CN108956790A - Application of one group of amino acid tags object in acute kidney injury diagnostic kit - Google Patents
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Abstract
The present invention relates to kit for diagnosing diseases field, specifically one group of amino acid tags object is preparing the application in acute kidney injury diagnostic reagent or kit, and the amino acid tags object is selected from kynurenin, one or more of hippuric acid and Pidolic Acid.3 amino acid tags objects proposed by the present invention can be in the generation of acute kidney injury early stage Accurate Diagnosis disease, and AUC value is all larger than 0.7 when being individually used for diagnosis acute kidney injury, and it is preferable that effect is distinguished in diagnosis.Wherein kynurenin and hippuric acid diagnosis capability are better than clinical common renal function marker creatinine and early diagnosis marker NGAL, and above-mentioned 3 amino acid is to the Diagnostic Time of AKI earlier than clinical renal function marker urea nitrogen.Targeting amino acid metabolism group detection method provided by the invention can be achieved at the same time the quantitative analyses of multiple amino acid, high sensitivity, as a result accurately and reliably.
Description
Technical field
The present invention relates to kit for diagnosing diseases fields, specifically, being three kinds of amino acid tags objects in acute kidney injury
Application in diagnostic kit.
Background technique
Acute kidney injury (AKI) is a kind of impaired morbid state of kidney structure and function, to happen suddenly and lasting kidney function
It can decline and be characterized, be as caused by different pathogeny, be related to the clinical syndrome of each systemic symptom of whole body.Slight injury of kidney can
Voluntarily to restore over time, even if AKI patient is relieved, long-term cardiovascular risk still be will increase.Severe
It must receive dialysis treatment when AKI patient's lesion progress to kidney failure.There are about 2,900,000 inpatients to suffer from AKI for China in 2013,
The death rate about 24%.AKI increases Hospitalization Expenses of Inpatients and the death rate, while causing the waste of medical resource.
Improve in the clinical practice guideline (KDIGO guide) that global kidney trouble prognosis tissue 2012 is formulated with serum creatinine and urine
Amount is standard, and AKI is defined as to the generation of following either case, is increased in SCr 48 hours up to >=0.3mg/dL (26.5 μm of ol/
L)；Or Scr increased in 7 days up to basic value >=1.5 times；Or urine volume < 0.5mL/kg/h continues 6 hours.Glomerular filtration rate
Rapid decrease can be increased by SCr and hypourocrinia is reacted, diagnosis effect be verified extensively.However, serum creatinine
Level variation is more slow, and urine volume is affected to the assessment of glomerular filtration rate by blood volume, based on serum creatinine and urine volume
Variation is difficult to accomplish early detection and accurate evaluation injury of kidney.Research shows that the damage of kidney is can to reverse after acute kidney injury
, the AKI diagnosis of early stage can accurately find that AKI patient makes it obtain timely therapeutic intervention, reduces the degree of injury of kidney.
Excavating new AKI early warning marker helps to solve the underdiagnosis of current AKI, makes patient can be by disease early stage
It was found that.
With the development of genomics, protein science and metabolism group, the excavation research of AKI early diagnosis marker is continuous
Deeply.The specific protein expressed and secreted when presently found AKI early warning marker is mostly renal damage, wherein neutral
Granulocyte gelatinase correlation apolipoprotein (NGAL) report is more, shows preferably to examine in experimental animal and clinical patient
The disconnected effect and time is earlier than conventional sign object creatinine, but its detection method is increasingly complex compared with the small molecules marker such as creatinine, adopts more
It is detected with enzyme-linked immunization.This method sensitivity and specificity are higher, but the cumbersome detection time of operating process is long, and matrix
In metabolin may occur cross reaction interference measurement result with antibody.
Kidney participates in the metabolism of a variety of amino acid and discharge process in vivo, tune of the vivo acid level by renal function
Section, renal function is damaged occurs disorder of amino acid metabolism often.Recent study discovery, a variety of amino acid levels and kidney
The renal function of Disease is significant related to prognosis, as Ng,Ng-Dimethylarginine, symmetrical dimethylarginine, S- adenosine are same
Type cysteine etc..It can be seen that complete renal function plays a significant role in maintaining amino acid cycle level, amino acid is made
A new target group of AKI early diagnosis are likely to become for the small molecule metabolites of metabolic chain terminal.Using targeting amino
Acid metabolic omics technology, which excavates effective amino acid tags object, has important meaning for clinical Rapid&Early diagnosis acute kidney injury
Justice.
