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CONVERSION OF MUSCLE TO MEAT | Glycogen
Glycogen E Puolanne and K Immonen, University of Helsinki, Helsinki, Finland r 2014 Elsevier Ltd. All rights reserved.
Glossary Glycogenolysis The break-down of glycogen to glucose-1phosphate and glucose. Glycogen debranching enzyme A double-function enzyme that moves three glucose units from the 1-6 branching point (transferase) and then 1,6-glucosidase removes the last glucose unit.
Introduction Most of the time, blood will provide enough nutrients for the metabolism, and especially, for the energy production of the muscles. The principal portion of the energy produced in a muscle cell originates from the main nutrients; carbohydrates, volatile and nonvolatile fatty acids plus glycerol, and proteins. Fat, as free fatty acids, is an important source of energy at rest and during light exercise. In light exercise, the plasma content of free fatty acids increases within a few minutes, whereas during heavy exercise it decreases. In ruminants, short-chain free fatty acids (mainly acetate, propionate and butyrate) form the major proportion of the energy substrates with carbohydrates having a minor role (5–15%). Oxygen is required, in long terms for all kinds of energy production. Muscles are able to produce/use 5 mmol of adenosine triphosphate (ATP) s−1 g−1 at maximum rate, and that appears to be universal across muscle tissues. The resting level of consumption is approximately 0.02–0.05 mmol ATP s−1 g−1, and is used for maintaining membrane potentials, and fuelling the calcium pump that returns Ca++ back to the sarcoplasmic reticulum, the occasional contractile reactions between actin and myosin, as well as the anabolic reactions of molecule syntheses. ATP consumption also generates body heat. The maximal rate of ATP consumption is at least 100 times greater than that of a resting muscle. Since the production and consumption of ATP must be equal at any given moment, the portion of ATP that cannot be provided by the oxidative metabolism must be produced anaerobically. This explains the existence of two mechanisms (aerobic and anaerobic) for ATP production. If indeed the aerobic production was to be able to cover the maximal level of energy consumption, the animal would, depending on species, need to have much larger organs (lungs, heart, circulatory system, organelles and enzymes for aerobic metabolism in fibres) than it currently has for obtaining and using oxygen. Since animals seldom need maximal energetic capacity over a sustained period of time in all of their muscles, a capacity for a full aerobic coverage would be an inefficient physiological ‘investment.’ Another mechanism has, therefore, developed for short bursts of energy. Depending on the oxidative capacity of a muscle, the oxidative production/usage of energy can increase due to increased activity. In pigs, for example, the increase is less than 10 times
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Glycogen phosphorylase A very fast enzyme that is capable to remove glucose-1-phosphate from nonbranched chains of glycogen, down to the fourth glucosyl unit from the 1-6-branching point. Glycolysis (from glycose, an older term for glucose+-lysis degradation) The metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO−+H+.
