31P NMR study of phosphorus metabolites in fast and slow muscles

1. 31P NMR was used to characterize phosphate pools in perchloric acid extracts of muscles with various composition of muscle fibre types. 2. The white m. pectoralis major (MPM) of chickens 15 min post mortem is characterized by 1.6-times higher relative content of phosphocreatine (PCr) in comparison with mixed leg muscle (LM) of this species. The glycerophosphorylcholine (GPC) does not occur in MPM at NMR detectable level in contrast to the leg muscles. Relative amounts of other phosphates are similar in both muscles. 3. The intermediate MPM of pigeons as well as mixed LM of this species contain 15 min post mortem a very small amount of PCr and ATP but a large amount of inorganic phosphate. Relative content of GPC is higher in leg muscles than in intermediate MPM. 4. Muscles with higher occurrence of white fibres contain relatively more PCr than muscles with lower occurrence of white fibres. 5. The occurrence of GPC seems to be connected with metabolism of red muscle fibres.     

Anaesthetic-induced Malignant Hyperpyrexia: A Suggested Method of Treatment

Experiments in susceptible Landrace pigs have shown that procaine blocks the initiation of anaesthetic-induced malignant hyperpyrexia by both halothane and succinylcholine. Pretreatment with curare prevents only the trigger action of succinylcholine. In a preliminary study procaine was used to treat the established syndrome in five pigs, two of which survived. On the basis of these findings a treatment regimen can be suggested for patients who develop malignant hyperpyrexia.     

Biochemical Basis of Malignant Hyperpyrexia

Pharmacologically-induced muscle contracture in vitro has been used as a model to study the biochemical basis of malignant hyperpyrexia. In 15 susceptible subjects halothane, succinylcholine, and potassium chloride all produced an abnormal muscle contracture, and the caffeine-induced contracture was greater than normal. The contractures were reproducible only in the presence of extracellular calcium ions. The fact that such dissimilar pharmacological stimuli all induced contracture in the affected muscle suggests that the essential abnormality in the muscle cell in malignant hyperpyrexia is an impaired binding of calcium ions to the membranes of the sarcoplasmic reticulum and the sarcolemma. Exposure of these membranes to halothane, succinylcholine, and other anaesthetic agents then leads to a rapid and abnormally large release of calcium into the myoplasm, which in turn gives rise to all the clinical features of the syndrome.     

Association of halothane sensitivity with growth and meat quality in pigs

Previous reports have indicated that a proportion of pigs, homozygous normal for the skeletal muscle ryanodine receptor gene (RYR1), was halothane sensitive, and this was associated with poor meat quality when pigs were handled aggressively. This study was conducted to evaluate halothane sensitivity in RYR1-normal pigs, managed under simulated commercial conditions, to ascertain the association of halothane sensitivity with growth rate and meat quality. A total of 363 pigs across four farrowing groups, from seven Landrace sires and 38 Yorkshire–Landrace F1 dams, were tested at 8 weeks of age for halothane sensitivity using a closed system that delivered 5% halothane at 2 l/min for 3 (group 1) or 2 (groups 2 to 4) min. After 1 min, limb rigidity, limb tremors and abdominal discoloration were evaluated on a binomial scale with 0 indicating no reaction and 1 indicating reaction. Testing was repeated 2 days later. At 10 weeks of age, pigs were moved to finishing pens and not moved again until marketing. Within farrowing group, pigs were harvested in one of two groups, and at marketing were moved a distance of 91 m, weighed, tattooed, loaded and transported a distance of 550 km to a commercial harvest plant. After overnight rest, pigs were harvested and the pH of the loin muscle was measured at 45 min (pH45) after stunning. After an 18-h chill, loin muscle pH (pHu), International Commission on Illumination (CIE) L*, a*, b*, color (1 to 6) and marbling (1 to 10) scores and fluid loss percent were collected. Generalized linear mixed models were used to estimate repeatabilities for response to halothane challenge. Repeatabilities for limb rigidity for the front right and left legs were 0.24 and 0.31, respectively, whereas rear right and left leg repeatabilities were 0.19 and 0.17, respectively. Repeatabilities for front right and left leg tremors were 0.16 and 0.20, respectively. Growth rate was not influenced by any measure of halothane sensitivity. Carcasses from pigs exhibiting limb rigidity tended to have lower pH45 (5.88 v. 5.97; P = 0.06), similar pHu (5.47 v. 5.49; P = 0.32), less pH decline from 45 min to 18 h (−0.40 v. −0.50; P = 0.04) and a tendency for greater fluid loss percent (5.01 v. 4.55; P = 0.08) than carcasses from pigs that did not exhibit limb rigidity during halothane challenge. A proportion of pigs normal for RYR1 did exhibit limb rigidity during halothane gas challenge, and subsequently tended to have lower 45 min pH and greater longissimus muscle fluid loss post harvest.     

