A rapid and sensitive high pressure liquid chromatography assay for deoxyribonucleoside triphosphates in cell extracts

Garrett, Charles; Santi, Daniel V.

doi:10.1016/s0003-2697(79)80005-6

Cited by 259 publications

(66 citation statements)

References 13 publications

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“…Peaks 1-5 correspond to dCTP, ara-CTP, dTTP, dATP and dGTP, respectively. Table 2 shows that under these modified reaction conditions recovery of the respective dNTPs was comparable to that reported in the literature (4,5), and that degradation of ribonucleoside triphosphates was complete. Ara-CTP was converted only to the corresponding base, cytosine.…”

Section: Analysis Of the Ara-ctp Breakdown Productsupporting

confidence: 82%

Determination of Pyrimidine Deoxynucleoside Triphosphates in Leukaemia Cell Extracts Containing 1-ß-D-Arabinofuranosylcytosine Triphosphate

Chiba

Szekeres²,

Jäger³

1991

Clinical Chemistry and Laboratory Medicine

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Summary: Deoxynucleoside 5'-triphosphates (dNTPs) can be determined in cell extracts by high performance liquid chromatography after prior selective degradation of ribonucleoside S'-triphosphates with sodium periodate and methylamine. When the method is used for the evaluation of deoxynucleoside triphosphates in 1-ß-D-arabinofuranosylcytosine triphosphate (ara-CTP)-containing cell extracts, an additional peak coeluting with thymidine triphosphate (dTTP) is observed. This peak is due to the formation of a carboxylic acid derivative of ara-CTP by periodate oxidation, and it can lead to considerable overestimation of dTTP. Formation of this peak can be avoided by using alkäline reaction conditions (pH 7.5) and by changing the sequence of addition of the reagents used in the periodation procedure. By employing this modified protocol, cellular dNTP and ara-CTP levels can be monitored in extracts of leukaemic blasts during cytosine arabinoside treatment in two separate HPLC runs.base. Subsequent ß-elirhination leads to removal of The measurement of deoxyribonucleoside-5'-triphos-the phosphate groups from the molecule and cleavage phates (dNTPs) has gaiiied widespread importance in of the N-glycosidic bond (6). Ribonucleotides are thus the study of anticancer and antiviral agents. An in-converted to the respective bases, which elute with direct assay üsing the respective endogerious deoxy-the void volume of the anion exchange column. nucleoside äs a limiting factor in a DNA-polyrneräse catalysed reaction has been described (1-3). This Pyrimidine dNTP levels have been shown to influence method caniiot be used, however, when the chemo^ the sensitivity of cells towards l-ß-/)-arabinofuranotherapeutic agents themselves or their metäbolites in-sylcytosine (ara-C) (7 -9). The determination of the terfere with the polymerase reaction. An alternative biologically active metabolite, l-ß-Z)-arabinofuranomethod uses high performance liquid chromatogra-sylcytosine triphosphate (ara-CTP), and of cellular phy to separate the deoxyribonucleotides (4, 5). Since dNTP levels in extracts prepared from blasts of the this method does not separate ribonucleotides same patient is, therefore, important, when studying (rNTPs) from deoxyribonucleotides, it is necessary to the mutual interference of drug and dNTP levels. first chemically.degrade the rNTPs, using periodate Although published high performance liquid chroin the presence of methylsunine to cleave the 2' -3' matography methods yield highly accurate and reprocarbon bond of ribose; the resulting dialdehyde de-ducible results for the determination of dNTPs in cell rivative reacts with the methylamine to form a Schiff extracts, samples containing ara-CTP cannol be anEur.

