title: Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes.
abstract: We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5'-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 μm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV = 4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 μmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5'-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.
authors.affiliation: Department Biomedical Sciences and Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Zinellu
authors.forename: Valeria
authors.initials: V
authors.lastname: Pasciu
authors.forename: Salvatore
authors.initials: S
authors.lastname: Sotgia
authors.forename: Bastianina
authors.initials: B
authors.lastname: Scanu
authors.forename: Fiammetta
authors.initials: F
authors.lastname: Berlinguer
authors.forename: Giovanni
authors.initials: G
authors.lastname: Leoni
authors.forename: Sara
authors.initials: S
authors.lastname: Succu
authors.forename: Ignazio
authors.initials: I
authors.lastname: Cossu
authors.forename: Eraldo Sanna
authors.initials: ES
authors.lastname: Passino
authors.forename: Salvatore
authors.initials: S
authors.lastname: Naitana
authors.forename: Luca
authors.initials: L
authors.lastname: Deiana
authors.forename: Ciriaco
authors.initials: C
authors.lastname: Carru
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis*; D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods; D005260:Female; D005453:Fluorescence; D007834:Lasers; D008297:Male; D009865:Oocytes / Q000737:chemistry*; D012756:Sheep; D013094:Spermatozoa / Q000737:chemistry*
chemical_list: D000255:Adenosine Triphosphate