Post mortem changes in skeletal muscle proton spin-lattice relaxation times

A previously published report indicated that there are early post mortem changes in the pulsed NMR proton spin-lattice relaxation time (T1) of skeletal muscle which must be taken into account in in vitro tissue analysis. We re-examined this subject. When T1 measurements were done by allowing the signal intensity to decay over two orders of magnitude from the original intensity, the T1 decay curves showed more than a single exponential relaxation time component as reported earlier. However, when T1 measurements were done by allowing signal intensity to decay to about one order of magnitude from the original intensity only a single exponential relaxation time component was found. We postulate that the latter method gives the average T1 value of the various macroscopic tissue components present and that this method gives a representative T1 value for the entire tissue. Using data obtained in this manner we could not find significant post mortem changes in the muscle T1 relaxation times during the first four hours but found change at a later time.     

Effect of rearing system and mixing at loading on transport and lairage behaviour and meat quality: comparison of outdoor and conventionally raised pigs

The effect of raising pigs outdoors or conventionally in a barren environment was investigated with respect to behaviour during a 3 to 4 h journey and a 2 h lairage, blood chemistry at slaughter and meat quality characteristics. Pigs were either kept in farm pen groups or were mixed at loading and kept in the groups, so formed, until slaughter. Non-mixed outdoor pigs settled more quickly during transport and lay down to a greater extent at the end of the journey and lairage period compared with conventionally raised pigs. Mixing led to fewer pigs sitting and lying during transport for conventionally raised pigs, where nearly 80% were still standing at the end of the journey. Mixing had no effect on pig posture in the lairage. Outdoor pigs were less aggressive than conventionally raised pigs especially during lairage and had a lower frequency of unacceptable skin damage in the rear and shoulder area. Aggressive interactions were almost exclusively confined to mixed groups and occurred mainly between pigs from different farm pens, i.e. between unfamiliar animals. Mixing at loading led therefore to higher levels of unacceptable skin damage. Cortisol concentrations in slaughter blood were not affected by rearing system or mixing, but mixed, conventionally raised pigs had higher plasma creatine kinase (CK) activities than non-mixed ones (1132 v. 761 U/l, respectively, P < 0.05). Outdoor pigs had similar CK activities, irrespective of mixing (682 and 771 U/l for mixed and non-mixed, respectively). Muscle pH early post mortem was highest in outdoor pigs and muscle temperature lower, but no pig showed pH values below 6.0. Ultimate pH values were both higher (Semimembranosus (S)) and lower (Semispinalis capitis (SC)) than in conventionally raised pigs, and outdoor pigs tended to have a lower frequency of higher than normal pH values. Internal reflectance (MQM) values in Biceps femoris (BF) were highest in outdoor pigs but the incidence of pale, soft and exudative (PSE) meat was low, varying between 0% and 1% for experimental groups. In general, the effects of rearing system and mixing on meat quality measurements taken early post mortem or the day after slaughter were slight, but the trends seen support the CK results, and show that conventionally raised pigs may have found mixing pre-slaughter to be more physically stressful than outdoor pigs did.     

The reproducibility of measurements of intramuscular magnesium concentrations and muscle oxidative capacity using <Superscript>31</Superscript>P MRS

³¹P magnetic resonance spectroscopy (³¹P MRS) has been used to measure intramuscular magnesium concentrations and muscle metabolism. Abnormal intramuscular magnesium has been reported in several patient populations with suspected metabolic disorders. The purpose of this study was to evaluate our ability to measure intramuscular magnesium and muscle metabolism in the quadriceps muscles of healthy subjects, and to test whether these measurements were influenced by prior exercise. Twelve normal, healthy male volunteers were tested in a 3 Tesla magnet on four separate days. Resting [Mg²⁺] was calculated from the heights and frequency shifts of the phosphate, phosphocreatine and ATP peaks. Phosphocreatine (PCr) recovery kinetics were measured after 30-39 second bouts of isometric exercise. Thirty minutes prior to the 3^rd test session the subjects completed a 2 hour treadmill walk at 40-60% of heart rate reserve. Resting [Mg²⁺] averaged 0.388 mM and had an interclass correlation coefficient between days (ICC) of 0.352. The mean end exercise PCr was 47.6% and the mean end exercise pH was 6.97. PCr recovery averaged 39 seconds (p = 0.892) and had an ICC of 0.819. Prior long duration exercise did not produce significant alterations in either PCr recovery kinetics or intracellular magnesium levels (p = 0.440). In conclusion, the reproducibility of Resting [Mg²⁺] was less than that of PCr recovery measurements, and may reflect the sensitivity of these measurements to phasing errors. In addition, prior exercise is unlikely to alter measurements of resting metabolites or muscle metabolism suggesting that rigorous control of physical activity prior to metabolic testing is unnecessary.     

