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.
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.
Karatzaferi
C
C
Neuromuscular Biology Research Group, Manchester Metropolitan University, Alsager, UK. C.Karatzaferi@mmu.ac.uk
De Haan
A
A
Offringa
C
C
Sargeant
A J
AJ
D000227:Adenine Nucleotides / Q000032:analysis*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003402:Creatine Kinase / Q000032:analysis*; D006801:Humans; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D000227:Adenine Nucleotides; D003402:Creatine Kinase