the resting level, whereas in cattle, it is 20-fold, and in horses, almost 50-fold. The above mentioned figures are of course approximates, because the oxygen consumption within species depends on the fibre type composition and the basic tone of each muscle. The oxygen consumption of various muscles depends on their activity at a given moment, as does the blood flow through the muscles, as well as the relative amount of oxygen left in the muscles. At rest, for example, the blood flow through muscles may be 20% of the total flow. At maximal activity, however, the cardiac output increases tenfold, and 80% of it is circulated through muscles, which then utilize up to 90% of the passing oxygen. Together, these values translate to a 25fold consumption of oxygen in comparison to a resting muscle. The oxidative capacity of a body's total muscle mass seems to be in balance with the capacity of blood circulation. Animals with a high oxidative capacity (e.g., horse, reindeer) have relatively big lungs and hearts, and muscles that have dense capillary networks, compared to animals with less oxidative capacity (e.g., pig, chicken). Furthermore, breeding has had a significant effect on the oxidative capacity of domestic animals. The proportion of oxidative fibres is higher in the muscles of the wild pig than in the domesticated pigs, as is the capillary density. In wild pigs, 1 g of heart tissue serves 85 g of muscular tissue, whereas in domestic pigs it has to serve 140 g. In poultry, severe oxygen deficiency within the poorly capillarized Pectoralis muscle results in deep pectoral myopathy. The crucial importance of sufficient oxygen supply for muscle tissues cannot, therefore, be overly emphasized. In a muscle fibre, glycogen is the energy reserve used in case the circulatory system cannot provide enough oxygen and nutrients. Glycogen breaks down to glucose phosphates that are utilised anaerobically or aerobically, depending on animal species and their level of physical and/or psychological stress. There is indirect evidence that in stress, triggered by adrenaline, glycogen is preferred even if there were other energy substrates available, i.e., even if the stress did not involve high physical activity. The lactate formed in the glycolysing fibres will be transported into the blood via monocarboxylate transporters (MCT) that move lactate through the cell membranes from the higher concentration toward the lower. Lactate is destined to be exported to the liver to be used as a substrate for the synthesis of glycogen or glucose-6-phosphate. While on its way, some of it will probably also be aerobically
Encyclopedia of Meat Sciences, Volume 1
doi:10.1016/B978-0-12-384731-7.00245-2
Conversion of Muscle to Meat | Glycogen utilized either by the oxidative muscle fibres or by the heart, if the lactate concentration in them is lower than that in blood.
for allowing a rapid release of glucose from its structure by glycogen phosphorylase (GP) (Figure 1). A 100 mM solution of glucose would create a high osmotic pressure, whereas the equivalent 0.002 mM of glycogen does not, the glycogen molecule being more a particle (granule) than an element in a solution. The maximum sized glycogen molecule/granule of a diameter of 40 nm contains approximately 55 000 glucose units, and 2100 nonreducing chain ends. The total molecular weight is approximately 107 Da. Each glycogen molecule has 40–50 GP dimers (or 20–25 tetramers) bound to it, facilitating the immediate and fast cleavage of the glucosyl units. Glycogen synthase (GS), glycogen branching enzyme (GBE), AMP-activated protein kinase (AMPK), as well as glycogen debranching enzyme (GDE) are also bound to the glycogen complex, and thus, readily available when needed. There are also several other proteins with regulatory roles bound to the complex (Figure 2). In the core of the glycogen molecule is glycogenin (molecular weight (MW) 37 000), an enzyme-acting protein (glucosyltransferase) that serves as a primer for the molecule. In glycogen formation, uridine diphosphate moves the first eight glucosyl units to glycogenin after which GS takes over and extends the chain of glucosyl units via α(1→4) linkages. Glycogen branching enzyme then transfers an A-chain of 6–7 glucosyl units forming an α(1→6) linkage between the original chain and the transferred chain. A new branch point must be at least 4 glucosyl units apart from the previous branch point. Theoretical calculations have shown that the optimal chain length is 13 (12–14) glucose units, and that it is optimal to have two branching points in each B-chain (Figure 1). Each level of chains forms a concentric tier, and all new tiers double the number of chains of the previous tier. After 12 tiers, the structure will turn self-limiting, eventually due to the spatial relations between the glycogen molecule and GS. The distance of four glucose units between the branching points thus leaves tails of 4 or 5 glucosyl units.
Structure of Glycogen Theoretical studies have revealed how the glycogen molecule has taken its form in the biological evolution. The glycogen molecule is optimized for storing a maximum amount of glucose in the smallest possible volume, and at the same time,
A-chain
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Figure 1 Glycogen molecule (5 tiers) according to Meléndez-Hevia and others. Each unbranched A (dotted line) and branched B (solid line) chain has 12–14 glucosyl residues. G ¼glycogenin. Adapted from Immonen, K., 2000. Bovine muscle glycogen concentration in relation to diet, slaughter and ultimate beef quality. Doctoral Thesis, University of Helsinki, EKT Series no 1203, 90 pp. Available at: https://helda. helsinki.fi/bitstream/handle/10138/20879/bovinemu.pdf?sequence ¼2 (accessed 15.08.13).
