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Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes

Zinellu, Angelo and Pasciu, Valeria and Sotgia, Salvatore and Scanu, Bastianina and Berlinguer, Fiammetta and Leoni, Giovanni Giuseppe and Succu, Sara and Cossu, Ignazio and Sanna Passino, Eraldo and Naitana, Salvatore and Deiana, Luca and Carru, Ciriaco (2010) Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes. Analytical and Bioanalytical Chemistry, Vol. 398 (5), p. 2109-2116. ISSN 1618-2642. eISSN 1618-2650. Article.

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DOI: 10.1007/s00216-010-4186-6

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.

Item Type:Article
ID Code:6169
Status:Published
Refereed:Yes
Uncontrolled Keywords:ATP, BODIPY FL EDA, capillary electrophoresis, spermatozoa, oocytes
Subjects:Area 07 - Scienze agrarie e veterinarie > VET/09 Clinica chirurgica veterinaria
Area 07 - Scienze agrarie e veterinarie > VET/02 Fisiologia veterinaria
Area 05 - Scienze biologiche > BIO/10 Biochimica
Area 05 - Scienze biologiche > BIO/12 Biochimica clinica e biologia molecolare clinica
Divisions:001 Università di Sassari > 02 Centri > Centro di eccellenza interdisc. sviluppo ricerca biotecnologica e studio biodiversità della Sardegna e dell'area mediterranea
002 Altri enti e centri di ricerca del Nord Sardegna > AGRIS Sardegna, Olmedo > Dipartimento di ricerca per l'incremento ippico
001 Università di Sassari > 01 Dipartimenti > Patologia e clinica veterinaria
001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
002 Altri enti e centri di ricerca del Nord Sardegna > Porto Conte Ricerche, Alghero
001 Università di Sassari > 01 Dipartimenti > Biologia animale
Publisher:Springer Berlin / Heidelberg
ISSN:1618-2642
eISSN:1618-2650
Copyright Holders:© Springer-Verlag 2010
Deposited On:22 Jul 2011 14:21

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