Fast and sensitive HPLC–MS/MS method for direct quantification of intracellular deoxyribonucleoside triphosphates from tissue and cells

Abstract

Deoxyribonucleoside triphosphates (dNTPs) are used in DNA synthesis and repair. Even slight imbalances can have adverse biological effects. This study validates a fast and sensitive HPLC-MS/MS method for direct quantification of intracellular dNTPs from tissue. Equal volumes of methanol and water were used for nucleotide extraction from mouse heart and gastrocnemius muscle and isolated cardiomyocytes followed by centrifugation to remove particulates. The resulting supernatant was analyzed on a porous graphitic carbon chromatography column using an elution gradient of ammonium acetate in water and ammonium hydroxide in acetonitrile with a run time of just 10 minutes. Calibration curves of all dNTPs ranged from 62.5–2,500 femtomole injections and demonstrated excellent linearity (r2>0.99). The within day and between day precision, as measured by the coefficient of variation (CV (%)), was <20% for all points, including the lower limit of quantification (LLOQ). The inter-day accuracy was within 12% of expected concentration for the LLOQ and within 7% for all other points on the calibration curve. The intra-day accuracy was within 22% for the LLOQ and within 11% for all points on the curve. Compared to existing methods, this study presents a faster and more sensitive method for dNTP quantification.

A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye, and inhibitor-resistant high-fidelity DNA polymerase

“…We speculate that the variation in dATP levels could be related to ischemia during sample collection and delayed quenching of enzymatic activity. Similar to two chromatographic quantifications (6,22), we found heart dGTP concentration to be 5 to 15-fold higher than that of other three dNTPs. Mitochondrial dNTP pools are extremely asymmetric with dGTP concentration being more than 10-fold higher than the concentration of other dNTPs (23).…”

“…In skeletal muscle samples, individual dNTP concentrations have ranged from less than 0.02 to 0.3 pmol/mg with the biggest variation seen in dATP levels (18,19). Interestingly, a HPLC-MS-based method has also shown surprisingly low dATP concentrations for mouse heart tissue (0.02 pmol/mg) (6). Although the ribonucleotide ATP is the physiological substrate for cellular ATPases, at least some of these enzymes can use dATP instead of ATP (21).…”

“…We have analysed more than 40 tissue samples in triplicates using our method in 384-well format (results to be published elsewhere). Only handful of studies have addressed the topic of tissue dNTP levels in health and disease (6,(17)(18)(19). As nucleotide metabolism is more and more studied in the field of cancer biology and in relation to metabolic diseases such as mitochondrial disorders (19,24) the availability of an easily applicable dNTP assay for use in the general laboratory is of the utmost importance.…”

“…As the starting point, we chose the well-validated 60% methanol extraction, which has been shown to give dNTP recoveries higher than 90% (6,7). We also adopted a 3-kDa cut-off centrifugal filtration from a previous study (7) to remove any remaining DNA and other macromolecules that could increase EvaGreen background fluorescence, and produce unspecific DNA polymerisation, or polymerase inhibition.…”

“…These methods have mainly allowed measurement of dNTPs from actively proliferating cultured cells with high dNTP concentrations and limited complexity of interfering biological matrix. Recently, a HPLC-MS-based method was developed that allows quantification of tissue dNTP levels from a feasibly small tissue amount (15 to 30 mg) (6). However, not every laboratory has suitable HPLC-MS equipment and expertise.…”