ion QC samples in example 1 of the present invention;
FIG. 14 is a ROC curve for the combination of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol and beta-cryptoxanthin in the differential metabolite of example 2 of the present invention;
FIG. 15 is a ROC curve of the combination of alpha-tocopherol and taurocholate in the differential metabolite according to example 2 of the present invention;
FIG. 16 is an X-ray diagram showing the position of the diagnostician A in example 2 of the present invention;
FIG. 17 is an X-ray diagram showing the position of the A frog of the diagnostician in example 2 of the present invention.
Detailed Description
The raw material information used in the examples of the present invention is shown in table 1:
TABLE 1 raw material information
The experimental instrument information used in the examples of the present invention is shown in table 2:
table 2 laboratory instrument information
The samples in example 1 of the present invention were grouped into a case group, which is a patient determined to have DDH, and a control group, which is a healthy population determined to have no DDH, using the Graf method as a diagnostic standard.
Example 1
The embodiment provides a screening method of differential metabolites, comprising the following steps:
(1) Sample preparation: the method comprises the steps of dividing the sample into a case group and a control group, wherein each group has 25 test cases and 50 samples, and the samples are serum samples;
(2) Extraction and analysis of metabolites: respectively transferring 50 mu L of test case samples of the case group or the control group in the step (1) to an EP tube (a plastic centrifuge tube), adding 200 mu L of extracting solution (methanol: acetonitrile=1:1 (V/V) containing isotope labeled compounds for use as internal standard), sequentially carrying out vortex mixing for 30 s, ice water bath ultrasonic treatment for 10 min and standing at-40 ℃ for 1 h to obtain a mixture A, centrifuging the mixture A under the conditions that the mixture A is centrifuged for 15 min at the rotation speed of 4 ℃ and 12000 rpm (the centrifugal force is 13800 (x g) and the radius is 8.6 cm), and taking supernatant after centrifugation for analysis by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry technology to obtain original data;
(3) And (3) data processing: processing the original data obtained in the step (2), and analyzing the processed data to determine the structure and name of the metabolite;
(4) Verification analysis: quantifying the diagnostic performance of the metabolite in step (3) by analysis of the subject's working characteristics curve (ROC curve), and when the area under the curve (Area under the curve, AUC) of the metabolite selected is 0.7.ltoreq.AUC.ltoreq.1.0, determining that the metabolite is a differential metabolite, the differential metabolite being a diagnostic marker for determining whether the diagnosed person has developmental hip dysplasia (DDH).
In this example, the differential metabolite selected includes 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-yl alcohol, beta-cryptoxanthin, alpha-tocopherol, taurocholate, glycocholic acid, 2- (3, 4-dihydroxybenzoyloxy) -4,6-dihydroxybenzoate, arabinosyl hypoxanthine, leucyl hydroxyproline, hypoxanthine, stigmastane-3,6-dione, tyramine ortho sulfate, phosphatidylcholine, 2' -dehydro-polyginositolxanthin, 1- (4-methoxyphenyl) -2-nitroethylene, hydroxypropionyl histidine, trimethylaminoacetone, creatinine, phenylpyruvic acid, 2-keto-3-deoxy-D-gluconic acid, n-acetyl-L-phenylalanine, D-cysteine hydrochloride monohydrate.
The ultra performance liquid chromatograph in the step (2) is a Vanquish (Thermo Fisher Scientific) ultra performance liquid chromatograph, wherein the detection conditions are as follows: a WatersACQUITY UPLCBEH Amide (2.1 mm ×100 mm, 1.7 μm) liquid chromatographic column is selected, the liquid chromatographic column A phase is an aqueous phase, 25 mmol/L ammonium acetate and 25 mmol/L ammonia water are contained, the B phase is acetonitrile, the temperature of a sample tray is 4 ℃, the sample injection volume is 2 μl, and the samples in the sample tray are the supernatant extracted in the step (2).