Effect of delay in cryofixation on the elemental composition of biopsies and post mortem specimens of the thyroid gland

The time between the excision and cryofixation of a biopsy is most important regarding its elemental composition as demostrated by an investigation of the thyroid glands of rats and pigs. Biopsies taken and cryofixed immediately served as control specimens. Biopsies that were allowed to stand at room temperature for 20 min before cryofixation and specimens cryofixed at 1 h post mortem were also investigated. Significant changes in the ion concentration of the cells and colloid were apparent in biopsies in which cryofixation was delayed for 20 min and in thyroids cryofixed 1 h post mortem. It was demonstrated that redistribution of electrolytes occurs within 1h post mortem and that similar changes occur in biopsies allowed to stand for 20 min at room temperature before cryofixation. The results stress the importance of immediate cryofixation after surgical excision of a biopsy. This is especially important since numerous elemental changes due to delayed cryofixation resemble those which occur in pathological processes.     

Some factors determining the PCr recovery overshoot in skeletal muscle

It has been proposed recently that the phosphocreatine (PCr) overshoot (increase above the resting level) during muscle recovery after exercise is caused by a slow decay during this recovery of the direct activation of oxidative phosphorylation taking place during muscle work. In the present article the factors determining the appearance and size of the PCr overshoot are studied using the computer model of oxidative phosphorylation in intact skeletal muscle developed previously. It is demonstrated that the appearance and duration of this overshoot is positively correlated with the value of the characteristic decay time of the direct activation of oxidative phosphorylation. It is also shown that the size of PCr overshoot is increased by low resting PCr/Cr ratio (what is confirmed by our unpublished experimental data), by high intensity of the direct activation of oxidative phosphorylation, by high muscle work intensity and by low rate of the return of cytosolic pH to the resting value during muscle recovery.     

POST MORTEM CHANGES IN EXTRACTABLE PROTEIN AND IN TISSUE pH IN THE GUINEA PIG BRAIN

—Guinea pigs were killed by asphyxiation with nitrogen and the soluble proteins were extracted from the brain at various times post mortem. The quantity of extractable brain protein decreased by 21 per cent when the animals remained at room temperature for 2 h post mortem. This decrease was not a consequence of extensive proteolysis or variations in blood volume but was probably a result of precipitation. After death, the pH of the brain fell rapidly to a minimum of ～6CE4 within about 35 min. Examination of the patterns of brain proteins after acrylamide gel electrophoresis showed a concomitant decrease in the content of several protein bands.     

Effect of halothane on the Ca2+-transport system of surface membranes isolated from normal and malignant hyperthermia pig skeletal muscle

Trapezius muscle from normal and malignant hyperthermia (MH) pigs was used to investigate the effects of halothane on contractile properties and on the calcium transport system of isolated surface membranes. We observed that (i) halothane, diluted in dimethyl sulfoxide, induced a higher isometric contracture response in MH muscle than in normal muscle, (ii) halothane had a more pronounced inhibitory effect on the sarcolemmal Ca2+-ATPase activity in MH membrane, and (iii) the actively accumulated calcium was released in higher amounts in MH muscle than in normal muscle. These results suggest that halothane might induce, in vivo, an important influx of extracellular calcium ions through the MH sarcolemmal membranes and this pool of intracellular calcium may constitute the trigger for the defective sarcoplasmic reticulum "calcium-induced calcium-release" system.     

Meat quality of the longissimus lumborum muscle of Casertana and Large White pigs: metabolomics and proteomics intertwined

Longissimus lumborum muscles from high fat-deposing Casertana and lean meat Large White pigs were assayed for meat quality parameters, including early and ultimate post mortem pH, water holding capacity and Minolta L*a*b*values. These parameters were correlated to results from differential proteomic and targeted metabolomic analyses. Higher levels of glycolytic enzymes and lactate accumulation were related to slow pH drop in Casertana pigs, albeit not to rapid pH lowering in LW counterparts. On the other hand, the individuation of pyruvate kinaseM1 and tropomyosin levels in LW were related to water holding capacity and Minolta values at 24h after slaughter. Bioinformatic analyses strengthened the correlation between over-expression of structural proteins in LW and more accentuated growth aptitude in this breed. Conversely, enzymes taking part into branching glycolytic reactions, such as glycerol 3-phosphate and creatine kinase M, were related to accentuated lipogenesis and slower albeit prolonged glycolytic rate in Casertana, respectively. Breed-specific differences at the protein level were not only related to growth performances and fat accumulation tendency in vivo, but they also affected post mortem performances through a direct influence on the forcedly anaerobic behavior of pig muscles after slaughter.     

