Human thiopurine methyltransferase activity varies with red blood cell age

Abstract

Aims Inherited differences in thiopurine methyltransferase (TPMT) activity are an important factor in the wide interindividual variations observed in the clinical response to thiopurine chemotherapy. The aim of this study was to establish a population range for red blood cell (RBC) TPMT activity in children with acute lymphoblastic leukaemia (ALL) at disease diagnosis. An additional aim was to investigate factors that can influence TPMT activity within the RBC. Methods Blood samples were collected from children with ALL at disease diagnosis, prior to any blood transfusions, as part of the nationwide UK MRC ALL97 therapeutic trial. RBC TPMT activity was measured by h.p.l.c. RBCs were age‐fractionated on Percoll density gradients. Results Pretreatment blood samples were received from 570 children within 3 days of venepuncture. TPMT activities at disease diagnosis ranged from 1.6 to 23.6 units/ml RBCs (median 7.9) compared with 0.654–18.8 units (median 12.9), in 111 healthy control children (median difference 4.5 units, 95% CI 3.9, 5.1 units, P < 0.001). A TPMT quality control sample, aliquots of which were assayed in 60 analytical runs over a 12 month period, contained a median of 11.98 units with a CV of 11.6%. Seven children had their RBCs age‐fractionated on density gradients. TPMT activities in the top gradient (young cells) ranged from 4.2 to 14.1 units (median 7.5) and in the bottom gradient (old cells) 1.5–12.6 units (median 4.7 units), median difference 2.3 units, 95% CI 0.7, 4.1, P = 0.035. Conclusions Circulating RBCs do not constitute a homogeneous population. They have a life span of around 120 days and during that time undergo a progressive ageing process. The anaemia of ALL is due to deficient RBC production. The results of this study indicate that RBC TPMT activities are significantly lower in children with ALL at disease diagnosis. This may be due, at least in part, to a relative excess of older RBCs.

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

“…Even though not specifically investigated in our study, the RBCs do not constitute a homogenous group and the RBCs have a life span of 120 days. Lennard et al (2001) showed that the RBC population at diagnosis was influenced by the disturbed haematopoiesis and the population consisted of an excess of old RBCs [14]. In that study, a density gradient centrifugation of the red blood cells showed that the population corresponding to the older cells displayed a lower TPMT enzyme activity compared to newer cells.…”

“…The TPMT enzyme activity in MOLT4 cells was determined by measuring the rate of formation of 6-methyl-mercaptopurine from 6-MP using 14 C-labeled S-adenosyl-Lmethionine as the methyl group donor [12]. An amount of 200 µg of the total cellular protein was incubated in duplicates for 2 hours at 37°C in 400 µl of 47 mmol/l potassium phosphate buffer (pH 7.…”

Thiopurine methyltransferase polymorphisms in a Brazilian population

“…18 TPMT phenotype has been assigned on the basis of its activity in erythrocytes as previously described, 19 but these measurements are laborious and vary with red blood cell age. 20 Furthermore, bimodal or trimodal activity patterns have been shown in several populations. 16,[21][22][23][24][25][26][27][28][29][30][31][32][33][34] Since ethnic differences have been demonstrated worldwide, it remains to be elucidated in Brazil, the fifth largest populated country in the world.…”

Rapid genotyping of common deficient thiopurine S-methyltransferase alleles using the DNA-microchip technique

“…5 Although TPMT deficiency can be diagnosed by assays of erythrocyte TPMT activity, different factors, including patient age, renal function, receiving of RBC transfusion and thiopurine administration, may alter these results. 15,16,21 Molecular genetic (PCR-based) methods are not affected by extraneous factors and offer a promising alternative to measurement of RBC TPMT activity. PCR-based methods require less than 1 mg of DNA, which is the amount contained in approximately 100 ml of whole blood.…”