Proton NMR studies of creatine in human erythrocytes

Proton spin-echo nuclear magnetic resonance spectroscopy was used to measure the relative levels of some metabolites in intact human erythrocytes that had been fractionated by density gradient centrifugation. Age dependent changes in the concentrations of free glycine, choline and ergothioneine were seen for the first time, while glutathione was essentially invariant. In addition, there was a 10-fold decrease in creatine levels from the youngest to oldest cells. This confirms earlier reports and provides a simple explanation for the variable creatine resonance intensities seen in spectra obtained from different erythrocyte samples prepared from the same donor. We demonstrated the different chemical shifts of the methylene resonances of creatine and creatine phosphate and hence confirmed that the bulk of the creatine in intact erythrocytes is not phosphorylated. The chemical shift difference enabled the monitoring of the creatine phosphokinase catalysed reaction in lysates to which the rabbit muscle enzyme had been added. This experiment indicated that the enzyme is not significantly inhibited by factors in the lysates, and introduced a new means of assaying the in situ activity of the enzyme.