title: Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ion-pair RP-HPLC/UV for the quantification of adenine and guanine pools.
abstract: Human African Trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. Although trypanosomes are well-studied model organisms, only little is known about their adenine and guanine nucleotide pools. Besides being building blocks of RNA and DNA, these nucleotides are also important modulators of diverse biochemical cellular processes. Adenine nucleotides also play an important role in the regulation of metabolic energy. The energetic state of cells is evaluated by the energy charge which gives information about how much energy is available in form of high energy phosphate bonds of adenine nucleotides. A sensitive and reproducible ion-pair RP-HPLC/UV method was developed and optimized, allowing the quantification of guanine and adenine nucleosides/nucleotides in T. brucei. With this method, the purine levels and their respective ratios were investigated in trypanosomes during logarithmic, stationary and senescent growth phases. Results of this study showed that all adenine and guanine purines under investigation were in the low mM range. The energy charge was found to decrease from logarithmic to static and to senescent phase whereas AMP/ATP, ADP/ATP and GDP/GTP ratios increased in the same order. In addition, the AMP/ATP ratio varied as the square of the ADP/ATP ratio, indicating AMP to be the key energy sensor molecule in trypanosomes.
authors.affiliation: Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Lausanne & University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland.
authors.forename: Patricia
authors.initials: P
authors.lastname: Graven
authors.affiliation: Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Lausanne & University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland.
authors.forename: Margherita
authors.initials: M
authors.lastname: Tambalo
authors.affiliation: Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Lausanne & University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Scapozza
authors.affiliation: Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, University of Lausanne & University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland. Electronic address: remo.perozzo@unige.ch.
authors.forename: Remo
authors.initials: R
authors.lastname: Perozzo
mesh_terms: D000227:Adenine Nucleotides / Q000032:analysis* / Q000378:metabolism; D000241:Adenosine / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D056148:Chromatography, Reverse-Phase / Q000379:methods / Q000592:standards; D006150:Guanine Nucleotides / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014346:Trypanosoma brucei brucei / Q000737:chemistry* / Q000254:growth & development*; D014466:Ultraviolet Rays
chemical_list: D000227:Adenine Nucleotides; D006150:Guanine Nucleotides; D000241:Adenosine
keywords: Energy charge; Growth phases; HPLC; Purine pool; Trypanosoma brucei