Summary of the invention
For overcome the deficiencies in the prior art, the first object of the present invention is that providing 3 can be used for diagnosing acute kidney
The amino acid tags object of damage；The second object of the present invention, which is to provide, a kind of is capable of amino acid tags object described in simultaneous quantitative
Analysis method.Above-mentioned purpose technical scheme is that: one group for diagnosing the plasma ammonia of acute kidney injury
Base acid marker, including kynurenin, hippuric acid and Pidolic Acid.A kind of method of amino acid tags object described in quantitative analysis,
Quantitative analysis carried out to the amino acid tags object by high performance liquid chromatography tandem mass spectrum technology, this method have it is high-throughput and
Highly sensitive feature can satisfy the quantitative detection of metabolin in biological sample.
The first aspect of the present invention provides one group for diagnosing the Amino Acid marker of acute kidney injury, including dog
Urinary ammonia acid, hippuric acid and Pidolic Acid.
The second aspect of the present invention provides one group of amino acid tags object and is preparing acute kidney injury diagnostic reagent or kit
In application, the amino acid tags object be selected from kynurenin, one or more of hippuric acid and Pidolic Acid.
Preferably, the diagnostic reagent or kit are serodiagnosis reagent or diagnostic kit.
Preferably, kynurenin in the diagnostic reagent or kit detection serum, in hippuric acid and Pidolic Acid
One or more kinds of contents.
Preferably, the diagnostic reagent or kit are to be detected in serum using high performance liquid chromatography tandem mass spectrum technology
Kynurenin, the combination of the reagent of one or more of hippuric acid and Pidolic Acid content.
The third aspect of the present invention, provides a kind of acute kidney injury diagnostic reagent or kit, the diagnostic reagent or
It include kynurenin in detection serum, the reagent of one or more of hippuric acid and Pidolic Acid content in kit.
The fourth aspect of the present invention provides kynurenin in detection serum, one of hippuric acid and Pidolic Acid or two
Kind or more the reagent of content preparing the application in acute kidney injury diagnostic kit.
Preferably, the reagent is to detect kynurenin in serum, horse using high performance liquid chromatography tandem mass spectrum technology
The reagent of one or more of uric acid and Pidolic Acid content.
The invention has the advantages that:
Amino acid tags object provided by the invention can be examined in acute kidney injury early stage Accurate Diagnosis disease, each marker
Disconnected value is evaluated with ROC curve analysis, and theoretically area AUC value is between 0.5 to 1 under ROC curve, and value is closer to 1
Diagnostic value is bigger, and AUC value indicates that result has clinical meaning between 0.7 to 0.9.3 amino acidity scales proposed by the present invention
Will object it is big can be individually used for AUC value when diagnosis acute kidney injury in the generation of acute kidney injury early stage Accurate Diagnosis disease
In 0.7, it is preferable that effect is distinguished in diagnosis.Wherein kynurenin and hippuric acid diagnosis capability are better than clinical common renal function marker flesh
Acid anhydride and early diagnosis marker NGAL, above-mentioned 3 amino acid urinate the Diagnostic Time of AKI earlier than clinical renal function marker
Plain nitrogen.Targeting amino acid metabolism group detection method provided by the invention can be achieved at the same time the quantitative analysis of multiple amino acid,
High sensitivity, as a result accurately and reliably.
Detailed description of the invention
Fig. 1 rat blood serum creatinine, urea nitrogen and NGAL concentration time curve；
Fig. 2 rat blood serum different amino acids Concentration-time change curve.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.The instrument or reagent used is not
What is elaborated is conventional instrument or reagent；The experimental working technique not specifically described is those of ordinary skill in the art
Well known conventional practices.
