TPMT Activity Research Articles

Genetic Determinants of the Pre- and Post-Azathioprine Therapy Thiopurine Methyltransferase Activity Phenotype

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Genetic Determinants of the Pre‐ and Post‐Azathioprine Therapy Thiopurine Methyltransferase Activity Phenotype

Thiopurine drug therapy has been reported to lead to a variable increase in red cell TPMT activity that may alter effective dose and therapeutic outcome. The aim of this study was to correlate Variable Number Tandem Repeat (VNTR) in the promoter region of the TPMT gene with induction of red cell TPMT activity in patients treated with azathioprine (AZA). In 58 patients, TPMT activity measured at 3 months was not significantly induced on average above pre‐therapy levels. Individual patients showed variation in TPMT activity pre‐ and post‐AZA therapy, however changes in TPMT activity were not predicted by VNTR configuration. In conclusion, TPMT promoter VNTRs are unlikely to play a significant role in changes in TPMT activity in response to AZA therapy.

Analytic Aspects of Monitoring Therapy with Thiopurine Medications

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THIOPURINE METHYL TRANSFERASE (TPMT) PHENOTYPE AS A PREDICTOR OF CLINICAL RESPONSE AND SIDE EFFECT PROFILE IN A POPULATION OF IBD PATIENTS

Purpose: TPMT is responsible for the conversion of 6-mercaptopurine (6-MP) to 6-methymercaptopurine, an inactive metabolite that has been associated with hepatotoxicity. Patients with decreased TPMT activity produce more 6-thioguannine (6-TG) which at high levels can lead to leukopenia. With the availability of TPMT enzyme activity testing, it is possible to screen patients with IBD starting therapy with 6-MP or azathioprine (AZA) in an attempt to limit drug side effects and toxicity. Aim: The purpose of this study was to determine if TPMT activity correlates with clinical response or the development of side effects in IBD patients starting therapy with 6-MP or AZA. Methods: 90 patients with IBD who had undergone TPMT phenotype testing were identified. Clinical response at 6 months could be assessed in 59 of these patients and was classified as complete response, non-response, partial response, or continued remission. Side effects were recorded including hepatitis, leukopenia, pancreatitis, allergic reactions, gastrointestinal intolerance, and opportunistic infection. Results: At 6 months, 22 patients were classified as complete responders, 14 as non-responders, 12 as partial responders, and 11 as continued remission. No significant difference was present in drug dosing for the 4 groups (1.11 – 1.16 mg/kg± 0.35–0.45). The 22 complete responders had a mean TPMT of 28.0 ± 10.4 EU, while the 14 non-responders had a mean TPMT of 31.8 ± 8.4 EU (p = 0.26). Mean TPMT values for partial responders and continued remission were 30.6 ± 4.6 and 31.0 ± 7.1 EU, respectively. Analysis of multiple cut points demonstrated no distinct cut off level of TPMT activity that correlated with clinical response. 52 patients reported 59 side effects. 31 patients (34.4%) had treatment discontinued due to side effects (mean TPMT 29.8 ± 9.5 EU). Leukopenia was the most common side effect occurring in 23 patients (15.6%). A TPMT level of <29.0 EU correlated with increased risk of leukopenia compared to a TPMT level ≥ 29.0 EU (p = 0.038). No distinct cut off level of TPMT activity correlated with the development of side effects. Conclusions: 1) TPMT activity did not correlate with clinical response or side effects other than leukopenia. 2) TPMT activity of <29.0 EU correlated with an increased risk of leukopenia.

Analytic aspects of monitoring therapy with thiopurine medications.

The thiopurine medications 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and azathioprine are used in treatment of childhood acute lymphoblastic leukemia, autoimmune diseases, and, in the case of azathioprine, in solid organ transplantation. They are converted in vivo to the active 6-thioguanine nucleotides (6-TGN). One person in 300 in white populations has low or undetectable TPMT activity and is at risk for accumulating 6-TGN with the consequence of severe, life-threatening myelosuppression. A rational therapeutic strategy for thiopurine drug use is to first determine TPMT phenotype/genotype and then to adjust the dosage on an individual basis. Determination of erythrocyte 6-TGN levels can further help to optimize therapy. TPMT activity (phenotype) is determined in erythrocytes using radiochemical or HPLC procedures. Recent HPLC procedures show good agreement with the original radiochemical method, while offering simplified sample pretreatment and improved precision. To date, 12 mutant alleles responsible for TPMT deficiency have been published. Restriction fragment length polymorphism PCR and allele-specific PCR have been used for detection of TPMT mutations. Genotyping methods that allow a higher throughput include real-time PCR (LightCycler) and denaturing HPLC. Numerous HPLC methods have been reported for quantification of 6-TGN. The majority involve acid hydrolysis to 6-TG at high temperature. There are substantial differences in the hydrolysis step, extraction procedure, chromatographic conditions and method of detection. Erythrocyte 6-TGN concentrations can vary up to 2.6-fold depending on the HPLC method. The method that has found the greatest application in clinical studies is that of Lennard. This has served as the basis for the establishment of treatment-related therapeutic ranges for thiopurine therapy. These ranges will not necessarily be applicable when other methodology is used. There is an urgent need to harmonize the analytic procedures for 6-TGN.

