Determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique.
A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 degrees C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/gHb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/gHb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/gHb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3-63.8 nmol 6-MTG/gHb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/gHb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.
Ford
Loretta Theresa
LT
Clinical Biochemistry Department, City Hospital, Dudley Road, Birmingham B18 7QH, UK.
Berg
Jonathan David
JD
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008780:Methyltransferases / Q000097:blood*; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013866:Thioguanine / Q000378:metabolism*
D008780:Methyltransferases; C022745:thiopurine methyltransferase; D013866:Thioguanine