In the step (2), the mass spectrum is a Orbitrap Exploris mass spectrometer, and the mass spectrometer performs primary and secondary mass spectrum data acquisition under the control of control software (Xcalibur, version: 4.4, thermo), and specific parameters are as follows: sheath gas flow rate:50 Arb, aux gas flow rate:15 Arb, capillary temperature:320 ℃, full MS resolution:60000, MS/MS resolution: 15000,Collision energy:10/30/60 in NCE mode, spray voltage:3.8 kV (positive) or-3.4 kV (negative).
The processing method in the step (3) includes that after original data is converted into mzXML format through Proteowizard software, the R program package (with the kernel of XCMS) is used for carrying out peak identification, peak extraction, peak alignment and integral processing, and then the R program package is matched with a BiotreeDB (V2.1) secondary mass spectrum database to annotate metabolites, wherein the Cutoff value of algorithm scoring is set to 0.3, and the Cutoff value is a critical point set according to statistical analysis.
As shown in FIG. 1, the metabolite heat maps of the case group and the control group show that the various metabolites in the case group and the control group are changed, and when the area under the curve (Area under the curve, AUC) of the selected metabolite is 0.7.ltoreq.AUC.ltoreq.1.0, the metabolite can be determined as a differential metabolite.
In step (4), the subject's working characteristics are plotted according to a series of different classification schemes (score=demarcation value or decision threshold), with true positive rate (sensitivity) on the ordinate and false positive rate (1-specificity) on the abscissa. ROC curves were plotted for each well-defined differential metabolite and the area under the curve (Area under the curve, AUC) was calculated. In this example, the names and corresponding AUC values of the differential metabolites selected are shown in table 3. Wherein the area under ROC curve (AUC) is between 1.0 and 0.5, the closer the AUC is to 1 when under AUC >0.5, the better the effect of the differential metabolite for use as an in vitro diagnostic marker of developmental hip dysplasia; the accuracy is lower when AUC is more than 0.5 and less than 0.7, and the accuracy is higher when AUC is more than or equal to 0.7 and less than 0.9; higher accuracy is achieved when AUC is greater than or equal to 0.9, and no value is achieved when auc=0.5.
TABLE 3 names of differential metabolites (21 total differential metabolites) and corresponding AUC values
In this example, as shown in FIG. 2, the ROC curve of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol in the differential metabolite, the ROC curve of beta-cryptoxanthin in the differential metabolite, the ROC curve of alpha-tocopherol in the differential metabolite, and the ROC curve of taurocholate in the differential metabolite are shown in FIG. 3, and the ROC curve of taurocholate in the differential metabolite are shown in FIG. 5.
In this embodiment, the process quality control is: equal volumes (50 μl each) of supernatants from all samples (50 test cases) were mixed to prepare Quality Control (QC) samples, mixed well, and 50 μl QC samples were removed to EP tubes (plastic centrifuge tubes) and 200 μl of extract (methanol: acetonitrile=1:1 (V/V), containing isotopically labeled compound, for use as internal standard), mixing by vortexing for 30 s, ice water bath ultrasound for 10 min, standing at-40 ℃ for 1 h to obtain mixture B, centrifuging the mixture B under such conditions that the mixture B is centrifuged at 4 ℃ and 12000 rpm (centrifugal force is 13800 (X g) and radius is 8.6 cm) for 15 min, collecting supernatant, analyzing by ultra-high performance liquid chromatography-tandem mass spectrometry analysis technique to obtain control data, wherein the control data is processed by the same method as the original data in step (3), namely, 1 group of control data is obtained after 10 groups of original data are obtained, 6 groups are counted, and the numbers are QC01, QC02, QC03, QC04, QC05 and QC06, and each group of control data is compared to obtain an internal standard positive ion EIC (fig. 6), an internal standard negative ion EIC (fig. 7), a positive ion PCA (fig. 8), a negative ion PCA (fig. 9), a one-dimensional ion PCA (fig. 12) in the QC sample, and a one-dimensional PCA (fig. 12) in the one-dimensional sample (fig. 12) Negative ion QC sample correlation analysis graph (fig. 13) to evaluate the stability of the system. The main component in the figure is the differential metabolite in this example.
