Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples

Abstract

Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.

Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery

“…In this case, the substrates were nonradioactive and the amount of product was measured by HPLC. The HPLC protocol is a variant of a previously described protocol for nucleoside triphosphates ( 47 ) but with a lower concentration of phosphate in the mobile phase in order to optimize it for the study of monophosphates instead of triphosphates and with methanol instead of acetonitrile in order to create larger spacing between the peaks. The assay itself was performed as described previously and included 200 μM of each of the five substrates and 100 ng G intestinalis thymidine kinase.…”

Zinc(II) Complex with Pyrazolone-Based Hydrazones is Strongly Effective against Trypanosoma brucei Which Causes African Sleeping Sickness

“…This fraction was used for quantification of nucleotides and deoxynucleotide diphosphates and triphosphates by a 150 × 3 mm C18-WP HPLC column (Chromanik Sunshell). 37 The analyses were performed on a 150 × 2.1 mm Sunshell C18-WP 2.6 μm column (ChromaNik Technologies Inc), at 30 °C using a mobile phase of 43% solution A and 57% solution B. Solution A contained 5.8% (v/v) acetonitrile, 23 g/L KH 2 PO 4 , and 0.7 g/L tetrabutylammonium bromide (TBA-Br) adjusted to pH 5.6 with KOH, while solution B contained 5.8% (v/v) acetonitrile and 0.7 g/L TBA-Br.…”

“…For the analysis, the method by Ranjbarian et al was applied. 37 The procedure started by adding 5 μM of the drug tested to 50 mL of a T. brucei culture (10 6 logarithmically growing cells per mL) and incubating the parasites for 1 h before extracting the NTPs, dNTPs, NDPs, and dNDPs from them with trichloroacetic acid and quantifying the nucleotides with HPLC. From these experiments, it was possible to evaluate changes in nucleotide pools between non-treated trypanosomes and those treated with H 2 L 1 and 1 (chromatographs in Figure S47 ).…”

“…brucei NTP, dNTP, and ADP pools were measured by HPLC and compared to the pools of parasites treated with H 2 L 1 and complex 1 . For the analysis, the method by Ranjbarian et al was applied . The procedure started by adding 5 μM of the drug tested to 50 mL of a T.…”

Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions

“…To analyze the metabolites consumed by TIR domains in red blood cell extracts, ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS) was used. Analyses were performed as previously published [ 66 ]. Briefly, the analytical platform employs a Vanquish UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA) coupled online to a Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA).…”