[HPLC determination and diversity analysis of thiopurine methyltransferase activity in human and pig].
OBJECTIVE
To develop an HPLC method for the determination of 6-methylmercaptopurine concentration and then numerate the thiopurine methyltransferase (TPMT) activity in erythrocyte via formula and analysis of the TPMT activity difference between human and pig.


METHODS
The chromatographic apparatus SHIMADZU GC-2010C was used. The stationary phase was a Symmetry C18 reverse-phase column (150 mm x 3.9 mm, 5 microm). The mobile phase was 0.1% acetic acid-methanol and the flow rate 1.0 mL/min. The samples were extracted by ethyl acetate and injected automatically. They were measured at UV 280 nm. 2-amino-6-methyl-mercaptopurine was used as the internal standard.


RESULTS
The retention times for 6-MP, AMMP and 6-MMP were 4.4 min, 7.7 min and 9.3 min respectively. The calibration curves were linear over the range of 6.25-100 nmol/mL. the methodology recovery was 98.08%-100.05%. The extraction recovery of 6-MMP was 88.1%-92.4%. The within-group RSD 1.0%-1.5% and inter-group RSD 1.0%-4.4%. The levels of TPMT activity of human, pig and Banna Minipig Inbred Line (BMI) were (17.45 +/- 3.62) U, (7.65 +/- 1.35) U and (8.73 +/- 1.55) U respectively.


CONCLUSIONS
The method is suitable for determination of TPMT activity in erythrocyte of different patients and species in that of clinical medicine and scientific research. TPMT activity of pig and BMI is obviously lower than human's.
Wang
Li-na
LN
Key Laboratory of Transplantation Engineering and Immunology, the Ministry of Health, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhang
Lin
L
Nan
Feng
F
Cheng
Jing-qiu
JQ
Bu
Hong
H
Liang
Mao-zhi
MZ
Lu
Yan-rong
YR
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D015122:Mercaptopurine / Q000031:analogs & derivatives* / Q000032:analysis; D008780:Methyltransferases / Q000378:metabolism*; D013045:Species Specificity; D013552:Swine; D013556:Swine, Miniature
C010240:6-methylthiopurine; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase