Use of superfused rat skeletal muscle for metabolic studies: assessment of pH by 31P n.m.r.

We developed a muscle superfusion system suitable for metabolic studies of small isolated rat muscle ex vivo in real time and in a non-destructive manner by n.m.r. spectroscopy. In order to determine biochemical stability of superfused extensor digitorum longus (EDL) muscle (from fasted 45 and 100 g rats), the energy state and the pH of muscle were continuously monitored by 31P n.m.r. spectroscopy. ATP and phosphocreatine remained stable during 2 h whatever the muscle size (20 or 45 mg). Neither metabolite was a sensitive probe of possible metabolic compartmentation within muscle under our experimental conditions. By contrast, the chemical shift of Pi by its sensitivity to pH was a discriminant factor in the assessment of muscle stability. Indeed, heterogeneity of pH was observed only in the 45 mg EDL muscle resulting from a core region with loss of glycogen. Together, these observations suggest deviations of energy metabolism to supply ATP. Consequently, pH may be considered as a new real-time criterion for monitoring a metabolic heterogeneity due to changes in energy metabolism of muscle preparations ex vivo.

Assessment of threonine metabolism in vivo by gas chromatography/mass spectrometry and stable isotope infusion.

The fractional contributions (FC) of threonine to glycine and 2-ketobutyrate (KB) fluxes in fed pigs have been assessed by the constant infusion of L-[1-13C]-threonine. The analysis of the enantiomeric purity of labeled threonine by gas chromatography/mass spectrometric (GC/MS) analysis is reported as the N-TFA isopropyl ester derivative. The commercially available [1-13C]threonine comprised 98.7% of the L-enantiomer, enriched at 99 atom percentage excess (APE), and 1.3% of L-allo-threonine contaminant, also enriched at 99 APE. The enantiomeric purity of threonine in plasma of pigs infused for 10 h with [1-13C]threonine showed that the L-allo contaminant did not accumulate. The t-butyl dimethylsilyl derivatives of threonine, glycine, and 2-aminobutyrate (ABA) were used to measure the enrichment of these compounds in plasma and liver samples by GC/MS/selected ion monitoring analysis. Analyses were performed on between 1 and 5 nmol of each amino acid extracted from biological fluids and a 1:10 split injection. GC/MS parameters were assessed with standards at similar quantities and found to be satisfactory; e.g., injection of 1-10 nmol of glycine did not significantly alter the slope and the precision of the standard curve. The coefficient of variation of enrichment determination was less than 10% for standards enriched at 0.4 APE or more and biological samples enriched at 0.6 APE or greater. Within-animal coefficients of variation for four plasma samples obtained at equal intervals between 8 and 10 h of [1-13C]threonine infusion were 4, 21, and 24% for threonine, ABA, and glycine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of in vivo protein synthesis in lamb tissues with [3H]valine flooding doses.

Week-old lambs received an intravenous injection of 4.3, 8.5, 12.8 or 17.1 mmol [3H]valine/5 kg body weight, i.e., 3.6-14.4-times the whole-body free valine content. To ensure that protein synthesis measurements in lambs are reliable within a 30-min period, these large amounts of valine must account for at least around 11-times the total free pool of valine. This amounted to 12.8 mmol valine/5 kg body weight. There were no significant variations in plasma insulin and plasma glucagon levels 5, 13 and 30 min after the injection of so much valine. The fractional rates of protein synthesis were determined in tissues of animals receiving either 12.8 or 17.1 mmol valine/5 kg body weight. The rates of protein synthesis in the jejunum (87.5%/day), liver (106.6%/day) and tensor fasciae latae muscle (18.8%/day) of lambs injected with the 12.8 mmol [3H]valine flooding dose, were in the range of data obtained in immature rats. Increasing the flooding amount of valine up to 17.1 mmol/5 kg body weight did not significantly alter protein synthesis rates in the jejunum, liver or skeletal muscle. This suggested that both the flooding-dose method in itself and valine had no effect on in vivo protein synthesis.