Embodiment 1: the screening of renal ischemia/reperfusion injury rat and sham-operation rat blood serum difference acidic amino acid
One, object and method
1. animal model preparation and grouping
Male Sprague-Dawley (SD) rat, weight 180-200g are mentioned by The 2nd Army Medical College Experimental Animal Center
For.Rat feeding is tested after a week to adapt to environment, and about 25 DEG C of animal house room temperature, relative humidity about 60%, during raising
Rat diet drinking-water freely, keeps each 12 hours light and shades round the clock, and testing preceding 8 hours Rat Fasts can't help water.Animal is divided at random
For control group and AKI group, every group 30, respectively 0 after Reperfu- sion, 2,4,6,10, take a blood sample for 24 hours through retroorbital venous clump, room temperature
Serum sample is separated after blood coagulation.AKI group rat is routinely opened with 10% chloraldurate 0.35mL/100g weight intraperitoneal injection of anesthesia
Abdomen successively exposes bilateral renal, and artery clamp folder closes bilateral renal arteries, unclamps artery clamp kidney after ischemic 50min and restore perfusion,
Layer-by-layer suture.Control rats are not pressed from both sides by above process exposure kidney and close the arteria renalis, sutured abdominal cavity after 50min.
2. main agents
Enzymic creatinine assay method creatinine assay kit (Nanjing is built up, article No. C011-2), urease method determination of urea nitrogen examination
Agent box (Nanjing is built up, article No. C013-2), rat NGAL detect Elisa kit (Wuhan doctor's moral, article No. EK0855), phosphoric acid
Salt buffer (PBS；Shanghai English fine horse biology), bovine serum albumin(BSA) (BSA；Win photo-biological company in Shanghai), hyptafluorobutyric acid (HFBA；
Adamas, Switzerland), formic acid (FA；U.S. world company)；Hippuric acid (Hip), kynurenin (Kyn), symmetrical dimethylarginine
(SDMA), Pidolic Acid (Oxo), glutamine (Gln), 6 kinds of amino acid standards such as amidomalonic acid (AMA) are purchased from Dalian beauty
Logical sequence biotechnology company；Remaining 17 kinds of amino acid standard is purchased from Chinese pharmaceutical biological product and identifies institute (batch number 140624-
200805), including lysine (Lys), tryptophan (Trp), leucine (Leu), isoleucine (Iso), phenylalanine (Phe),
Methionine (Met), threonine (Thr), valine (Val), glycine (Gly), alanine (Ala), arginine (Arg), silk
Propylhomoserin (Ser), L-aminobutanedioic acid (Asp), tyrosine (Tyr), cysteine (Cys), histidine (His), glutamic acid (Glu)；Deuterium
For alanine (L-Ala-d4；North York, Canada), deuterated methionine (L-Met-d3；North York, adds and takes
Greatly), deuterated phenylalanine (L-Phe-d5；North York, Canada), analyze pure methanol and acetonitrile (Merk, Germany).
3. serum amino acid and the detection of other renal function index
The screening of 3.1 serum different amino acids
3.1.1 sample preparation
Using precipitation of protein, precipitating reagent is containing 0.2% formic acid and each interior target acetonitrile solution of 0.4 μ g/mL.Take 50 μ L blood
The precipitating reagent 150 μ L of pre-cooling is added in 1.5mL centrifuge tube in sample, and vortex 30s is placed on ultrasound 5min in ice water and sufficiently precipitates egg
It is white.Then 13000rpm high speed centrifugation is centrifuged 10min.Supernatant liquor is taken to carry out LC-MS/MS analysis, all operations are on ice
It carries out.
3.1.2 LC-MS/MS testing conditions
Chromatography uses ultra performance liquid chromatography (UHPLC, Agilent 1290, USA).Chromatographic column is Agilent
ZORBAX SB-C18 column (5 μm, 3.0mm × 150mm), 4 DEG C of autosampler temperature, 50 DEG C of column temperature, 2 μ L of sampling volume, flowing
It is mutually A (methanol solution), B (aqueous solution containing 0.2% formic acid and 0.02% hyptafluorobutyric acid), flow velocity 0.4mL/min；Gradient is washed
De- condition: 0~1min is 98~85%B phase, and 1~4min keeps 85%B phase to elute, and 4~5min is 85-80%B phase, 5-
9.5min is 80-20%B phase.Data acquisition time is 9.5min, rear runing time 3.5min, when completing primary analysis always operation
Between 13min.