Malin
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Figure 2 A schematic summary of proteins known to interact with glycogen. GDE, glycogen debranching enzyme; AMPK, AMP-activated protein kinase; GBE, glycogen branching enzyme; GM and PP1R6, regulatory subunits of protein phosphate 1 PP1; GN, glycogenin; GNIP, glycogenininteracting protein; GS, glycogen synthase; GSK, glycogen synthase kinase; GP, glycogen phosphatase; PhK, phosphorylase kinase; PTG, protein targeting to glycogen. Reproduced from Graham, T.E., Yuan, Z., Hill, A.K., Wilson, R.J., 2010. The regulation of muscle glycogen: The granule and its proteins. Review. Acta Physiologica 199, 489–498.
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Until recently, glycogen was thought to exist as two separate forms: proglycogen and macroglycogen. Particles with molecular weight of 400 kDa are called proglycogen, which due to its protein content of approximately 10%, is insoluble in acidic solutions. Glycogen molecules of a much higher molecular weight are called macroglycogen, which in turn maintain its water-solubility even in acidic solutions due to the dominant abundance of carbohydrate. It has been postulated that proglycogen is utilised in severe stress as well as postmortem, whereas macroglycogen is used during endurance exercise, and less so postmortem. The existence of different glycogen pools has, however, been challenged. It has been concluded that instead of existing as two separate forms, there is a continuum of glycogen molecules of different size. It has also been claimed that muscle fibres accommodate glycogen in multiple sites; some free in cytosol, and some bound to the sarcoplasmic reticulum. There are, therefore, differences in both solubility and availability for use. Microscopically, the granules are most abundant between the myofibrils, but also inside the fibrils. In humans, the average diameter of a glycogen granule is 25 nm, i.e., tier 8 is mainly in use, and less than 20% of the granules have built all 12 tiers. In conclusion, glycogen particles are not of fixed molecular weight, but instead, are subject to on-going resynthesis or degradation depending on the level of physical activity and/or stress, as well as the nutritional status of the animal.
Metabolism of Glycogen The main function of glycogen is to serve as a source of glucose for anaerobic glycolysis (includes glucose-1-P to pyruvate and, with the help of muscle-type lactate dehydrogenase, from pyruvate to lactate). Anaerobic glycolysis is a very rapid metabolic pathway producing 3 ATP of energy per glucosyl unit, the yield being much lower, however, than if pyruvate was utilised aerobically. The biochemical role of lactate formation is to regenerate NAD+ for glycolysis, and one proton will be simultaneously bound to pyruvate when lactate is formed. There has been an intensive discussion over the source of such protons, and it seems that physiologists, dealing with the pH range of 7.2–6.4 of a dynamic living muscle, and meat and animal scientists, working with a broader range of pH, i.e., from 7.2 all the way down to the pH 5.0 of a more stable postmortem muscle, see the matter quite differently. The protons derive from earlier stages of glycolysis, so that different pKa-values of glycolytic metabolites and phosphates at different postmortem pH-values relate to free proton formation. Irrespective of the sources of protons, and/or the buffering capacity of meat, there is an abundance of data confirming the linear negative correlation between lactate content and pH-value. In the breakdown of glycogen, there are two enzymes directly involved: GP that releases glucose is strongly controlled by various factors, including hormonal, and GDE that disassembles the branch points. The rate of the process is limited by the availability of free HPO42−. The pKa value of H2PO4 is 7.2, and at pH 5, the pool of HPO42− is virtually nonexistent. GP (842 amino acids and a MW of 97.4 kDa) amounts to approximately 2% of the soluble proteins of a muscle, and has
