Method of determining thiopurine methyltransferase activity
View Patent ↗The present invention provides a method of determining thiopurine methyltransferase (TPMT) activity in a subject. The method includes the steps of reacting sample obtained from the subject with a thiopurine derivative that is not 6-mercaptopurine to produce a methylated purine product; contacting the reacted sample with acid, thereby precipitating proteinaceous material from the reacted sample; separating supernatant from the precipitated proteinaceous material; and detecting in the supernatant the methylated purine product, where the amount of the methylated purine product indicates a level of thiopurine methyltransferase activity in the subject. In a method of the invention, the subject can be, for example, an inflammatory bowel disease patient. In one embodiment, the acid used to precipitate proteinaceous material is perchloric acid, for example, 70% perchloric acid. In another embodiment, the thiopurine derivative used as a substrate is 6-thioguanine. In a further embodiment, the methylated purine product is detected by fluorescence, which can be combined, if desired, with high performance liquid chromatography (HPLC).
1. A method for determining a subject's thiopurine methyltransferase (TPMT) genotype, said method comprising:
(a) reacting a sample obtained from said subject with a thiopurine derivative to produce a methylated purine product, provided that said thiopurine derivative is not 6-mercaptopurine;
(b) contacting said reacted sample with an acid, thereby precipitating proteinaceous material from said reacted sample;
(c) separating supernatant from said precipitated proteinaceous material;
(d) detecting in said supernatant said methylated purine product, wherein the amount of said methylated purine product indicates a level of TPMT activity in said subject; and
(e) determining said subject's TPMT genotype based upon said level of TPMT activity.
2. The method of claim 1 , wherein said subject is an inflammatory bowel disease patient.
3. The method of claim 1 , wherein said acid is perchloric acid.
4. The method of claim 3 , wherein said perchloric acid is 70% perchloric acid.
5. The method of claim 1 , wherein said thiopurine derivative is 6-thioguanine.
6. The method of claim 5 , wherein said methylated purine product is detected by fluorescence.
7. The method of claim 1 , wherein said methylated purine product is detected by high performance liquid chromatography (HPLC).
8. The method of claim 1 , wherein said methylated purine product is detected by capillary electrophoresis, gas chromatography, gas chromatography-mass spectroscopy, or thin layer chromatography.
9. The method of claim 1 , wherein said level of TPMT activity greater than 23.60 nmoles 6-methylthioguanine (6-mTGN)/g hemoglobin (Hb)/hr indicates that said subject has a wild-type TPMT genotype.
10. The method of claim 9 , further comprising recommending a standard initial dose of 6-mercaptopurine (6-MP) or azathioprine (AZA) to said subject.
11. The method of claim 1 , wherein said level of TPMT activity between 6.76 and 23.60 nmoles 6-mTGN/g Hb/hr indicates that said subject has a heterozygous TPMT genotype.
12. The method of claim 11 , further comprising recommending a low initial dose of 6-MP or AZA to said subject.
13. The method of claim 1 , wherein said level of TPMT activity less than 6.76 nmoles 6-mTGN/g Hb/hr indicates that said subject has a homozygous low TPMT genotype.
14. The method of claim 13 , further comprising recommending that said subject not receive 6-MP or AZA.