Variability of energy metabolism and nuclear T3-receptors within the skeletal muscle tissue of pigs different with respect to the halothane gene.

Energy metabolism of skeletal muscle tissue of pigs growing from approximately 12 to 18 kg (12 homozygous halothane negative, HH; 16 heterozygotes, Hh; 17 homozygous halothane susceptible, hh) was measured in vivo using 31P nuclear magnetic resonance (NMR) spectroscopy. Data for intracellular pH, phosphocreatine (PCr), phosphomonoesters (PME), and ATP were analyzed by canonical discriminant analysis, an artificial neural network approach, and analysis of variance. Within the hh pigs, two subpopulations could be distinguished before the application of halothane treatment. Some of the hh pigs had a high PME concentration in the biceps femoris muscle (hh(pme+)), whereas others had a low concentration (hh(pme-)) (2.18 +/- .12 for hh(pme+) vs 1.68 +/- .12 mM for hh(pme-), P < .004). The hh(pme+) pigs were statistically different from HH pigs for pH (P < .03), PME (P < .004), and PCr (P < .008) before halothane treatment. The hh(pme-) pigs were not different from the Hh and HH pigs with respect to PME when measured before halothane treatment (P > .05). However, intracellular pH (P < .03) and PCr (P < .008) of the hh(pme-) pigs were different from those of HH pigs (7.15 vs 7.19 for pH and 38.7 vs 35.1 for PCr, respectively). When combining intracellular pH, PME, and PCr within a canonical discriminant analysis, all were measured before halothane treatment, Hh pigs were found to be different from HH pigs (Mahalanobis distance different from zero, P < .02). In a second experiment, growth rate, depth of longissimus muscle, and maximal binding capacity of nuclear T3-receptors of skeletal muscle tissue were different (P < .05, P < .002, and P < .02, respectively) among pigs selected from the same genetic lines. Of the variability in depth of the longissimus muscle, 22% was explained by variability in maximal binding capacity of nuclear T3-receptors. These results, if confirmed with a large number of pigs, might open new possibilities for selection procedures for leanness because, with respect to halothane susceptibility, a shift between genotypic and phenotypic variability was observed.

Correlation of rapid changes in the average water diffusion constant and the concentrations of lactate and ATP breakdown products during global ischemia in cat brain.

Rapid changes in the average water diffusion constant, Dav = 1/3[Dxx+Dyy+Dzz], and in the concentrations of lactate and purine nucleotides and nucleosides were measured upon global ischemia (cardiac arrest) in cat brain, at a combined time resolution of 36 s (n = 7). At this time resolution, the normalized time curves of 1 - Dav and the increase in ATP breakdown product did not coincide, with the changes in Dav being most rapid. The normalized curves of 1 - Dav and the lactate increase coincided for the first 2-2.5 min after which the change in Dav was more rapid. After this time point, an excellent correlation was found between the drop in Dav and the decrease in energy utilization rate, which was calculated from the measured time curves of lactate formation and ATP breakdown, and from the time curve for phosphocreatine use reported in the literature. These results are in agreement with the expected biphasic changes in ion and water homeostasis during ischemia and with the model of diffusional changes being a consequence of a water shift from interstitial to intracellular space.

Variability within intramuscular fat content of pigs as measured by gravimetry, FTIR and NMR spectroscopy.

In order to determine in vivo intramuscular fat content of pigs' biceps femoris, three methods were compared. Gravimetry and FTIR spectroscopy after total fat extraction from a biopsy (about 400 mg skeletal muscle tissue) and in vivo (1)H NMR spectroscopy after imaging and volume of interest selection were used. Mean values (g fat/100 g fresh tissue) were, respectively, 1·47 ± 0·35 (gravimetry), 1·26 ± 0·33 (FTIR) and 0·51 ± 0·19 (NMR); but NMR-values represented only triglycerides. Within an intramuscular fat range from 1·1 to 2·7 g per 100 g fresh muscle tissue, possible to estimate a calibration line between the in vitro and in vivo data for hybrid piglets of about 18 kg. Repeated in vivo NMR measurements on the same muscle volume showed a mean coefficient of variation of 5·5 ± 2·7%. The coefficient of variation of measurements on different volumes within the same muscle was 14 ± 10%. The mean intramuscular fat content of 18 kg or 100 kg pigs was, respectively, 1·64 ± 0·46 (biceps femoris) and 1·32 ± 0·1 (longissimus dorsi) g per 100 g fresh muscle tissue.