an immense acting capacity. GP is activated both by allosteric interactions as well as reversible phosphorylation. The inactive form b, of GP can be found in resting muscles, and is activated by a cascade involving epinephrine, calcium, cyclic AMP, AMPactivated protein kinase (which activates phosphorylase kinase), and inhibited, however, by glucose-6-P and ATP. GP is biologically active as a dimer of two identical subunits, but can also be found as a tetramer. There are dimeric GP molecules attached to each glycogen molecule/particle. Each GP monomer consists of two domains: the C-terminal domain (the one responsible for catalysing the reaction), and the N-terminal domain (responsible for regulation of the enzyme). The binding of phosphate or AMP at the effector sites leads to changes in the conformation of the protein, and increases the acceptance of glycogen at the active site. One subunit is being active whereas the other, as attached to the molecule, is regulating the activity. When the outermost tier is full of glycosyl units, 50% of all glucose within a glycogen particle lies in the unbranched A-chains, whereas the remaining 50% constructs the B-chains, irrespective of the total number of tiers. GP can only cut 9 out of the 13 consecutive 1–4-linked glucosyl units of a linear A-chain. Thus, according to theoretical calculations, GP can initially cleave 34.6% of the glucosyl units of any glycogen particle. At the fourth glucosyl unit from the 1–6-linked branch, the activity of GP ceases. The cascade for the activation of glycogenolysis is very effective. One epinephrine molecule bound to a receptor on a muscle fibre results in 400 000 glucose-1-P units cleaved by GP per second. This means that one molecule of GP can disassemble glycogen at a speed of 30 000 degradations per particle per second. Blood epinephrine concentration of 10−10 M generates an intercellular cyclic adenosine monophosphate (cAMP) concentration of 10−6 M. The relative concentrations of the three successive enzymes of the cascade; cAMPdependent protein kinase, phosphorylase kinase and GP, occur as molar ratios of 1:10:240 including adrenalin at a ratio of 0.0001, indicating an effective amplification of the signal. At the onset of muscle contraction, GP is activated from form b to form a by the increasing content of free cytosolic Ca++, as well as by norepinephrine secreted from the motorend-plates at the sarcolemma. GP then remains active in high concentrations of AMP and phosphate, and will be inhibited again when ATP and glucose-6-P levels have increased. Decreasing levels of ATP and glucose-6-P will start glycogenolysis. In case of nonintensive exercise, the increased blood supply of nutrients and oxygen quickly take over, and GP a is phosphorylated back to b. In a postmortem muscle of the pig, for example, GP stays in the form b for the first 10 min. It should be noted, however, that in a stressed animal, when creatine phosphate has been utilised, glycolysis starts earlier, even before slaughter. In this case, the reaction CP2−+ADP3−+H+ → C+ATP4− will not generate the initial buffering effect. For the continuation of glycogenolysis, GDE must remove the 1–6 linkages. The rate of GDE is not more than 10% of that of GP. The biological significance for this may be that a controlling system is needed to stop excessive breakdown which would cause damages in the fibre. Also GDE is bound to the glycogen particle, and thus, always available. It has a
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Figure 3 The relationship between muscle glycogen concentration and the rate of resynthesis in young bulls. Reproduced with permission from Tarrant, P.V., 1988. Animal behaviour and environment in the dark-cutting condition. In: Fabiansson, S.U., Shorthose, W.H., Warner, R.D. (Eds.), Dark-Cutting in Cattle and Sheep, pp. 8−18. Australian Meat and Live-stock Research & Development Corporation.