Determination of thiopurine methyltransferase activity in isolated human erythrocytes does not reflect putative in vivo enzyme inhibition by sulfasalazine.

Determination of thiopurine S -methyltransferase (TPMT; EC 2.1.1.67) activity is intended to screen patients before therapy with thiopurine drugs (6-mercaptopurine, 6-thioguanine, and azathioprine) to rule out a deficiency in this enzyme. The enzyme catalyzes the S-methylation of these medicinal agents, a metabolic pathway that competes with the formation of pharmacologically active 6-thioguanine nucleotides (6-TGNs), thereby modulating their therapeutic and toxic effects (1)(2). Individuals with low TPMT activity are known to be at high risk for severe thiopurine-induced myelodepression. In addition to a genetic polymorphism of TPMT alleles, which is responsible for the wide interindividual differences in TPMT activity, cotherapy with various drugs, including aminosalicylates, has been shown to influence enzyme activity (1)(2). In vitro kinetic studies with recombinant human TPMT demonstrated that sulfasalazine, 5-aminosalicylic acid, balsalazide, and olsalazine (3)(4)(5) inhibit the enzyme activity in a noncompetitive manner. Therefore, simultaneous therapy with both thiopurines and aminosalicylates was postulated to create a “phenocopy” of individuals with a low TPMT activity genotype (1). In line with this hypothesis, Lewis et al. (3) described a case of a patient with Crohn disease and TPMT activity in the low normal range who had severe bone marrow dysfunction while receiving treatment with 6-mercaptopurine and olsalazine. The authors speculated that the TPMT activity determined ex vivo represented the patient’s baseline TPMT activity in vivo because the inhibitory effect of olsalazine would be removed by dilution in the assay. Subsequently, several groups (6)(7)(8) investigated this drug–drug interaction in clinical studies with more cases. As indicators of an inhibitory effect, ex vivo determination of erythrocyte TPMT activity and erythrocyte 6-TGNs were chosen. No changes in TPMT activity, but significantly increased 6-TGN concentrations, were observed under combined thiopurine/aminosalicylate therapy (6)(8). Whereas Lowry et al. (6) speculated …

Phenotypic and genotypic analysis of thiopurine s-methyltransferase polymorphism in the bulgarian population.

Genetic polymorphism of TPMT activity is an important factor responsible for large individual differences in thiopurine toxicity and therapeutic efficacy. The aim of this study was to determine the distribution of TPMT activity as well as the types and frequencies of mutant alleles in a Bulgarian population sample. TPMT activity was measured in 313 Bulgarians, using an established HPLC procedure. All individuals with TPMT activity less than 12.0 nmol/(mL Ery.h) (n = 76) were additionally genotyped using a color multiplex hybridization assay. The samples were tested for TPMT*2, *3A, *3B, *3C, *3D, *4, and *6 mutant alleles. TPMT activities varied from 1.1 to 24.0 nmol/(mL Ery.h) [mean 14.2 +/- 3.2 nmol/(mL Ery.h)]: 92.3% of the individuals investigated had high TPMT activity [>10 nmol/(mL Ery. h)], whereas 7.4% were intermediate [2.8-10 nmol/(mL Ery.h)], and 0.3% were low metabolizers [< 2.8 nmol/(mL Ery.h)]. A significant gender-related difference in TPMT activity (P = 0.02) was observed with 6.2% higher values in men than in women. There was no significant correlation between age and enzyme activity (r = 0.06, P = 0.27). Genotype analysis revealed three mutant TPMT alleles: 2, 3A, and 3C. The frequency of these alleles among the TPMT-deficient individuals was 2.17%, 30.4%, and 2.17%, respectively. These data show a similar distribution of TPMT activity among the Bulgarian population investigated as in most other white populations with the frequency of intermediate metabolizers being somewhat lower (7.4% versus approximately 11%) in the Bulgarians. The most common variant allele was TPMT-3A, as in other white populations.