In situ 13C NMR quantification of hepatic glycogen.

We report on the 13C NMR visibility of the C-1 glycosidic carbon of alpha-particulate glycogen in perfused rat liver. We used rats fed ad libitum, animals refed after a 48 h fast with a sucrose supplement with or without glucocorticoid treatment, and gsd/gsd rats with a hepatic glycogen storage disease due to phosphorylase kinase deficiency. Thus we studied a wide range of glycogen levels (25-140 mg/g liver). All livers were perfused with 15 mM glucose, to maintain constant glycogen levels. Failure to activate glycogen phosphorylase ensures stable glycogen levels in gsd/gsd livers. Natural abundance 13C NMR signals were calibrated against a phantom containing a fixed amount of glycogen. Accumulated free induction decays were analysed after Fourier transformation by numerical integration, or by direct analysis of the signal in the time domain using a non-iterative method based on singular value decomposition. NMR quantification of the glycogen correlated well with the chemical determination over the whole concentration range. However, the precision (reproducibility) of glycogen determinations (be it by chemical methods or by NMR spectroscopy) may pose problems. Authors should be encouraged to report systematically on the precision of their methods.

Metabolic response to halothane in piglets susceptible to malignant hyperthermia: an in vivo 31P-NMR study.

Using in vivo 31P-nuclear magnetic resonance spectroscopy, we studied the skeletal muscle metabolism of 17 anesthetized malignant hyperthermia-susceptible piglets and 25 control piglets during and after a halothane stress test. At rest, the phosphocreatine- (PCr) to-ATP ratio was 12% higher in the anesthetized piglets than in the control piglets, which may reflect a higher proportion of fast glycolytic fibers in the former. About 15 min of halothane administration sufficed to provoke onset of a reaction, which was characterized by a reciprocal drop in PCr and an increase in Pi with commencing intracellular acidosis. Halothane was withdrawn after a 20% drop in PCr. Within the next few minutes, intracellular pH dropped sharply and phosphomonoesters (PME) accumulated excessively. ATP was observed to decrease in 8 of the 17 animals. Halothane inhalation provoked a switch of metabolism toward glycolysis. Accumulation of PME suggests a mismatch between glycogenolysis and glycolysis. Despite severe acidification, glycolysis was not completely halted. Recovery of PCr and Pi started approximately 5 min after halothane withdrawal, with a longer time constant for recovery of the former. PME and intracellular pH aberrations lingered and started to recover later. Lost ATP was never restored within the observed recovery period of approximately 20 min.

Identification of halothane gene carriers by use of in vivo 31P nuclear magnetic resonance spectroscopy in pigs.

In vivo muscle 31P nuclear magnetic resonance spectroscopy was performed on 12 homozygous halothane-nonsensitive female pigs and 13 female pigs heterozygous with respect to the halothane gene. Fifteen female pigs of a third line, consisting of heterozygotes and halothane-nonsensitive homozygotes, were also available. Body weight ranged from 12 to 18 kg. Mean decrease in phosphocreatine concentration in the biceps femoris of anesthetized pigs was significantly lower for heterozygous vs homozygous pigs (3.46% vs 5.94%, P less than 0.01) after 40 minutes of halothane exposure (3%; oxygen flow, 3 L/min). Also, a statistically significant difference, with respect to the initial (7.21 vs 7.11, P less than 0.008) and end muscle pH values (7.18 vs 7.06, P less than 0.0002), was observed for homozygous vs heterozygous pigs. By means of canonical discriminant analysis, it was possible to distinguish nonsensitive homozygotes from heterozygotes (P less than 0.0001). When applying this classification method to pigs of the same strain, 2 populations (nonsensitive homozygotes, heterozygotes) emerged, with a proportion of pigs corresponding to the expected value on the basis of breeding records. In contrast to the phenotypic expression of muscular rigidity related to the malignant hyperthermia syndrome, the expression of metabolic variables (phosphocreatine, pH) was shown to be dominant.

