Reference intervals for thiopurine S-methyltransferase activity in red blood cells using 6-thioguanine as substrate and rapid non-extraction liquid chromatography.
BACKGROUND
Although widely used, thiopurine drugs have a narrow therapeutic index and treatment can result in life-threatening toxicity, the basis being pharmacogenetic variation in thiopurine metabolism by thiopurine S-methyltransferase (TPMT). We recently developed a modified phenotyping assay to determine TPMT activity in red blood cells. Here we describe improvements to the method and establish reference intervals in a large prospective study.


METHODS
A modified enzyme assay for TPMT activity is reported. It uses 6-thioguanine as substrate with heat treatment of the incubate to stop the reaction and precipitate protein prior to high-performance liquid chromatographic (HPLC) analysis. Measurement of the reaction product, 6-methylthioguanine (6-MTG), uses HPLC with fluorimetric detection.


RESULTS
The assay shows excellent characteristics, with clear discrimination of patients who are deficient in TPMT activity (< 5 nmol 6-MTG per g Hb per h) from heterozygotes (5-24 nmol 6-MTG per g Hb per h) and patients with normal activity (>25 nmol 6-MTG per g Hb per h).


CONCLUSIONS
A modified TPMT assay is described which is suited for routine analysis in a regional centre. The method overcomes the need for extraction and has speeded up the chromatographic determination of 6-MTG, enabling large numbers of samples to be analysed. A prospective study of 1000 individuals has established the distribution of TPMT activity using the assay.
Clinical Biochemistry Department, City Hospital, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, B18 7QH, UK.
Loretta T
LT
Ford
Sheldon C
SC
Cooper
Matthew J V
MJ
Lewis
Jonathan D
JD
Berg
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001774:Blood Chemical Analysis; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D006801:Humans; D008297:Male; D008780:Methyltransferases / Q000097:blood* / Q000235:genetics; D008875:Middle Aged; D010641:Phenotype; D013379:Substrate Specificity; D013866:Thioguanine / Q000031:analogs & derivatives* / Q000097:blood
C035366:6-methylthioguanine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D013866:Thioguanine