Polymorphism in the MTHFR gene affects TPMT activity

Polymorphism in the MTHFR gene affects TPMT activity

Genetic determinants of the thiopurine methyltransferase intermediate activity phenotype in British Asians and Caucasians.

Polymorphisms in the TPMT gene open reading frame (ORF) are associated with reduced TPMT activity. Variable number tandem repeats (VNTR*3 to VNTR*9) in the promoter region of the gene consisting of combinations of Type A, B and C repeat units, may modulate TPMT activity. Here we present the allele frequencies of genetic modifiers of TPMT activity in a British Asian population, as well as the concordance between intermediate TPMT activity and ORF and VNTR genotypes in a predominantly Caucasian population. VNTR type and ORF mutations were determined in two selected TPMT activity ranges, intermediate activity (4-8 U, 108 patients), normal (12-15 U, 53 patients) and in 85 British Asians. In British Asians, TPMT*3C was the prevalent mutant allele (four heterozygotes). One patient was heterozygous for TPMT*3A. Overall VNTR frequencies did not differ from Caucasians. Three new VNTR alleles were designated VNTR*6c, VNTR*6d, and VNTR*7c. Forty-one percent of patients with intermediate activity were heterozygous for a TPMT ORF mutation (3A, 2B, 1C). Marked linkage disequilibrium was noted between VNTR*6b - TPMT*3A (D' = 1), VNTR*4b - TPMT*3C (D' = 0.67) and VNTR*6a - TPMT*1 (D' = 1) alleles. As a result, significant differences (P < 0.05) in the distribution of Type A, B or the total number of repeats summed for both alleles, were found between the ORF heterozygous intermediate activity group and the wild-type intermediate or normal activity groups. No significant difference was found between the two wild-type groups. Our results suggest that TPMT gene VNTRs do not significantly modulate enzyme activity.

Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children.

Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity. We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate. The K(m) values calculated for 6MP were 0.54 (RBC), 0.85 (pMNC) and 0.65 (Molt-F4 cells) mmol/L. The K(m) values for S-adenosylmethionine were 11.9 (RBC), 16.4 (pMNC) and 6.65 (Molt-F4 cells) micro mol/L. The assay variation was 8.2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean +/- standard deviation): female children and young adults, 15.1 +/- 4.8 pmol/10(7) cells per h (n = 44); male children and young adults, 15.8 +/- 6.4 pmol/10(7) cells per h (n = 59). No gender or age differences were found. The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.

The thiopurine S-methyltransferase gene locus -- implications for clinical pharmacogenomics.

Thiopurine methyltransferase catalyzes the S-methylation of azathioprine (AZA), 6-mercapto-purine (6-MP) and thioguanine, medications widely used to treat malignancies, rheumatic diseases, dermatologic conditions, inflammatory bowel disease and solid organ transplant rejection. TPMT activity exhibits a genetic polymorphism in 10% of Caucasians, with 1/300 individuals having complete deficiency. Patients with intermediate or deficient TPMT activity are at risk for excessive toxicity, including fatal myelosuppression, after receiving standard doses of thiopurine medications. The molecular basis for low TPMT activity has been elucidated, leading to the development of assays for the three signature mutations, which account for the majority of mutant alleles. TPMT genotype is correlated with erythrocyte and leukemia blast cell TPMT activity and associated with a risk of toxicity after thiopurine therapy. Recent studies defined target starting doses for mercaptopurine based on TPMT genotypes. This polymorphism is one of the best models for the translation of genomic information to guide patient therapeutics.

Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay

A new liquid–liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH 4Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5–250 ng ml −1 ( r≥0.997, slope=1.497, intercept=−0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml −1, respectively. The coefficients of variation were ≤6.1% for within-day assay ( n=6) and ≤9.5% for between-day assay precision ( n=6; 14 days). TPMT activity was determined in a French adult Caucasian population ( n=70). The results ranged from 7.8 to 27.8 nmol h −1 ml −1 packed red blood cells and the frequency distribution histogram is similar to that previously published.

Genotypic and phenotypic analysis of the polymorphic thiopurine S-methyltransferase gene (TPMT) in a European population.