Whether the detection system is stable or not can be judged through the difference of response peak heights of internal standards among QC samples, and the retention time and the response intensity stability of the internal standards in the QC samples can be seen from the figures 6 and 7, so that the instrument data acquisition stability is good; from FIGS. 8 and 9, it can be seen that QC samples (circled portions in the figures) have good aggregation, which indicates that the screening method has good stability; from FIGS. 10 and 11, it can be seen that 6 QC samples (QC 01, QC02, QC03, QC04, QC05, QC 06) are all within.+ -. 2 std, which indicates that the experimental data of this example is very high in quality; it can also be seen from FIGS. 12 and 13 that QC samples have high correlation (QC samples have correlation close to 1), further demonstrating that the whole screening method has good stability and high data quality.
Example 2
This example is an experiment performed without knowing whether or not the diagnosed person a has DDH:
wherein the age of diagnosed person A is 1 year old.
The step of judging whether the diagnosed person A has DDH is:
(1) Sample preparation: taking serum of a diagnosed person A as a sample;
(2) Extraction and analysis of markers: transferring 50 mu L of the serum sample in the step (1) into an EP tube, adding 200 mu L of extracting solution (methanol: acetonitrile=1:1 (V/V) containing isotope labeled compounds for use as internal standard), sequentially carrying out vortex mixing for 30 s, ice water bath ultrasonic treatment for 10 min and standing for 1 h at minus 40 ℃ to obtain a mixture A, centrifuging the mixture A under the conditions that the mixture A is centrifuged for 15 min at the temperature of 4 ℃ and the rotation speed of 12000 rpm (the centrifugal force is 13800 (x g) and the radius is 8.6 cm), and analyzing the supernatant after centrifugation by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry analysis technology to obtain original data;
(3) And (3) data processing: processing the raw data obtained in the step (2), selecting a differential metabolite from metabolites, and analyzing the processed data to determine the structure and name of the corresponding differential metabolite, wherein in the embodiment, the differential metabolite is the combination of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol and beta-cryptoxanthin, and the combination of alpha-tocopherol and taurocholate;
(4) Verification analysis: validation of the assay for differential metabolites using subject work profile (ROC profile), auc=0.91 for the combination of 4- β -hydroxymethyl-4- α -methyl-5- α -cholest-7-en-3- β -ol and β -cryptoxanthin (fig. 14), auc=0.935 for the combination of α -tocopherol and taurocholate (fig. 15), conclusions were drawn that diagnosed a had DDH;
(5) Rechecking results: in order to ensure the correctness of the results, the present embodiment further analyzes the diagnosed person a by using X-ray, and the analysis results are shown in fig. 16 (righting position indication) and fig. 17 (frog position indication), so that it can be seen that the bilateral hip joint of the diagnosed person a is dislocated, the bilateral femoral head development is delayed, and the rechecking conclusion is that the diagnosed person a has DDH.
In summary, in the application of the differential metabolite provided by the invention as the in-vitro diagnostic marker of developmental hip dysplasia, the AUC value of a single differential metabolite in an ROC curve is more than 0.8, the accuracy is certain, when two differential metabolites are used together, the AUC values in the ROC curve are more than 0.9, namely, one or more of the differential metabolites can be optionally combined, the more the types of the differential metabolites are selected, the more accurate the result is, compared with the condition that the Graf method in the prior art easily causes incorrect diagnostic results due to inaccurate or nonstandard images, misdiagnosis or missed diagnosis is caused, the accuracy is greatly improved, and especially, whether the infant suffering from DDH is detected in early stage or not can be detected, and the method has important value for intervention and treatment of the infant suffering from DDH in time.
It will be understood that the invention is not limited to what has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the invention is limited only by the appended claims.