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Section: Analysis Of the Ara-ctp Breakdown Productsupporting

confidence: 82%

Determination of Pyrimidine Deoxynucleoside Triphosphates in Leukaemia Cell Extracts Containing 1-ß-D-Arabinofuranosylcytosine Triphosphate

Chiba

Szekeres²,

Jäger³

1991

Clinical Chemistry and Laboratory Medicine

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“…They were destroyed by periodate oxidation [12] to limit potentially cross-reacting contaminations of RIA samples. Alkaline phosphatase activity is inhibited by inorganic phosphates present in the elution buffer.…”

Section: Discussionmentioning

confidence: 99%

A Method for the Quantification of Intracellular Zidovudine Nucleotides

Küster

Vogt

Joos

et al. 1991

Journal of Infectious Diseases

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An assay to quantify the phosphorylation products of zidovudine (AZT) in peripheral blood mononuclear cells (PBMC) was developed. Extracts of PBMC were separated by high-performance liquid chromatography. Eluted AZT mono-(MP), di-(DP), and triphosphate (TP) were collected in separate portions. Treatment with alkaline phosphatase yielded equimolar amounts of AZT, which after solid-phase enrichment were assayed by radioimmunoassay. Detection limit was 0.1 pmol/f O" PBMC for each nucleotide. Recoveries of 102%-118% were observed. AZT nucleotides were measured in samples from three patients receiving 250 mg of AZT every 12 h. Intracellular concentrations of AZT-MP after 1-2 h ranged from 0.9 to 1.4 pmol/Hl" PBMC and then declined to 0.3-1.1 pmol/LO" PBMC after 4 h. AZT-DP and AZT-TP reached concentrations of 0.3-0.5 pmol/Iu" PBMC after 1-2 h and could not be detected after 4 h in any of the three patients. Duplicate determinations deviated by <20%.Zidovudine (3'-azido-3'-deoxythymidine; AZT) has been shown to improve survival in patients with AIDS and advanced AIDS-related complex [I]. The compound is a thymidine analog that is efficiently converted intracellularly to AZT-5'-monophosphate (AZT-MP) by thymidine kinase. AZT-MP is then further phosphorylated by thymidylate kinase to the 5'-diphosphate (DP) form and finally to AZT-5'-triphosphate (AZT-TP) [2]. AZT-TP is assumed to be the active form of the drug as it competitively inhibits viral reverse transcriptase [2-4] and further causes proviral DNA chain termination [5].Serum pharmacokinetics of AZT have been studied extensively [6]; however, no data about the extent and kinetics of in vivo phosphorylation are available. To date the intracellular anabolism of AZT and of other dideoxynucleosides has been examined only in vitro using radiolabeled compounds [2,[7][8][9]. A detailed knowledge about the in vivo phosphorylation is important for several reasons. First, there is a documented variability of AZT phosphorylation in various cell systems [8], and data from in vitro experiments cannot necessarily be extrapolated to the in vivo situation. Second, interindividual differences in drug metabolism are well known in clinical medicine for a variety of compounds, and it is conceivable that differences in phosphorylation may at least Informed consent was obtained from all study subjects: guidelines of the University Hospital Ethical Committee were followed.Grant support: Swiss National Science Foundation partly explain the observed differences in the toxicity profile and the clinical and virologic response. Finally, a better understanding of the in vivo pools and pharmacokinetics of intracellular AZT-TP might lead to improved drug schedules for individual patients. Thus we developed a method to measure the intracellular anabolites of AZT in whole blood from patients treated with this drug. Materials and MethodsPreparation of cell extracts. From patients receiving 250 mg of AZT every 12 h or from healthy human immunodeficiency virus (HIV)-seronegative controls, peripheral...

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“…Subsequently, the samples were injected immediately on the same HPLC-system, 34 using the same elution conditions as described above. Response factors were calculated using an external standard containing the various (deoxy) ribonucleotides.…”

Section: (Deoxy)ribonucleotide Analysis By Anion-exchange Hplcmentioning

confidence: 99%

Cyclopentenyl cytosine increases the phosphorylation and incorporation into DNA of 1‐β‐D‐arabinofuranosyl cytosine in a human T‐lymphoblastic cell line

