Assay of thioinosinic acid, an active metabolite of azathioprine, in human lymphocytes.

Fletcher, Lynda; Maddocks, J L

doi:10.1111/j.1365-2125.1980.tb01757.x

Cited by 18 publications

(2 citation statements)

References 12 publications

(18 reference statements)

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“…Briefly, this involved extracting the nucleotide from 100pl of packed RBCs (containing -8 x108 cells) by a modification of the 6-thioinosinic acid assay of Fletcher and Maddocks (1980). This was then hydrolysed to the parent purine, 6-thiogianine, which was assayed fluorimetrically.…”

Section: Methodsmentioning

confidence: 99%

Childhood lymphoblastic leukaemia: Sex difference in 6-mercaptopurine utilization

Lilleyman¹,

Lennard²,

Morgan³

et al. 1984

Br J Cancer

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Summary Twelve boys and 10 girls on similar long term remission maintenance treatment for lymphoblastic leukaemia had 79 random assays of their red cell 6 thioguanine nucleotide (6TGN) concentrations performed as an index of cytotoxic activity generated by oral 6-mercaptopurine (6MP).Correlation between the dose of 6MP and 6TGN was statistically significant in the girls (r=0.58, P<0.001) but not in the boys (r=0.15). Additionally, as a group the boys tolerated more 6MP (P<0.05), despite similar prescribing criteria, but this did not result in a higher mean 6TGN concentration or increased myelotoxicity.It appears that girls develop 6MP cytotoxicity at lower doses and more predictably than boys. If so, this may be relevant to the as yet unexplained but marked sex difference in prognosis apparent in some studies.The cytotoxic effect of 6-mercaptopurine (6MP) can be related to incorporation of 6MP derived 6-thioguanine nucleotide (6TGN) into DNA (Tidd & Paterson, 1974). We have been measuring the 6TGN found in red cells from children taking 6MP as part of remission maintenance treatment for acute lymphoblastic leukaemia (ALL), and somewhat to our surprise, have found a sex difference in the relationship between 6MP dose, 6TGN concentration and myelosuppression. This may be relevant to the higher late relapse rate frequently seen in boys and explain why such a sex difference in prognosis is found with some treatment schedules but not others. Patients and methodsConsecutive children with ALL treated at the Sheffield Children's Hospital were studied. The children had all been in complete remission for at least 6 months, and were treated with an identical maintenance schedule (the Medical Research Council regimen UKALL VIII). This consisted of daily 6MP (75 mg m 2) and weekly methotrexate (20 mg m -2), both oral, and both prescribed as a target dose based on body surface area. Doses were reduced to 75%, 50% and 0% of the target on the basis of neutropenia or thrombocytopenia at the time of prescription and always in parallel. Both of these drugs were taken as a single early morning dose on an empty stomach. Monthly pulses of a single dose of i.v. vincristine (1.5mgm 2) and 5 days oral prednisone (40 mgm-2) were also given to all patients irrespective of blood counts.Blood for 6TGN assay was obtained at the time of venepuncture for vincristine administration, and so monthly in most children for the study period. The children had been taking 6MP for between 7 months and 2 years, and were in good health at the time of assay. As co-trimoxazole has been found to interfere with 6MP metabolism, (Rees et al., 1984) it was ensured that no patients were taking this drug at the time of study, though all received it for the first 6 months of remission maintenance treatment as per protocol.Red blood cell (RBC) 6TGN was assayed by the method of Lennard & Maddocks (1983). Briefly, this involved extracting the nucleotide from 100pl of packed RBCs (containing -8 x108 cells) by a modification of the 6-thioinosinic acid assay of Fletche...

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Section: Methodsmentioning

confidence: 99%

Childhood lymphoblastic leukaemia: Sex difference in 6-mercaptopurine utilization

Lilleyman¹,

Lennard²,

Morgan³

et al. 1984

Br J Cancer

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“…Intracellular thiopurine ribonucleotides have been identified as the active metabolites of AZA (71, unfortunately, the assay procedures required to measure these compounds have proven difficult (8)(9)(10).…”

Section: Erdmann Chan and Canafaxmentioning

confidence: 99%

HPLC Determination of 6-Thiouric Acid and 6-Mercaptopurine in Organ Transplant Patient Serum

Erdmann

Chan

Canafax

1988

Journal of Liquid Chromatography

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A sensitive high performance liquid chromatographic method f o r the simultaneous determination of 6-thiouric acid and 6-mercaptopurine in serum is described. Our intent was to develop a procedure that could be used for pharmacokinetic studies and therapeutic drug monitoring in organ transplant patients taking azathioprine. Serum samples were precipitated with acetonitrile containing 6-n-propyl-2-thiouracil as the internal standard. The chromatographic separation was performed with an octadecylsilane column and gradient solvent system consisting of acetonitrile and 0.01 M sodium dihydrogen phosphate, pH 6.1. An initial acetonitrile concentration of I% was used to elute 6-thiouric acid but was increased to 16% to recover the 6-mercaptopurine and internal standard. The flow rate was increased from 1.3 ml/min to 1.5 ml/min during the analysis. The column effluent was monitored at 353 nm and 323 nm for detection of 6-thiouric acid and 6-mercaptopurine, respectively. Statistical analysis of standard curve data showed good intra-and inter-day accuracy, precision and reproducibility throughout a concentration range of 10 -2500 ng f o r 6-thiouric acid and 10 -500 ng f o r 6-mercaptopurine/ml of serum.The method has been applied to the quantification of 6-thiouric acid and 6-mercaptopurine in serum from two kidney allograft recipients.

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A reversed phase high performance liquid chromatography approach in determining total red blood cell concentrations of 6‐thioguanine, 6‐mercaptopurine, methylthioguanine, and methylmercaptopurine in a patient receiving thiopurine therapy

Erdmann

France

Bostrom

et al. 1990

Biomedical Chromatography

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A reversed phase high performance liquid chromatographic procedure was developed to quantify 6-thioguanine, 6-mercaptopurine, methylthioguanine, and methylmercaptopurine in red blood cells. The free base of each thiopurine was liberated from the respective nucleoside and nucleotide moiety by acid hydrolysis, which allowed for a determination of the total thiopurine present. 6-Thioguanine and 6-mercaptopurine were analyzed on an octadecylsilane column using methanol + 20 mM sodium phosphate (15:85), pH 7.5, containing 0.07% tetrabutylammonium chloride. Detection was by potassium permanganate oxidation and fluorescence detection at 290 nm excitation and 400 nm emission. Methylmercaptopurine and methylthioguanine were analyzed on a cyanopropylsilane column using methanol + 40 mM sodium phosphate (18:82), pH 2.7, and then ultraviolet absorption at 314 nm and 290 nm, respectively. The method was used to quantify the four primary thiopurines present in red blood cells of an acute lymphoblastic leukemia patient. The procedure may be a therapeutic monitoring technique that quantifies the cytotoxic drug burden in patients receiving azathioprine or 6-mercaptopurine therapy.

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Assay of thioinosinic acid, an active metabolite of azathioprine, in human lymphocytes.

Cited by 18 publications

References 12 publications

Childhood lymphoblastic leukaemia: Sex difference in 6-mercaptopurine utilization

Childhood lymphoblastic leukaemia: Sex difference in 6-mercaptopurine utilization

HPLC Determination of 6-Thiouric Acid and 6-Mercaptopurine in Organ Transplant Patient Serum

A reversed phase high performance liquid chromatography approach in determining total red blood cell concentrations of 6‐thioguanine, 6‐mercaptopurine, methylthioguanine, and methylmercaptopurine in a patient receiving thiopurine therapy

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