Reverse-phase ion-pairing high-performance liquid chromatography of phosphocreatine, creatine and creatinine in equine muscle.
Abstract: A simple, robust and reproducible analytical method for the determination of phosphocreatine (PCr), creatine (Cr) and creatinine (Cn) in equine skeletal muscle is presented. The technique used isocratic reverse-phase ion-pairing high-performance liquid chromatography. Neutralized perchloric acid extracts of equine muscle biopsies were analysed and the values obtained were compared with determinations from an established enzymic procedure. Good resolution of all three metabolites was achieved within a retention time of less than 11 min. Linearity for each metabolite within the concentration range in the samples was demonstrated. Peak purity was specifically addressed. The abolition of each creatine in a pooled extract by enzymic incubation showed no underlying peaks. It was concluded that peaks were free of co-eluents which would otherwise lead to an overestimation of PCr, Cr and Cn concentrations.
Publication Date: 1991-04-01 PubMed ID: 2042018DOI: 10.1080/00365519109091099Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research describes a reliable and simple analytical method for measuring the concentrations of phosphocreatine, creatine, and creatinine in horse muscle using reverse-phase ion-pairing high-performance liquid chromatography.
Methodology and Technique
- The researchers present a method of using isocratic reverse-phase ion-pairing high-performance liquid chromatography to measure levels of phosphocreatine, creatine, and creatinine in horse muscle tissue. This technique has been chosen because it provides accurate results quickly – in less than 11 minutes.
- Isocratic reverse-phase and ion-pairing are terms used to describe the type of liquid chromatography used. With ‘isocratic,’ the same solvent is used throughout the chromatographic run. ‘Reverse-phase’ and ‘ion-pairing’ refer to the nature of the stationary phase and the mobile phase, respectively, and the interactions between them which allow for the separation of different molecules.
Sample Preparation and Analysis
- The muscle tissues were first processed using neutralized perchloric acid to extract the metabolites. The perchloric acid extracts were then analysed using the liquid chromatography technique.
- The results obtained through this method were compared to those obtained from a previously established enzymatic detection method. This comparison served as a form of cross-validation to ensure the results were accurate.
Results and Conclusion
- The liquid chromatography method provided good resolution of the three metabolites, showing peak purity. In other words, the peaks were free of co-eluting substances that could potentially lead to falsely high measurements of phosphocreatine, creatine, and creatinine.
- This was confirmed by using enzyme incubation to selectively eliminate each creatine from a pooled extract, which showed no underlying peaks.
- In conclusion, the liquid chromatography method presented in this study offers a quick, simple, and accurate way to measure phosphocreatine, creatine, and creatinine levels in equine muscle tissue.
Cite This Article
APA
Dunnett M, Harris RC, Orme CE.
(1991).
Reverse-phase ion-pairing high-performance liquid chromatography of phosphocreatine, creatine and creatinine in equine muscle.
Scand J Clin Lab Invest, 51(2), 137-141.
https://doi.org/10.1080/00365519109091099 Publication
Researcher Affiliations
- Department of Comparative Physiology, Animal Health Trust, Newmarket, UK.
MeSH Terms
- Animals
- Chromatography, High Pressure Liquid / methods
- Chromatography, High Pressure Liquid / statistics & numerical data
- Creatine / analysis
- Creatinine / analysis
- Horses
- Muscles / chemistry
- Phosphocreatine / analysis
Citations
This article has been cited 7 times.- Teng H, Mi Y, Deng H, He Y, Wang S, Ai C, Cao H, Chen L. Acylated anthocyanin inhibited the formation of heterocyclic amines in hybrid chemical model system and its underlying mechanism.. Food Chem X 2023 Mar 30;17:100559.
- Teng H, Mi Y, Deng H, He Y, Wang S, Ai C, Cao H, Zheng B, Chen L. Inhibitory effect of acylated anthocyanins on heterocyclic amines in grilled chicken breast patty and its mechanism.. Curr Res Food Sci 2022;5:1732-1739.
- Roberts PA, Fox J, Peirce N, Jones SW, Casey A, Greenhaff PL. Creatine ingestion augments dietary carbohydrate mediated muscle glycogen supercompensation during the initial 24 h of recovery following prolonged exhaustive exercise in humans.. Amino Acids 2016 Aug;48(8):1831-42.
- Bhatt DP, Houdek HM, Watt JA, Rosenberger TA. Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis.. Neurochem Int 2013 Feb;62(3):296-305.
- Edner AH, Nyman GC, Essén-Gustavsson B. Metabolism before, during and after anaesthesia in colic and healthy horses.. Acta Vet Scand 2007 Nov 15;49(1):34.
- Rebaudo R, Melani R, Carità F, Rosi L, Picchio V, Ruggeri P, Izvarina N, Balestrino M. Increase of cerebral phosphocreatine in normal rats after intracerebroventricular administration of creatine.. Neurochem Res 2000 Nov;25(11):1493-5.
- Greenhaff PL, Harris RC, Snow DH, Sewell DA, Dunnett M. The influence of metabolic alkalosis upon exercise metabolism in the thoroughbred horse.. Eur J Appl Physiol Occup Physiol 1991;63(2):129-34.
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