Liquid chromatography–tandem mass spectrometry method for quantification of thymidine kinase activity in human serum by monitoring the conversion of 3′-deoxy-3′-fluorothymidine to 3′-deoxy-3′-fluorothymidine monophosphate
Highlights
► A LC–MS/MS for measuring thymidine kinase activity in human serum is described. ► Phosphorylation of 3′-deoxy-3′-fluorothymidine indicates activity. ► The monophosphorylated product is measured quantitatively. ► The method uses protein precipitation and on-line column switching. ► The method was FDA validated and applied to hepatocellular carcinoma patients.
Introduction
Thymidine kinase 1 (TK1, ATP; thymidine 5′-phosphotransferase; EC.2.7.1.21) is a key cellular enzyme in DNA synthesis which catalyzes the one step pyrimidine salvage pathway [1]. Specifically, TK1 catalyzes the transfer of terminal phosphate from ATP to the 5′ hydroxyl group of deoxythymidine (dThd) to produce deoxythymidine monophosphate (dTMP). Increased activity of serum or plasma TK1 has been reported in diseases involving DNA metabolism, e.g., viral infections [2], [3], vitamin B12 deficiency [3] and in a variety of malignant diseases including acute and chronic leukemia [4], Hodgkin's disease [5], non-Hodgkin's lymphoma [6], [7], lung cancer [6] and ovarian cancer [6], [8]. TK1 also phosphorylates derivatives of thymidine or deoxyuridine (dUrd). In particular, TK1 can phosphorylate nucleotide analogs with modifications at the 5′-position of the pyrimidine ring, and the 3′-position of the ribose including 5-fluoro-2′-dideoxythymidine and 3′-azido-3′-deoxythymidine (Zidovudine) [9], [10]. These nucleotide analogs can therefore be used to monitor the activity of TK1 [3], [6], [11]. For example, 18F-3′-deoxy-3′-fluorothymidine (18F-FLT), has been used to monitor tumor proliferation via positron emission tomography [12], [13], [14]. Phosphorylation of FLT by TK1 leads to cellular trapping and accumulation of FLT-monophosphate (FLT-MP) without incorporation into DNA. Since FLT has low affinity for TK2 in comparison with FLT-MP, it acts as a selective substrate to monitor TK1 activity [10], [15].
Serum TK1 activity can reflect tumor burden and proliferation, and is most commonly measured using a commercially available thymidine kinase radioenzymatic assay (TK-REA), which uses 125I-deoxyuridine as a substrate to measure thymidine kinase 1 activity [3]. This assay is time-consuming and requires radioactive waste management. A competitive enzyme-linked immunosorbent assay (ELISA) has also been used for measuring serum TK1 activity for measuring phosphorylation of the selective TK1 substrate 3′-azido-2′-deoxythymidine (AZT) [11]. Alternatively, 5-bromodeoxyuridine (BrdU) incorporation into DNA can be monitored by ELISA [16]. After phosphorylation by TK1, bromodeoxyuridine monophosphate (BrdUMP) is processed to bromodeoxyuridine triphosphate (BrdUTP) by yeast enzymes, and the BrdUTP is immobilized by incorporation into an immobilized DNA strand using a recombinant reverse transcriptase. Further, the amount of BrdUTP incorporated in DNA is estimated using an ELISA. This assay is more sensitive than the assay using AZT as a substrate [16]. However, complex sample processing, reagent expense, and selectivity issues associated with ELISA methods are limitations to its application.
Quantification of nucleotide analogs can be performed using high performance liquid chromatography (HPLC) with ultraviolet or fluorescence detection [17], [18]. In the last decade, mass spectrometric (MS) detection has increasingly been used for quantification of nucleoside and nucleotide analogs [19], [20]. Mass spectrometry allows extremely specific and sensitive quantification of nucleotide analogs from biological matrices including serum. Thus, LC–MS/MS can be used as a tool to measure the conversion of FLT to FLT-MP in serum.
In our previous work, a method was developed to quantify the intracellular conversion of FLT to FLT-MP in cell lysates using LC–MS/MS [21]. Separation of the analytes was achieved using C18 column and detection using Applied Biosystems SCIEX API 4000 QTrap mass spectrometer. This method was able to monitor proliferating cell TK1 activity in as few as 500 cells per well in LNCaP prostate cancer cells. In the present report, we describe the development and analytical validation of a suitable non-isotopic, non-immunologically based assay for quantitative monitoring of FLT to FLT-MP in human serum. This method uses a labeled isotope internal standard, and column trapping to improve assay ruggedness.
Section snippets
Chemicals and reagents
HPLC grade acetonitrile and methanol were purchased from Honeywell Burdick & Jackson (Muskegon, MI, USA). 3′-Deoxy-3′-fluorothymidine was purchased from Sigma–Aldrich (St. Louis, MO, USA). 3′-Deoxy-3′-fluorothymidine-5′-monophosphate disodium salt (FLT-MP), 3′-deoxy-3′-fluorothymidine-d3 (FLT-d3) and 3′-deoxy-3′-fluorothymidine-5′-monophosphate-d3 (FLT-MP-d3) were purchased from Toronto Research Chemicals Inc. (TRC, Toronto, ON, Canada). Citric acid, formic acid, sodium acetate, adenosine
Results and discussion
This serum TK1 activity assay monitors the phosphorylation of FLT, a thymidine analog and selective TK1 substrate, to its metabolite FLT-MP. The reaction is performed at 37 °C in a 0.1 M sodium acetate phosphorylation buffer supplemented with enzyme co-factors and stabilizing reagents [11], [23]. ATP serves as a phosphate donor in the reaction. The reducing agent DTE liberates TK1 from inhibitory serum protein complexes, and UMP serves as a FLT-MP degradation inhibitor [11]. The FLT
Conclusions
Serum TK1 activity is a potentially useful biomarker for monitoring hematological malignancies and solid tumors. A sensitive, non-radiometric LC–MS/MS assay for was developed and validated for monitoring the conversion of FLT to FLT-MP in human serum. Based on established, radiometric TK1 activity assays, this novel method shows good linearity and selectivity in human serum samples. Stability studies demonstrated adequate intermediate processing and post-preparative analyte stability.
The
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