double function. In one end of the molecule, there is a transferase removing three glucosyl units and placing them at the end of another chain, most probably B, whereas in the other end of the enzyme complex there is 1,6-glucosidase activity cleaving the last remaining glucosyl unit as free glucose, which is then immediately phosphorylated. Finally, GDE will move further away from the A-chain thus allowing GP to function again. Very little is known about the control of GDE, except that it can only process A-chains of a length of four glucosyl units. It seems that glycogenolysis is independent on GDE at high glycogen concentrations, but at lower glycogen contents, GDE is of more relevance. GDE works optimally at a temperature range of 39–42 °C, and is not particularly sensitive to a lowering of pH. GDE is, however, very sensitive to low temperatures, and has hardly any activity at below 10 °C. It is, therefore, possible that postmortem, at low levels of glycogen, and in cold meat, GDE is rate-limiting. GS is allosterically activated by glucose-6-phosphate. Contrary to GP, GS is thus active when high blood glucose concentrations lead to elevated intracellular glucose-6phosphate. GS is inactivated by an on-going glycogenolysis, as the cAMP cascade ceases glycogen synthesis. GS is phosphorylated by protein kinase A as well as by phosphorylase kinase. Phosphorylation of GS promotes the ‘b’ (less active) conformation. The cAMP cascade with AMPK having a central role thus controls the levels of glycogen. Also the intake of glucose into a fibre is controlled by AMPK. In the liver, glucose-1-phosphate may also, instead of being converted to glycogen, be converted into glucose-6-phosphate, which is then dephosphorylated for the release into the blood stream (from muscle fibres the charged glucose phosphates do not escape). Once degraded, it takes a relatively long time to replenish the glycogen stores. There are, however, marked differences between species. In beef animals, the resynthesis is particularly slow, 1–2 mmol kg−1 h−1, increasing only at very low glycogen stores (Figure 3). In race horses, during three consecutive bouts within 2 h, the glycogen content first decreases approximately 30%, and interestingly, an additional 20% during 4 h at rest. The recovery takes 3 days indicating that the
consumption of carbohydrates is high after repetitive intensive exercises.
Effect of Glycogen Content on Meat Quality The glycogen contents of animals at rest or just after slaughter have mostly been estimated with the glycolytic potential, i.e., the sum of glycogen, glucose, glucose-6-P and lactate, expressed as lactate equivalents. This is, in principle, a good indicator for antemortem glycogen levels. However, as one connects the relative contents of the above variables with the time postmortem, and especially, with the pH and buffering capacity, it soon becomes evident that in many cases it has not been all that significant of an indicator for meat quality due to complexity of the events. An alternative simple procedure has not, however, at least to date, been introduced for the retrospective determination of preslaughter glycogen contents. The analyses are indeed challenging, especially because glycogenolysis and glycolysis can proceed very rapidly during sample preparation and analysis. It is, therefore, very important to immediately freeze the fresh muscle samples in liquid nitrogen. A resting muscle biopsy taken before any measures of transportation, or just before slaughter, would make the most representative sample, provided that sampling and freezing are, again, carried out promptly. The glycogen contents of resting muscles are usually at the levels of 80–100 mmol kg−1, given as glucose, yet lower levels are frequently found at slaughter due to ante-mortem stress. In pigs, higher levels (of 120 or even 150 mmol kg−1) are found, especially in the Hampshire, and Hampshire crossbreeds. Also in cattle, high resting levels (110– 120 mmol kg−1) can be found in animals that have first been depleted of glycogen, and then allowed to recover on a high energy diet. Pre-slaughter stress, such as transportation, invariably reduces glycogen contents. Although the glycogen levels of mammals are controlled by AMPK through PRKAG3 gene, the mechanism in poultry may be different. In chicken, the glycogen contents of the light Pectoralis major muscles are lower (below 100 mmol kg−1) than in the light
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6.8 6.6 A Model B Model C Model D Model
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Figure 4 Ultimate pH and glycogen content immediately before slaughter in pig M. longissimus dorsi. A–D, various exercise intensities before slaughter. Reproduced from Henckel, P., Karlsson, A., Jensen, M.T., Oksbjerg, N., Søholm Petersen, J., 2002. Metabolic conditions in Porcine longissimus muscle immediately pre-slaughter and its influence on peri- and post mortem energy metabolism. Meat Science 62 (2), 145–155.