In vivo muscle 31P nuclear magnetic resonance spectroscopy during treatment of halothane-sensitive and halothane-nonsensitive pigs.

In vivo muscle 31P nuclear magnetic resonance spectroscopy was performed on 10 female pigs originating from a homozygous halothane-sensitive line and on 10 female pigs from a homozygous halothane-nonsensitive line. The mean concentration of phosphocreatine in the biceps femoris muscle of the anesthetized pigs decreased to 86% of the initial value after 11 minutes of halothane exposure (3%, oxygen flow 3 L/min). After the next 5.6 minutes, phosphocreatine concentration reached a minimal value of 52%, followed by a mean recovery to 76% of the initial value during the ensuing 11 minutes. Response was not observed in anesthetized homozygous halothane-nonsensitive pigs. Thus, a decrease to 86% of the initial value of phosphocreatine was 100% predictive for homozygous halothane-sensitive pigs with body weight ranging from 10 to 18 kg.

Modulation of maximal glycogenolysis in perfused rat liver by adenosine and ATP.

Rat livers perfused at constant flow via the portal vein with dibutyryl cyclic AMP produced glucose equivalents at a steady maximal rate (6 mumol/min per g of liver). Addition of adenosine (150 microM) caused a biphasic effect. (i) First, the glycogenolytic rate rose transiently, to a mean peak of 150% of control levels after 2 min. This glycogenolytic burst was reproduced by two P1-receptor agonists, but not by ATP, and was blocked by a P1-antagonist (8-phenyltheophylline), as well as by inhibitors of eicosanoid synthesis (indomethacin, ibuprofen or aspirin). It did not occur in phosphorylase-kinase-deficient livers. The adenosine-induced glycogenolytic burst coincided with moderate and transient changes in portal pressure (+6 cmH2O) and O2 consumption (-20%), but it could not be explained by an increase in cytosolic Pi, since the n.m.r. signal fell precipitously. (ii) Subsequently, the rate of glycogenolysis decreased to one-third of the preadenosine value, in spite of persistent maximal activation of phosphorylase. The decrease could be linked to the decline in cytosolic Pi: both changes were prevented by the adenosine kinase inhibitor 5-iodotubercidin, whereas they were not affected by ibuprofen or 8-phenyltheophylline, and were not reproduced by non-metabolized adenosine analogues. In comparison with adenosine, ATP caused a slower decrease of Pi and of glycogenolysis. The fate of the cytosolic Pi was unclear, especially with administered ATP, which did not increase the n.m.r.-detectable intracellular ATP.

The cytosolic concentration of phosphate determines the maximal rate of glycogenolysis in perfused rat liver.

Glycogenolysis was studied in glycogen-rich perfused livers in which glycogen phosphorylase was fully converted into the a form by exposure of the livers to dibutyryl cyclic AMP. We monitored intracellular Pi by 31P n.m.r. Perfusion with Pi-free medium during 30 min caused a progressive decrease of the Pi signal to 50% of its initial value. In contrast, exposure of the livers to KCN and/or 2,4-dinitrophenol resulted in a rapid doubling of the Pi signal. Alterations in the intracellular Pi coincided with proportional changes in the rate of hepatic glycogenolysis (measured as the output of glucose plus lactate). The results indicate that the rate of glycogenolysis catalysed by phosphorylase a depends linearly on the hepatic Pi concentration. Hence the Km of phosphorylase a for its substrate Pi must be considerably higher than the concentrations that occur in the cytosol, even during hypoxia.