Mass spectral analysis uses triple quadrupole rods tandem mass spectrometries (Agilent 6460QQQ, USA).Electric spray ion source
(ESI), scanning mode is multiple-reaction monitoring (MRM) mode, positive ion mode monitoring, spray voltage 5000V；Atomizing pressure is
50psi；Dry 325 DEG C of flow velocitys of temperature degree are 10L/min；350 DEG C of flow velocity 12L/min of sheath temperature degree.23 components to be measured and 3
The detection parameters of internal standard compound are shown in Table 1, including F value (Fragmentor), CE value (Collisine Energy), parent ion
(Precusor Ion), daughter ion (Product Ion).
Each amino acid standard curve range are as follows: Cys and Hip (0.008-12 μ g/mL)；Kyn, Asp, SDMA, Arg (0.08-
8μg/mL)；AMA, His, Met, Ser (0.05-12 μ g/mL)；Glu, Thr, Gln (0.8-40 μ g/mL)；Iso, Leu, Tyr,
Phe, Lys, Val (2-48 μ g/mL)；Gly, Oxo (2-60 μ g/mL)；Ala(4-120μg/mL)
The MS detection parameters of table 1 23 kinds of amino acid and 3 internal standard compounds
3.2 creatinine urea nitrogens and NGAL detection
Serum creatinine level is measured using enzymic creatinine assay method, urease method measures serum urea nitrogen level, enzyme linked immunological
It is horizontal that method measures serum N GAL.The detection of each index is all made of commercial kit, and detection process requires to carry out to specifications
Operation.
4. data process&analysis
Statistical analysis uses SPSS19.0 software, since each Testing index disobeys normal distribution, with median ± tetra- point
Position is away from expression.Mean value compares using non-parametric rank sum test between two groups, and p < 0.05 thinks that difference has statistical difference.Difference ammonia
The screening of base acid is carried out using 13.0 software of SIMCA, by establishing OPLS-DA (orthogonal partial least squares discriminant analysis) model, is sought
It looks between acute kidney injury rat and sham-operation rat and contributes larger (VIP > 1.0；P < 0.05) different amino acids.
Two, result
Two groups of rat baseline renal function index are consistent, and the postoperative serum creatinine of AKI group rat and urea nitrogen levels increase immediately,
2 hours serum creatinines of Reperfu- sion and urea nitrogen are significantly higher than control group, hereafter continue to be increased to reach maximum value in 24 hours.By above
Renal ischemia/reperfusion injury known to renal function index variation causes rat acute injury of kidney, Animal Model success.AKI group rat
The horizontal progressive of serum N GAL increases (Fig. 1) consistent with document report.
Screening obtains 3 different amino acids, respectively kynurenin, hippuric acid and Pidolic Acid altogether；Above-mentioned amino acid exists
The selected different amino acids of each detection time point after AKI occurs.Above-mentioned 3 serum different amino acids press horizontal mobile after AKI occurs
Fig. 2 is shown in state variation.Kynurenine levels increase immediately after kidney injury, reach peak within Reperfu- sion 1 hour, hereafter gradually return
It falls but is consistently higher than control group.Hippuric acid level is significantly higher than control group in 1 hour after kidney injury, and hereafter 24 hours
Interior lasting raising.Serum Pidolic Acid level gradually rises after AKI occurs, until Reperfu- sion 10 hours begin to decline；Control group is big
The horizontal Reperfu- sion of mouse Pidolic Acid is presented in 4 hours and is increased by a small margin, then begins to decline, until increasing again after 12 hours.By
This can be used in acute kidney injury as it can be seen that above-mentioned 3 different amino acids levels show significantly to change after acute kidney injury
Early diagnosis.