mammalian muscles, the buffering capacity, however, being higher. This may be because chicken use the Pectoralis muscles mainly for single intensive bursts for flight, followed then by an aerobic recovery. The threshold glycogen content resulting in ultimate pH-values above the normal of 5.5 is 53 mmol kg−1 in pigs (Figure 4) and 57 in cattle. This means that any induced stress can last for quite some time without affecting the ultimate pH value, even with significant glycogen depletion. Especially in beef, the pyruvate resulting from glycolysis may also be immediately used aerobically by the fibre in question, or by another fibre of better aerobic status. In a young bull, the average rate of glycogen breakdown is 0.18 mmol kg−1 min−1, the maximum being 0.39. The corresponding hourly rates would therefore be 11 and 23 mmol kg−1, should the exercise truly last for hours. There is an abundance of literature on the effects of animal stress on meat quality, and especially on the incidence of the pale, soft and exudative (PSE) meat, as well as the dark, firm and dry (DFD) meat. These aspects will be dealt elsewhere in this book (preslaughter treatment), and are therefore not discussed here in detail. However, the quality effects of preslaughter stress manifest themselves through glycogenolysis, and consequently, through the rate of pH fall, as well as the ultimate pH. When the muscles of pigs and poultry, and more seldom cattle, coming to slaughter have a high content of glycogen accompanied with low levels of creatine phosphate, low oxygen saturation, as well as high body temperature, glycogenolysis and glycolysis will start premortally, and remain rapid for the first few hours postmortem. This will result in a low pH at temperature still being high, and usually in a very low ultimate pH, causing PSE meat. On the contrary, when glycogen content is low already at rest, or has been consumed in a long-term (hours) stress, the consequent low level at slaughter results in a lower rate of pH fall and an elevated ultimate pH. High pH lowers the keeping quality of meat, as well as causes a nontypical taste to it, while
increasing the water-holding capacity and tenderness. Glycogen reserves are hardly ever totally exhausted; approximately 10–20 mmol kg−1 always being left over, even in severely stressed cattle. The glycogenolysis may also cease at a point where, at least in theory, a sufficient amount is left yet to accommodate an additional pH fall of substantial size (Figure 5). It is not known whether the lower keeping quality is related to pH exclusively, or if also the low content of carbohydrate contributes to the alteration of the bacterial flora and/or its metabolism. The liver can store 10% or more of its weight as glycogen, and release it into the blood as glucose. In a slaughter-weight pig, this equates to 150 g of glucose, or 2.4 MJ of energy, and corresponds to approximately 20% of the needs of basic daily metabolism.
Concluding Remarks Livestock live normally a peaceful life without having to deal with frequent, long-lasting physical stress. Stress, namely, the secretion of epinephrine, increases glycogenolysis, and if psychological stress/excitement is accompanied with physical strain, it results in a very rapid use of glycogen. Various preslaughter treatments (collecting, regrouping with fighting and withdrawal of feed, marking, transport, slaughterhouse lairage, and finally, driving to stunning) involve potential physical and psychological stress factors that an animal may not have ever previously experienced. Furthermore, exposure to these treatments may continue from hours to several days, and thus, depending on species as well as the length and type of the logistic channel of each individual case, seriously jeopardize both animal welfare as well as meat quality. The quantity of research in carbohydrate metabolism is overwhelming, but the majority concerns humans and/or laboratory animals, and has been focused on living organisms, i.e., at pH 7.0 and temperature 37 °C. The decreasing pH and
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Figure 5 A suggestion (A) for beef quality categories based on the residual (48 h postmortem) glycogen+glucose content and ultimate pH (B). A I: low-stress, unproblematic; A II: medium-stress, potentially problematic; A III: high-stress, problematic. B shows that substantial amounts of glycogen may be found in chilled beef. Reproduced from Immonen, K., Puolanne, E., 2000. Variation of residual glycogen-glucose concentration at ultimate pH values below 5.75. Meat Science 55, 279–283.
temperature, however, induce changes in enzyme activities, and in the net charges of, for example, phosphates and glycolytic intermediates, thus greatly influencing the regulation of glycogenolysis and glycolysis. As examples, the scientific community does not fully agree from where protons are derived from postmortem, and what it is that stops glycolysis with more or less glycogen still left in the muscle. More research is needed to focus on postmortem biochemistry where pH and temperature are ‘one-way downwards’ variables, and the metabolic profile (glycogen, lipid and protein metabolisms in relation to energy supply and recovery) resulting from the various forms of animal stress needs to be studied in more detail.
