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.
Ford
Loretta T
LT
Clinical Biochemistry Department, City Hospital, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, B18 7QH, UK.
Cooper
Sheldon C
SC
Lewis
Matthew J V
MJ
Berg
Jonathan D
JD
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