1. Characterization of allelic variants of the TPMT gene (TPMT) responsible for changes in TPMT activity, and elucidation of the mechanism by which these alleles act, are required because of the clinical importance of this polymorphism for patients receiving thiopurine drugs. 2. We defined the mutational and allelic spectrum of TPMT in a group of 191 Europeans. Using PCR-SSCP, we screened for mutation the entire coding sequence, the exon-intron boundaries, the promoter region and the 3'-flanking region of the gene. Six mutations were detected throughout the ten exons and seven TPMT alleles were characterized. Four of them, TPMT*2, *3A, *3C and *7, harbouring the known mutations, G238C, G460A, A719G or T681G, were nonfunctional and accounted for 0.5, 5.7, 0.8 and 0.3% of the allele totality, respectively. 3. Within the promoter region, six alleles corresponding to a variable number of tandem repeats (VNTR), were identified. VNTR*V4 and *V5a which harbour four or five repeats of a 17-18 bp unit, were the most frequent (55% and 34%, respectively). The other VNTR alleles, having from five to eight repeats, were rarer. 4. The TPMT phenotype was correctly predicted by genotyping for 87% of individuals. A clear negative correlation between the total number of repeats from both alleles and the TPMT activity level was observed, indicating that VNTRs contribute to interindividual variations of TPMT activity. Therefore, additional analysis of the promoter region of TPMT can improve the phenotype prediction rate by genotyping.

Detection of known and new mutations in the thiopurine S-methyltransferase gene by single-strand conformation polymorphism analysis.

To detect mutations in the thiopurine S-methyltransferase gene (TPMT), we have developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The sensitivity of the method was first evaluated by analyzing DNA samples from five individuals, including two high methylators (HMs), two intermediate methylators (IMs), and one deficient methylator (DM). TPMT alleles and mutations in each of these individuals had previously been characterized by conventional PCR-based assays and direct sequencing analysis. All mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments, allowing their identification. We further tested the efficiency of the strategy to detect new TPMT mutations. For this purpose, additional DNAs from 15 IMs and 15 HMs were submitted to PCR-SSCP analysis. A total of 7 alleles were characterized, including two new alleles. The first one, termed TPMT*1A, harbors a single mutation C-->T at nucleotide -178 in exon 1 and was detected in a HM subject. The second one, termed TPMT*7, was characterized by a T-->G transversion at nucleotide 681 in exon 10. This allele should be a nonfunctional allele of the TPMT gene since it was observed in combination with a wild-type allele in an intermediate methylator. We conclude that the PCR-SSCP strategy we developed could be advantageously used to fully characterize the extent of allelic variation at the TPMT gene locus in populations and thus to improve our understanding of the genetic polymorphism of TPMT activity, which has considerable consequences for the toxicity and efficacy of therapeutically important and widely used drugs.

1-34-03 Azathioprine-induced pancytopenia in a patient of refractory myasthenia gravis with low TPMT activity

1-34-03 Azathioprine-induced pancytopenia in a patient of refractory myasthenia gravis with low TPMT activity

Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity.

TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT*2 and TPMT*3A), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units/ml pRBC (rs = 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT*1) and mutant (TPMT*2 and TPMT*3A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with in vitro translation in rabbit reticulocyte lysates. In contrast, pulse-chase experiments revealed significantly shorter degradation half-lives for TPMT*2 and TPMT*3A ( approximately 0.25 hr) compared with wild-type TPMT*1 (18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT*2 and TPMT*3A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.

A single point mutation leading to loss of catalytic activity in human thiopurine S-methyltransferase.

Thiopurine S-methyltransferase (TPMT; S-adenosyl-L-methionine:thiopurine S-methyltransferase, EC 2.1.1.67) activity exhibits genetic polymorphism, with approximately 0.33% of Caucasians and African-Americans inheriting TPMT deficiency as an autosomal recessive trait. To determine the molecular genetic basis for this polymorphism, we cloned the TPMT cDNA from a TPMT-deficient patient who had developed severe hematopoietic toxicity during mercaptopurine therapy. Northern blot analysis of RNA isolated from leukocytes of the deficient patient demonstrated the presence of TPMT mRNAs of comparable size to that in subjects with high TPMT activity. Sequencing of the mutant TPMT cDNA revealed a single point mutation (G238-->C), leading to an amino acid substitution at codon 80 (Ala80-->Pro). When assessed in a yeast heterologous expression system, this mutation led to a 100-fold reduction in TPMT catalytic activity relative to the wild-type cDNA, despite a comparable level of mRNA expression. A mutation-specific PCR amplification method was developed and used to detect the G238-->C mutation in genomic DNA of the propositus and her mother. This inactivating mutation in the human TPMT gene provides insights into the genetic basis for this inherited polymorphism in drug metabolism.

Pharmacogenetics of human erythrocyte thiopurine methyltransferase activity in a French population.

