Stability of High-Energy Substrates in Fast- and Slow-Twitch Muscle: Comparison of Enzymatic Assay of Biopsy with in Vivo31P Nuclear Magnetic Resonance Spectroscopy

Abstract

The purpose of this study was to investigate the stability of ATP and PCr levels in stored muscle samples and extracts. ATP and PCr levels were measured by fluorimetric analysis in freeze-clamped biopsies of soleus, extensor digitorum longus, and gastrocnemius muscles of the rat after storage in a freezer at −70°C as (i) intact wet muscle, (ii) freeze-dried muscle, and (iii) an extract of freeze-dried muscle. Assays were performed within 24 h of taking the biopsy and after variable periods of storage from 1 to 4 weeks. The data for the gastrocnemius muscles were compared with those obtained, in the same rat, by in vivo31P NMR spectroscopy. In the biopsies, the ATP levels were stable irrespective of the duration or method of storage. The PCr levels fell by 13-16% compared with the values obtained from the assay done within 24 h of taking the biopsy, irrespective of the method of storage, but could be corrected in the freeze-dried stored muscle by expressing the data in relation to the total creatine levels. The fluorimetrically measured PCr, in whole muscle extracts of the gastrocnemius, assayed within 24 h, were comparable to those obtained from 31P NMR spectroscopy. We concluded that PCr levels in muscle are not stable during storage at −70°C and should be assayed within 24 h of taking a muscle biopsy to ensure that the values are the same as those obtained by 31P NMR.