Verschuur

Gennip

Leen

et al. 2002

Intl Journal of Cancer

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The cytotoxic effect of 1-␤-D-arabinofuranosyl cytosine (araC) depends on the intracellular phosphorylation into its active compound araCTP, on the degree of degradation of araCTP and on the incorporation of araCTP into DNA. Deoxycytidine triphosphate (dCTP) inhibits the phosphorylation of araC (by feedback inhibition of the enzyme deoxycytidine kinase) and the incorporation of araCTP into DNA (by competition for DNA polymerase). In a T-lymphoblastic cell line, we studied whether the cytotoxicity of araC (2 nM-50 M) could be enhanced by decreasing the concentration of dCTP, using the nucleoside-analogue cyclopentenyl cytosine (CPEC), an inhibitor of the enzyme CTP synthetase. Preincubation of the cells with CPEC (100 -1,600 nM) for 2 hr increased the concentrations of araCMP 1.6 -9.5-fold, which was significant for each concentration of CPEC used. The concentration of araCDP remained low, whereas the concentration of araCTP changed depending on the concentration of araC used. With 2-15 M of araC and a preincubation with 400 nM of CPEC, the araCTP concentration increased by 4 -15% (not significant), and the total amount of araC nucleotides increased significantly by 21-45%. When using a concentration of araC of 2 nM after a preincubation with CPEC of 100 nM, the concentration of araCMP increased by 60% (p ‫؍‬ 0.015), whereas that of araCTP decreased by 10% (p ‫؍‬ 0.008). This was compensated by an increase of 41% (p ‫؍‬ 0.005) of araCTP incorporation into DNA, which represented 43% of all araC metabolites. Moreover, by performing pulse/chase experiments with 400 nM of CPEC and 2 M of araC, the retention of cytosolic araCTP and the incorporated amount of araCTP into DNA were increased by CPEC. The modulation by CPEC of araC metabolism was accompanied by a synergistic increase of araC-induced apoptosis and by an additive effect on the araC-induced growth inhibition. © 2002 Wiley-Liss, Inc. Key words: cytarabine; deoxycytidine kinase; dCTP; CTP synthetase; cyclopentenyl cytosine 1-␤-D arabinofuranosyl cytosine (araC) is a nucleoside analogue that constitutes the main drug of virtually all treatment protocols for AML and proved its benefit in precursor-B acute lymphocytic leukemia (ALL) and T-ALL. 1-3 One can distinguish low-dose (LD-araC) regimens, applying doses of 75-200 mg/m 2 , from high-dose (HD-araC) regimens where 1-3 g/m 2 are used. The cytotoxic effect of araC depends on several intracellular mechanisms: araC is metabolized into arabinofuranosyl cytosine triphosphate (araCTP) by 3 consecutive phosphorylating steps, catalyzed by deoxycytidine kinase (dCK), nucleoside monophosphate (NMP) kinase and nucleoside diphosphate (NDP) kinase, respectively (Fig. 1). AraCTP is incorporated into DNA as a deoxynucleotide-analogue, where it exerts its cytotoxic properties by inhibiting DNA replication. 4 -6 The phosphorylation of araC into araCTP can be counteracted by the enzyme (deoxy)cytidine deaminase, which converts araC into arabinofuranosyl uracil (araU), dCMP deaminase, converting arabinofuranosyl cytosine mon...

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A rapid and sensitive high pressure liquid chromatography assay for deoxyribonucleoside triphosphates in cell extracts

Cited by 259 publications

References 13 publications

Determination of Pyrimidine Deoxynucleoside Triphosphates in Leukaemia Cell Extracts Containing 1-ß-D-Arabinofuranosylcytosine Triphosphate

Determination of Pyrimidine Deoxynucleoside Triphosphates in Leukaemia Cell Extracts Containing 1-ß-D-Arabinofuranosylcytosine Triphosphate

A Method for the Quantification of Intracellular Zidovudine Nucleotides

Cyclopentenyl cytosine increases the phosphorylation and incorporation into DNA of 1‐β‐D‐arabinofuranosyl cytosine in a human T‐lymphoblastic cell line

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