PMID- 21036136
OWN - NLM
STAT- MEDLINE
DCOM- 20110620
LR  - 20101223
IS  - 1096-0309 (Electronic)
IS  - 0003-2697 (Linking)
VI  - 409
IP  - 2
DP  - 2011 Feb 15
TI  - A simplified method for rapid quantification of intracellular nucleoside 
      triphosphates by one-dimensional thin-layer chromatography.
PG  - 249-59
LID - 10.1016/j.ab.2010.10.029 [doi]
AB  - Quantification of nucleotides is an important part of metabolomics but has been 
      hampered by the lack of fast, sensitive, and reliable methods. We present a less 
      time-consuming, more sensitive, and more precise method for the quantitative 
      determination of nucleoside triphosphates (NTPs), 5-ribosyl-1-pyrophosphate 
      (PRPP), and inorganic pyrophosphate (PP(i)) in cell extracts. The method uses 
      one-dimensional thin-layer chromatography (TLC) and radiolabeled biological 
      samples. Nucleotides are resolved at the level of ionic charge in an optimized 
      acidic ammonium formate and chloride solvent, permitting quantification of NTPs. 
      The method is significantly simpler and faster than both current two-dimensional 
      methods and high-performance liquid chromatography (HPLC)-based procedures, 
      allowing a higher throughput while common sources of inaccuracies and technical 
      problems are avoided. For determination of PP(i), treatment with inorganic 
      pyrophosphatase (PPase) of the radiolabeled phosphate is employed for removal of 
      contaminating pyrophosphate. Biological examples performed in triplicates showed 
      standard deviations of approximately 10% of the mean for the determined 
      concentrations of NTPs.
CI  - Copyright (c) 2010 Elsevier Inc. All rights reserved.
FAU - Jendresen, Christian Bille
AU  - Jendresen CB
AD  - Center for Systems Microbiology, Department of Systems Biology, Technical 
      University of Denmark, Lyngby, Denmark.
FAU - Kilstrup, Mogens
AU  - Kilstrup M
FAU - Martinussen, Jan
AU  - Martinussen J
LA  - eng
PT  - Journal Article
DEP - 20101029
PL  - United States
TA  - Anal Biochem
JT  - Analytical biochemistry
JID - 0370535
RN  - 0 (Nucleosides)
RN  - 0 (Nucleotides)
RN  - EC 3.6.1.- (Pyrophosphatases)
SB  - IM
MH  - Chromatography, Thin Layer/*methods
MH  - Lactococcus lactis/metabolism
MH  - Nucleosides/analysis
MH  - Nucleotides/*analysis
MH  - Pyrophosphatases/analysis/metabolism
EDAT- 2010/11/03 06:00
MHDA- 2011/06/21 06:00
CRDT- 2010/11/02 06:00
PHST- 2010/08/17 00:00 [received]
PHST- 2010/10/18 00:00 [revised]
PHST- 2010/10/25 00:00 [accepted]
PHST- 2010/11/02 06:00 [entrez]
PHST- 2010/11/03 06:00 [pubmed]
PHST- 2011/06/21 06:00 [medline]
AID - S0003-2697(10)00685-8 [pii]
AID - 10.1016/j.ab.2010.10.029 [doi]
PST - ppublish
SO  - Anal Biochem. 2011 Feb 15;409(2):249-59. doi: 10.1016/j.ab.2010.10.029. Epub 2010 
      Oct 29.