Measurement of high-energy phosphates in tiny fragments of human muscle fibres

Standard

This research method was  developed in the group headed by Professor Anthony J Sargeant in Amsterdam. Arnold de Haan had developed the basis of the technique in the 1980s as part of his own PhD work. This was subsequently refined to enable very small fragments of human muscle fibre obtained by needle biopsy to be analysed. The present research paper describes that refined techniques and its sensitivity. The work formed part of the PhD thesis the outstanding Greek PhD student, Christina Karatzaferi, who was supervised by Tony Sargeant and Arnold de Haan.

Improved high-performance liquid chromatographic assay for the determination of “high-energy” phosphates in mammalian skeletal muscle. Application to a single-fibre study in man

Christina Karatzaferi, Arnold de Haan, Carla Offringa, Anthony J Sargeant.

Journal of Chromotography

J Chromatogr B Biomed Sci Appl. 1999 Jul 9;730(2):183-91
Abstract
A sensitive and reproducible method for the determination of adenine nucleotides (ATP, IMP) and creatine compounds [creatine (Cr), phosphocreatine (PCr)] in freeze-dried single human muscle fibre fragments is presented. The method uses isocratic reversed-phase high-performance liquid chromatography of methanol extracts. Average retention times (min) of ATP, IMP and PCr, Cr from standard solutions were 10.6+/-0.42, 2.11+/-0.06 (n=6) and 10.5+/-0.31 and 1.19+/-0.02 (n=9), respectively. Detection limits in extracts from muscle fibre fragments were 2.0, 1.0, 3.0 and 2.0 mmol/kg dm, respectively. The assay was found successful for analysis of fibre-fragments weighing > or = 1 microg.
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