See also: Conversion of Muscle to Meat: Color and Texture Deviations; Glycolysis; Rigor Mortis, Cold, and Rigor Shortening. Electrical Stimulation. Growth of Meat Animals: Physiology. Modeling in Meat Science: Meat Quality. Muscle Fiber Types and Meat Quality. Preslaughter Handling: Preslaughter Handling; Welfare including Housing Conditions; Welfare of Animals
Further Reading Fabiansson, S.U., Shorthose, W.R., Warner, R.D. (Eds.), 1989. Dark-cutting in cattle and sheep. Sydney: Australian Meat & Live-stock Research & Development Corporation. Fernandez, X., 1991. A review of the causes of variation in muscle glycogen content and ultimate pH in pigs. Journal of Muscle Foods 2, 209–235. Graham, T.E., Yuan, Z., Hill, A.K., Wilson, R.J., 2010. The regulation of muscle glycogen: the granule and its proteins. Review. Acta Physiologica 199, 489–498. Hamm, R., 1977. Postmortem breakdown of ATP and glycogen in ground muscle: A review. Meat Science 1, 15–39. Henckel, P., Karlsson, A., Jensen, M.T., Oksbjerg, N., Søholm Petersen, J., 2002. Metabolic conditions in Porcinelongissimus muscle immediately pre-slaughter and its influence on peri- and postmortem energy metabolism. Meat Science 62 (2), 145–155. Hocquette, J.F., Ortigues-Marty, I., Pethick, D., Herpin, P., Fernandez, X., 1998. Nutritional and hormonal regulation of energy metabolism in skeletal muscles of meat-producing animals. Livestock Production Science 56, 115–143. Immonen, K., 2000. Bovine muscle glycogen concentration in relation to diet, slaughter and ultimate beef quality. Doctoral Thesis, University of Helsinki, EKT Series no 1203, 90 pp. Available at: https://helda.helsinki.fi/bitstream/handle/ 10138/20879/bovinemu.pdf?sequence=2 (accessed 15.08.13). Immonen, K., Puolanne, E., 2000. Variation of residual glycogen-glucose concentration at ultimate pH values below 5.75. Meat Science 55, 279–283.
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Johnson, L.N., Barford, D., 1990. Glycogen phosphorylase. Minireview. The Journal of Biological Chemistry 265, 2409–2412. Meléndez-Hevia, E., Waddelm, T.G., Shelton, E.D., 1993. Optimization of molecular design in the evolution of metabolism: the glycogen molecule. Biochemical Journal 295, 477–483. Scheffler, T.L., Park, S., Gerard, D.E., 2011. Lessons to learn about post-mortem metabolism using the AMPKy3R200Q mutation in pig. Meat Science 89, 244–250.
Tarrant, P.V., 1988. Animal behaviour and environment in the dark-cutting condition. In: Fabiansson, S.U., Shorthose, W.H., Warner, R.D. (Eds.), Dark-Cutting in Cattle and Sheep, pp. 8−18. Austratian Meat and Live-stock Research & Development Corporation. Ylä-Ajos, M., 2006. Glycogen debranching enzyme activity in the muscles of meat producing animals. Doctoral Thesis, University of Helsinki, EKT Series no 1363, 64 pp. Available at: https://helda.helsinki.fi/bitstream/handle/10138/20823/ glycogen.pdf?sequence=2 (accessed 15.08.13).
Glossary Glycogenolysis The break-down of glycogen to glucose-1phosphate and glucose. Glycogen debranching enzyme A double-function enzyme that moves three glucose units from the 1-6 branching point (transferase) and then 1,6-glucosidase removes the last glucose unit.