Embodiment 2: building ROC curve compares the ability of 3 serum different amino acids diagnosis acute kidney injury
Using the assessment of the above-mentioned different amino acids diagnosis capability of receiver operating curves (ROC) progress, and and creatinine, urea
Nitrogen and NGAL are compared.Above-mentioned 3 different amino acids, traditional renal function marker creatinine and urea nitrogen, and early diagnosis
Marker NGAL, which is individually used for diagnosis AKI, the results are shown in Table 1.The results show that kynurenin, 3 ammonia of hippuric acid and Pidolic Acid
The ability that base acid marker is individually used for diagnosis acute kidney injury is stronger, and area (AUC) is all larger than 0.7 under ROC curve, has and faces
Bed diagnostic significance.1 hour 3 amino acid tags object of beginning and creatinine and NGAL level are shown preferably after kidney injury
AKI diagnosis capability, diagnosis capability is by being followed successively by kynurenin, hippuric acid, NGAL, creatinine and Pidolic Acid to weak by force；AUC value point
It Wei 1.00,0.97,0.91,0.82 and 0.79.Urea nitrogen levels more lag the diagnosis of AKI, open within 2 hours after kidney injury
Begin that there is diagnostic effect.It can be seen that kynurenin and hippuric acid are to the diagnosis capability of AKI better than common kidney in clinic at present
Functional parameter creatinine and early sign object NGAL, three different amino acids are on Diagnostic Time earlier than the common renal function of clinic
Index urea nitrogen.Therefore, above-mentioned 3 amino acid can be used as acute kidney injury diagnosis marker.
The diagnosis situation of 1 rat blood serum amino acid tags object of table, creatinine, urea nitrogen and NGAL to AKI
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (7)
1. one group of amino acid tags object is preparing the application in acute kidney injury diagnostic reagent or kit, the amino acidity scale
Will object is selected from kynurenin, one or more of hippuric acid and Pidolic Acid.
2. one group of amino acid tags object according to claim 1 is in preparing acute kidney injury diagnostic reagent or kit
Using, which is characterized in that the diagnostic reagent or kit is serodiagnosis reagent or diagnostic kit.
3. one group of amino acid tags object according to claim 1 is in preparing acute kidney injury diagnostic reagent or kit
Using kynurenin in, which is characterized in that the diagnostic reagent or kit detection serum, in hippuric acid and Pidolic Acid
One or more kinds of contents.
4. one group of amino acid tags object according to claim 1 is in preparing acute kidney injury diagnostic reagent or kit
Using, which is characterized in that the diagnostic reagent or kit is to detect serum using high performance liquid chromatography tandem mass spectrum technology
Middle kynurenin, the combination of the reagent of one or more of hippuric acid and Pidolic Acid content.
5. a kind of acute kidney injury diagnostic reagent or kit, which is characterized in that include in the diagnostic reagent or kit
Detect kynurenin in serum, the reagent of one or more of hippuric acid and Pidolic Acid content.
6. detecting kynurenin in serum, the reagent of one or more of hippuric acid and Pidolic Acid content is anxious in preparation
Application in property injury of kidney diagnostic kit.
7. kynurenin in detection serum according to claim 6, one or both of hippuric acid and Pidolic Acid with
The reagent of upper content is preparing the application in acute kidney injury diagnostic kit, which is characterized in that the reagent is using high
Effect liquid phase chromatogram tandem mass spectrometry detects kynurenin in serum, and one or more of hippuric acid and Pidolic Acid contain
The reagent of amount.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907569A (en) * | 2019-12-25 | 2020-03-24 | 山东英盛生物技术有限公司 | Method for simultaneously detecting 4 protein-bound uremia toxins in blood sample |
| CN112180017A (en) * | 2019-11-20 | 2021-01-05 | 南京品生医学检验实验室有限公司 | Method and kit for detecting ADMA (adenosine triphosphate) and SDMA (space division multiple Access) in plasma by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN114487214A (en) * | 2022-01-24 | 2022-05-13 | 广州市番禺区中心医院 | Biomarker for distinguishing benign prostatic hyperplasia and prostatitis and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112180017A (en) * | 2019-11-20 | 2021-01-05 | 南京品生医学检验实验室有限公司 | Method and kit for detecting ADMA (adenosine triphosphate) and SDMA (space division multiple Access) in plasma by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN110907569A (en) * | 2019-12-25 | 2020-03-24 | 山东英盛生物技术有限公司 | Method for simultaneously detecting 4 protein-bound uremia toxins in blood sample |
| CN114487214A (en) * | 2022-01-24 | 2022-05-13 | 广州市番禺区中心医院 | Biomarker for distinguishing benign prostatic hyperplasia and prostatitis and application thereof |
| CN114487214B (en) * | 2022-01-24 | 2024-05-14 | 广州市番禺区中心医院 | Biomarker for distinguishing benign prostatic hyperplasia and prostatitis and application thereof |
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Application publication date: 20181207 |