Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry.
Nitrogen-containing bisphosphonates (N-BPs) are shown to inhibit a key enzyme of intracellular mevalonate pathway, FPP synthase, leading to intracellular accumulation of pathway metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In our previous studies we have shown that a new type of ATP analog, ApppI (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester), is also formed in addition to IPP and DMAPP accumulation. ApppI has cytotoxic effects leading to direct apoptosis of various cancer cells. In this study we present a validated method based on ion-pair LC-MS(2) for the analysis of isomeric mevalonate pathway metabolites and ATP analogs in cell culture samples. Limit of quantitation for IPP and DMAPP was 0.030microM (1.35fmol on-column) and for ApppI and ApppD 0.020microM (0.9fmol on-column). Acceptable accuracies and precision were also obtained for quality control samples in low and high concentrations of the calibration curve. In addition, we present a new method for quantitation of each coeluting isomer utilizing the peak intensity ratios of two characteristic fragment ions of each compound. For IPP and DMAPP, fragment ions m/z 177 and m/z 159 in the MS(2) were monitored, whereas for ATP analogs, ApppI and ApppD (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-2-enyl) ester), the same fragments in the MS(3) spectra were followed. IPP and DMAPP accumulation as well as ApppI and ApppD formation was demonstrated using MCF-7 breast cancer cells. Cells were treated with 25muM zoledronic acid (an N-BP) for 24h, conditions found to induce significant production of the metabolites. We found that the total amount of IPP and DMAPP was 2.4nmol/mg of protein and amount of ApppI and ApppD was 1.1nmol/mg protein. Relative portions of the isomers were approximately 1:4 IPP:DMAPP and 3:7 ApppI:ApppD. Untreated control samples did not contain IPP, DMAPP, ApppI or ApppD.
Jauhiainen
Marjo
M
Department of Pharmaceutics, University of Kuopio, Box 1627, FI-70211 Kuopio, Finland. Marjo.Jauhiainen@uku.fi
Mönkkönen
Hannu
H
Räikkönen
Johanna
J
Mönkkönen
Jukka
J
Auriola
Seppo
S
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D001943:Breast Neoplasms / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D045782:Hemiterpenes / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D016014:Linear Models; D008798:Mevalonic Acid / Q000032:analysis / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C543712:triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; D000255:Adenosine Triphosphate; D008798:Mevalonic Acid