Claims (8)
1. Use of a differential metabolite comprising one or more of 4-beta-hydroxymethyl-4-alpha-methyl-5-alpha-cholest-7-en-3-beta-ol, beta-cryptoxanthin, alpha-tocopherol, taurocholate, glycocholic acid, 2- (3, 4-dihydroxybenzoyloxy) -4,6-dihydroxybenzoate, arabinosyl hypoxanthine, leucyl hydroxyproline, hypoxanthine, stigmastane-3,6-dione, tyramine ortho-sulfate, phosphatidylcholine, 2' -dehydro-inositol yellow, 1- (4-methoxyphenyl) -2-nitroethylene, hydroxypropionyl histidine, trimethylaminoacetone, creatinine, phenylpyruvic acid, 2-keto-3-deoxy-D-gluconic acid, n-acetyl-L-phenylalanine, D-cysteine hydrochloride monohydrate as a diagnostic marker.
2. Use of a differential metabolite according to claim 1 as a diagnostic marker, characterized in that the screening method of the differential metabolite comprises the steps of:
(1) Sample preparation: dividing into a case group and a control group, wherein the sample is a serum sample;
(2) Extraction and analysis of metabolites: respectively transferring samples of the case group and the control group in the step (1), and adding an extracting solution, wherein the volume ratio of the samples to the extracting solution is 1: (4-5), the extracting solution comprises methanol and acetonitrile, the extracting solution is subjected to vortex mixing for 30-60 seconds and ice water bath ultrasonic treatment for 10-20 minutes in sequence, a mixture A is obtained after standing for 1-2 hours at the temperature of minus 40 ℃, the mixture A is centrifuged for 15-30 minutes at the rotating speed of 12000 rpm at the temperature of 4 ℃, and the supernatant after centrifugation is analyzed by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry analysis technology to obtain original data;
(3) And (3) data processing: processing the original data obtained in the step (2), and analyzing the processed data to determine the structure and name of the metabolite;
(4) Verification analysis: quantifying the diagnostic performance of the metabolite in the step (3) by ROC curve analysis, and determining the metabolite to be a differential metabolite when the AUC of 0.7-1.0 of the selected metabolite is less than or equal to the 0.7, wherein the differential metabolite is used as a diagnostic marker for determining whether a diagnosed person suffers from developmental hip dysplasia.
3. The use of a differential metabolite as diagnostic marker according to claim 2, characterized in that in step (1) the case group and the control group are grouped using the Graf method as diagnostic standard.
4. The use of a differential metabolite according to claim 2 as a diagnostic marker, wherein the volume ratio of methanol to acetonitrile in the extract of step (2) is 1:1.
5. the use of a differential metabolite according to claim 2 as a diagnostic marker, characterized in that the AUC of 4- β -hydroxymethyl-4- α -methyl-5- α -cholest-7-en-3- β -ol in the differential metabolite is equal to or greater than 0.882, or the AUC of β -cryptoxanthin is equal to or greater than 0.886, or the AUC of α -tocopherol is equal to or greater than 0.875, or the AUC of taurocholic acid is equal to or greater than 0.866, or the AUC of glycocholic acid is equal to or greater than 0.8656, or the AUC of 2- (3, 4-dihydroxybenzoyloxy) -4,6-dihydroxybenzoate is equal to or greater than 0.864, or the AUC of arabinosyl hypoxanthine is equal to or greater than 0.8624, or the AUC of leucyl hydroxyproline is equal to or greater than 0.8592, or the AUC of hypoxanthine is equal to or greater than 0.8592, or the AUC of stigmastane-3,6-dione is equal to or greater than 0.8544, or the AUC of tyramine ortho-sulfate is equal to or greater than 0.8496, or the AUC of phosphatidylcholine is equal to or greater than 0.8496, or the AUC of 2' -dehydro-polyinositole is equal to or greater than 0.8464, or the AUC of 1- (4-methoxyphenyl) -2-nitroethylene is equal to or greater than 0.8368, or the AUC of hydroxypropionyl histidine is equal to or greater than 0.8352, or the AUC of trimethylaminoacetone is equal to or greater than 0.8288, or the AUC of creatinine is equal to or greater than 0.8288, or the AUC of phenylpyruvic acid is equal to or greater than 0.816, or the AUC of 2-keto-3-deoxy-D-gluconic acid is equal to or greater than 0.808, or the AUC of n-acetyl-L-phenylalanine is equal to or greater than 0.8064, or the AUC of D-cysteine hydrochloride monohydrate is equal to or greater than 0.8032.