Changes in cytoplasmic free calcium caused by halothane. Role of the plasma membrane and intracellular Ca2+ stores

Malignant hyperthermia is a muscle disease characterized by an abnormal response to anaesthetics, stress, and exercise. It is typified by muscle contracture and a dramatic elevation in body temperature. A defect in the regulation of the concentration of cytoplasmic free calcium, [Ca2]i, is thought to underlie this disease, but the actual [Ca2+]i was not measurable until recently. We have shown that the anaesthetic halothane increases [Ca2+]i in isolated lymphocytes from malignant hyperthermia-susceptible humans and pigs but not in the normal counterparts. In this report we extend these observations to a larger number of cases and analyze the molecular mechanisms responsible for the increase. The halothane-mediated rise in [Ca2+]i required external Ca2+ and was prevented by nifedipine, an inhibitor of the voltage-sensitive Ca2+ channels of the cell membrane. In addition, the effect of halothane on the releasable Ca2+ from intracellular stores was determined by measuring the size of the releasable pool before and after addition of the anaesthetic. After addition of halothane, about 73% of this Ca2+ pool was still available for release by the Ca2+ ionophore ionomycin in cells from normal humans and pigs. In contrast, only about 45% of the free Ca2+ in intracellular stores was left after treatment with halothane in cells from malignant hyperthermia-susceptible humans and swine. These results indicate that halothane acts both at the cell membrane and at intracellular organelles, and that this action results in a net increase in [Ca2+]i in malignant hyperthermia, but not in normal cells. The action at the cell membrane appears to be on the voltage-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of delayed freezing on content of phosphagens in human skeletal muscle biopsy samples

The concentrations of ATP, phosphocreatine (PCr), creatine, and lactate were determined in muscle biopsy samples frozen immediately or after a delay of 1-6 min. During the delay the samples were exposed to normal air or a gas mixture of 6.5% CO2-93.5% O2. The ATP content was unchanged, but PCr increased significantly from 72 mmol after rapid freezing to 85 mmol X kg dry muscle-1 during the 1st min in air. The lactate concentration increased (2.8 to 5.2 mmol X kg-1). If muscles were made anoxic by circulatory occlusion for 4-6 min before sampling, no increase in PCr was observed. Direct homogenization of fresh tissue in perchloric acid gave the same ATP, PCr, and lactate contents as frozen samples. It is concluded that the ATP and PCr contents in muscle are unaffected by freezing but that the biopsy procedure activates the energy utilization processes resulting in PCr decrease. It is suggested that the muscle PCr content after a 1-min delay in tissue freezing corresponds to the level in resting fresh muscle.     

Changes in enzymes associated with energy metabolism during the early post mortem period in longissimus thoracis bovine muscle analyzed by proteomics

Changes in metabolic protein levels in biopsies during the early post mortem period in the bovine longissimus thoracis muscle were investigated by 2-DE based proteome analyses. Nine NRF (Norwegian Red) dual purpose bulls were included in the study. Twenty-four proteins underwent changes between the two sampling times and were classified into two major groups: metabolic proteins and heat shock proteins. Of the metabolic proteins, 5 enzymes involved in the glycolytic pathway and the tricarboxylic acid (TCA) cycle, increased in intensities during the post mortem period. In addition, the NADP-dependent enzyme 3-hydroxyisobutyrate dehydrogenase, associated with the TCA cycle in muscle, was increased. This documents that an increased aerobic energy metabolism occurs immediately after slaughter, with the aim to replenish the ATP levels in the muscle.     

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm

We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.     

The reliability of histochemical fibre typing of human necropsy muscles

The reliability of muscle fibre typing of post mortem specimens was investigated with special reference to the influence of time and temperature. In specimens stored at +4 degrees C, muscle fibre typing could be reliably performed up to at least ten and fifteen days post mortem for the masseter and biceps brachii muscles respectively. The corresponding figures for storage at room temperature were three and six days. The difference in the preservation of enzyme activity between masticatory and limb muscles might be related to the demonstrated difference in the fibre type composition and thus the enzyme content and energy sources.     

The reliability of histochemical fibre typing of human necropsy muscles

Summary The reliability of muscle fibre typing of post mortem specimens was investigated with special reference to the influence of time and temperature. In specimens stored at +4° C, muscle fibre typing could be reliably performed up to at least ten and fifteen days post mortem for the masseter and biceps brachii muscles respectively. The corresponding figures for storage at room temperature were three and six days. The difference in the preservation of enzyme activity between masticatory and limb muscles might be related to the demonstrated difference in the fibre type composition and thus the enzyme content and energy sources.