1. A genetic polymorphism in human erythrocyte thiopurine methyltransferase activity (RBC TPMT) resulting in a trimodal phenotypic distribution has been demonstrated both in a North American population and in British children. 2. We studied whether such a polymorphism may be also present in a white French population by testing RBC TPMT activity in 303 randomly selected blood donors. 3. We found a large inter-individual variation in RBC TPMT activity which ranged from 2 to 40 nmol ml-1 packed RBC h-1, with a mean value of 15.4 +/- 7.0 nmol ml-1 packed RBC h-1. The enzyme activity was not significantly influenced by the sex and age of the subjects. 4. In our population sample, we found no subject with undetectable enzyme activity. However, the probit plot of the log RBC TPMT activity showed a highly significant change in slope at a TPMT activity of 7.5 nmol ml-1 packed RBC h-1. Thirty four subjects (11% of our population) had TPMT activities below 7.5 nmol ml-1 packed RBC h-1. 5. These data are consistent with the view that the genetic polymorphism of TPMT activity described in populations from North America and the United Kingdom is also present in a French population, with about 89% of subjects exhibiting a high activity and 11% an intermediate activity.

Thiopurine Methyltransferase in Humans: Development and Tissue Distribution

Thiopurine methyltransferase (EC 2.1.1.67, TPMT) was studied with 6-mercaptopurine as substrate in the cytosolic fraction from 18 human fetal liver, 16 placental and 22 adult liver specimens. TPMT activity (pmol x min-1 x mg-1; mean +/- SD) was 33.2 +/- 15.8 (fetal liver), 19.5 +/- 11.1 (placenta) and 105 +/- 57.1 (adult liver). Fetal liver activity of TPMT is one third that in adult liver suggesting that this enzyme is well developed in the mid-gestational human fetus. The distribution of TPMT seems to be ubiquitous both in the fetus and adult subject. The kidney is an important site of methylation as suggested by the renal activity of TPMT (197 +/- 70 pmol x min-1 x mg-1) which is twice as high as the hepatic one. Fetal and adult hepatic TPMT obey nonmichaelian kinetics. Two phases, one with lower and one with higher affinity for 6-mercaptopurine, were observed. The average Km for the high affinity phase was 0.12 mmol/l (fetus) and 0.13 mmol/l (adult), whereas the Km for the lower affinity phase was 1.79 mmol/l (fetus) and 1.42 mmol/l (adult). This paper shows that TPMT develops before the second trimester of gestation in human fetus, that it has an ubiquitous distribution in the human fetus and adult subjects and the kinetic pattern of this enzyme is consistent in fetal and adult liver.

Human erythrocyte histamine N-methyltransferase: radiochemical microassay and biochemical properties

A radiochemical assay for the measurement of histamine N-methyltransferase (HNMT) activity in human erythrocytes (RBCs) has been developed. This assay was developed as a first step toward testing the hypothesis that the biochemical properties and regulation of HNMT in an easily obtainable human cell, the RBC, might reflect those of the enzyme in less accessible cells and tissues. The Michaelis ( K m ) constant in the RBC for histamine, the methyl acceptor substrate for the reaction, was 5.0 × 10 −5 mol/l. The K m constant for S- adenosyl- l- methionine , the methyl donor, was 2.8 × 10 −6 mol/l. The assay was performed at a reaction pH of 7.4 with a potassium phosphate buffer. The product of the reaction was identified as N T-methylhistamine by high performance liquid chromatography. The K ii for inhibition of the RBC enzyme by amodiaquine, an HNMT inhibitor, was 1.O × 10 −7 mol/l, while the K is value was 0.48 × 10 −7 mol/l. Blood samples obtained from 39 randomly selected adult white subjects had a mean activity of 130 ± 30 U/ml of packed RBCs (mean ± SD). Enzyme activities varied over a range from 74–213 U. There were no differences between men and women in mean activities, nor was there a significant correlation between RBC HNMT activity and age. The results of experiments in which lysates with ‘low’ and ‘high’ activities were mixed gave no indication that individual variations in RBC HNMT activities were due to the effects of endogenous enzyme inhibitors or activators. RBC HNMT activities measured in blood samples from 17 individual subjects four times over 6 wk were quite constant in each subject, with an average coefficient of variation of 6.2%. The availability of this assay will make it possible to test the hypothesis that individual variations in determined variations in human RBC COMT activity [31]are directly correlated with variations in COMT activity in other human tissues [8,9]. Those variations are also significantly correlated with large individual differences in the O-methylation by COMT of orally administered catechol drugs such as l-dopa and methyldopa [32,33]. RBC TPMT activity in man is also under genetic control [34], and the properties and levels of activity of RBC TPMT reflect those of the enzyme in other human tissues and cells [10,11]. The results of our experiments with RBC HNMT will now make it possible to determine whether individual variations in this human RBC methyltransferase may help us to understand and predict one factor responsible for individual variations in the N T-methylation of histamine and related compounds in man.