HPLC-FLD-DAD analysis method of simultaneously assaying two aristolochic acid-DNA adducts in tissue

A technique of aristolochic acid, analysis method, applied in the field of biotechnology analysis, to achieve the effect of high sensitivity

Inactive Publication Date: 2016-12-07
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chan [17] etc. once administered a mixture of AA-I and AA-II (1:1) to rats and analyzed it by H...

Method used

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  • HPLC-FLD-DAD analysis method of simultaneously assaying two aristolochic acid-DNA adducts in tissue
  • HPLC-FLD-DAD analysis method of simultaneously assaying two aristolochic acid-DNA adducts in tissue
  • HPLC-FLD-DAD analysis method of simultaneously assaying two aristolochic acid-DNA adducts in tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Detection of AA-DNA adducts in kidney tissue of rats administered with Guan Mu Tong

[0026] 1. Materials

[0027] 1.1 Reagents and instruments

[0028] 2-Deoxyadenosine (dA) and calf thymus DNA (CT-DNA) were purchased from Sigma. Deoxyribonuclease I (DNaseI) and phosphodiesterase I were purchased from Worthington Company. Alkaline phosphatase was purchased from Shanghai Sangon Biotechnology Co., Ltd. Acetonitrile and methanol were HPLC grade, Thermo Fisher Company. Acetic acid, ethyl acetate, zinc powder, sodium chloride, and magnesium chloride were all analytically pure, from Beijing Chemical Plant. Tris (Tris) was purchased from Beijing Dongsheng Taibo Technology Co., Ltd. AA-DNA adducts (dA-AA-I, dA-AA-II), the structure of which was synthesized by our research group and confirmed by mass spectrometry.

[0029] Agilent 1200 liquid chromatograph (DAD detector, FLD detector, column thermostat, binary pump, autosampler, degasser). Phenomenex Luna C 18...

Embodiment 2

[0046] Example 2 Detection of AA-DNA adducts in the liver tissue of rats administered with Guan Mu Tong

[0047] 1. Materials

[0048] 1.1 Reagents and instruments

[0049] With embodiment 1.

[0050] 1.2 Medicinal materials and water decoction

[0051] With embodiment 1.

[0052] 1.3 Experimental animals

[0053] With embodiment 1.

[0054] 2. Method

[0055] 2.1 Animal experiments

[0056] The operation is the same as in Example 1, and the tissue obtained is rat liver tissue.

[0057] 2.2 HPLC-FLD-DAD detection of AA-DNA adducts in tissues

[0058] Operation is the same as embodiment 1

[0059] 3. Results

[0060] dA-AA-I could be detected in rat liver after administration for 5 days, but dA-AA-II was not detected, as figure 2 D, its content is 7.24±2.24×10 -5 .

[0061] Validation Example Method validation for detection of AA-DNA adducts and deoxyadenosine by HPLC-FLD-DAD

[0062] 1. Method validation for the determination of AA-DNA adducts by HPLC-FLD

[00...

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Abstract

The invention relates to an HPLC-FLD-DAD analysis method for detecting a biomarker of aristolochic acid nephropathy (AAN), namely, aristolochic acid-DNA (AA-DNA) adducts. In the method, according to fluorescent characters of the AA-DNA adducts, HPLC-FLD is used for detecting the AA-DNA adducts while HPLC-DAD is used for detecting deoxynucleosides, thereby measuring the content of the adducts. The method can simultaneously assay two most main adducts of AAN: a deoxyadenosine-aristolochic acid I adduct (dA-AA-I) and a deoxyadenosine-aristolochic acid II adduct (dA-AA-II), and mainly includes the steps of DNA extraction, DNA digestion, enrichment of the adducts, HPLC-FLD for assaying the AA-DNA adducts, HPLC-DAD for detecting the deoxynucleosides, and calculation of the content of the AA-DNA adducts. The method is used for detecting the content of the AA-DNA adducts in animal tissue (e.g. liver or kidney tissue of a mouse) that is dosed with drugs or foods that contain the AAs, such as caulis aristolochiae manshuriensis.

Description

[technical field] [0001] The invention belongs to the field of biotechnology analysis, and in particular relates to an HPLC-FLD-DAD method for detecting aristolochic acid nephropathy biomarkers (i.e., aristolochic acid-DNA adducts), which can be used for aristolochic acid nephropathy-related research work. [Background technique] [0002] Aristolochic acid nephropathy (AAN) is a tubulointerstitial disease caused by ingestion of Chinese herbal medicines containing aristolochic acids (AAs) (such as Guan Mu Tong, Guang Fang Ji, Qing Mu Xiang, etc.). Current literature reports that about 50% of AAN patients also suffer from upper tract urothelial cell carcinoma (UTUC) [1] , 70% of UTUC patients have AA-DNA adducts [2] . AA-DNA adducts prevent transcription or DNA replication, promote cell cycle arrest or lead to apoptosis by causing mutations at the gene site AT→TA [3] , causing tumors. AA-DNA adducts as biomarkers of AA exposure [2，4] , it is important to detect [5] . ...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 王璇易莉炜蔡少青高志
Owner PEKING UNIV
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