Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes.
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.
Zinellu
Angelo
A
Department Biomedical Sciences and Centre of Excellence for Biotechnology Development and Biodiversity Research, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy.
Pasciu
Valeria
V
Sotgia
Salvatore
S
Scanu
Bastianina
B
Berlinguer
Fiammetta
F
Leoni
Giovanni
G
Succu
Sara
S
Cossu
Ignazio
I
Passino
Eraldo Sanna
ES
Naitana
Salvatore
S
Deiana
Luca
L
Carru
Ciriaco
C
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*
D000255:Adenosine Triphosphate