6. The use of a differential metabolite according to claim 5 as diagnostic marker, characterized in that the AUC of the combination of 4- β -hydroxymethyl-4- α -methyl-5- α -cholest-7-en-3- β -ol and β -cryptoxanthin in the differential metabolite is not less than 0.91.
7. The use of a differential metabolite according to claim 5 as diagnostic marker, characterized in that the AUC of the combination of α -tocopherol and taurocholate in the differential metabolite is not less than 0.935.
8. A kit comprising the differential metabolite of any one of claims 1-7.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU1817854C (en) * | 1990-03-05 | 1993-05-23 | Республиканский Научно-Исследовательский Центр Охраны Здоровья Матери И Ребенка Минздрава Республики Казахстан | Method of predication of congenital pathology of children |
| WO2004045369A2 (en) * | 2002-11-14 | 2004-06-03 | Nura, Inc. | Nuclear receptor-based diagnostic, therapeutic, and screening methods |
| RU2296337C1 (en) * | 2005-10-11 | 2007-03-27 | Федеральное государственное учреждение "Нижегородский научно-исследовательский институт травматологии и ортопедии Федерального агентства по здравоохранению и социальному развитию" | Method for differential predicting the complications of inborn femoral dislocation in children and teenagers |
| US20110212090A1 (en) * | 2008-07-23 | 2011-09-01 | Dako Denmark A/S | Combinatorial Analysis and Repair |
| CN105194690A (en) * | 2015-11-03 | 2015-12-30 | 上海交通大学医学院附属新华医院 | Application of long-chain non-coding RNA (ribonucleic acid) in treatment of DDH (developmental dysplasia of hip) disease |
| US20210239711A1 (en) * | 2020-02-05 | 2021-08-05 | The Curators Of The University Of Missouri | Biomarkers for early diagnosis of chronic hip displasia and methods of using the same |
-
2023
- 2023-07-06 CN CN202310822021.0A patent/CN116539901A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU1817854C (en) * | 1990-03-05 | 1993-05-23 | Республиканский Научно-Исследовательский Центр Охраны Здоровья Матери И Ребенка Минздрава Республики Казахстан | Method of predication of congenital pathology of children |
| WO2004045369A2 (en) * | 2002-11-14 | 2004-06-03 | Nura, Inc. | Nuclear receptor-based diagnostic, therapeutic, and screening methods |
| RU2296337C1 (en) * | 2005-10-11 | 2007-03-27 | Федеральное государственное учреждение "Нижегородский научно-исследовательский институт травматологии и ортопедии Федерального агентства по здравоохранению и социальному развитию" | Method for differential predicting the complications of inborn femoral dislocation in children and teenagers |
| US20110212090A1 (en) * | 2008-07-23 | 2011-09-01 | Dako Denmark A/S | Combinatorial Analysis and Repair |
| CN105194690A (en) * | 2015-11-03 | 2015-12-30 | 上海交通大学医学院附属新华医院 | Application of long-chain non-coding RNA (ribonucleic acid) in treatment of DDH (developmental dysplasia of hip) disease |
| US20210239711A1 (en) * | 2020-02-05 | 2021-08-05 | The Curators Of The University Of Missouri | Biomarkers for early diagnosis of chronic hip displasia and methods of using the same |
Non-Patent Citations (1)
| Title |
|---|
| "Developmental dysplasia of the hip: Risk markers, clinical screening and outcome", PEDIATRICS INTERNATIONAL, vol. 50, no. 03, pages 341 - 345 * |
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