Introduction Most of the time, blood will provide enough nutrients for the metabolism, and especially, for the energy production of the muscles. The principal portion of the energy produced in a muscle cell originates from the main nutrients; carbohydrates, volatile and nonvolatile fatty acids plus glycerol, and proteins. Fat, as free fatty acids, is an important source of energy at rest and during light exercise. In light exercise, the plasma content of free fatty acids increases within a few minutes, whereas during heavy exercise it decreases. In ruminants, short-chain free fatty acids (mainly acetate, propionate and butyrate) form the major proportion of the energy substrates with carbohydrates having a minor role (5–15%). Oxygen is required, in long terms for all kinds of energy production. Muscles are able to produce/use 5 mmol of adenosine triphosphate (ATP) s−1 g−1 at maximum rate, and that appears to be universal across muscle tissues. The resting level of consumption is approximately 0.02–0.05 mmol ATP s−1 g−1, and is used for maintaining membrane potentials, and fuelling the calcium pump that returns Ca++ back to the sarcoplasmic reticulum, the occasional contractile reactions between actin and myosin, as well as the anabolic reactions of molecule syntheses. ATP consumption also generates body heat. The maximal rate of ATP consumption is at least 100 times greater than that of a resting muscle. Since the production and consumption of ATP must be equal at any given moment, the portion of ATP that cannot be provided by the oxidative metabolism must be produced anaerobically. This explains the existence of two mechanisms (aerobic and anaerobic) for ATP production. If indeed the aerobic production was to be able to cover the maximal level of energy consumption, the animal would, depending on species, need to have much larger organs (lungs, heart, circulatory system, organelles and enzymes for aerobic metabolism in fibres) than it currently has for obtaining and using oxygen. Since animals seldom need maximal energetic capacity over a sustained period of time in all of their muscles, a capacity for a full aerobic coverage would be an inefficient physiological ‘investment.’ Another mechanism has, therefore, developed for short bursts of energy. Depending on the oxidative capacity of a muscle, the oxidative production/usage of energy can increase due to increased activity. In pigs, for example, the increase is less than 10 times
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Glycogen phosphorylase A very fast enzyme that is capable to remove glucose-1-phosphate from nonbranched chains of glycogen, down to the fourth glucosyl unit from the 1-6-branching point. Glycolysis (from glycose, an older term for glucose+-lysis degradation) The metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO−+H+.
the resting level, whereas in cattle, it is 20-fold, and in horses, almost 50-fold. The above mentioned figures are of course approximates, because the oxygen consumption within species depends on the fibre type composition and the basic tone of each muscle. The oxygen consumption of various muscles depends on their activity at a given moment, as does the blood flow through the muscles, as well as the relative amount of oxygen left in the muscles. At rest, for example, the blood flow through muscles may be 20% of the total flow. At maximal activity, however, the cardiac output increases tenfold, and 80% of it is circulated through muscles, which then utilize up to 90% of the passing oxygen. Together, these values translate to a 25fold consumption of oxygen in comparison to a resting muscle. The oxidative capacity of a body's total muscle mass seems to be in balance with the capacity of blood circulation. Animals with a high oxidative capacity (e.g., horse, reindeer) have relatively big lungs and hearts, and muscles that have dense capillary networks, compared to animals with less oxidative capacity (e.g., pig, chicken). Furthermore, breeding has had a significant effect on the oxidative capacity of domestic animals. The proportion of oxidative fibres is higher in the muscles of the wild pig than in the domesticated pigs, as is the capillary density. In wild pigs, 1 g of heart tissue serves 85 g of muscular tissue, whereas in domestic pigs it has to serve 140 g. In poultry, severe oxygen deficiency within the poorly capillarized Pectoralis muscle results in deep pectoral myopathy. The crucial importance of sufficient oxygen supply for muscle tissues cannot, therefore, be overly emphasized. In a muscle fibre, glycogen is the energy reserve used in case the circulatory system cannot provide enough oxygen and nutrients. Glycogen breaks down to glucose phosphates that are utilised anaerobically or aerobically, depending on animal species and their level of physical and/or psychological stress. There is indirect evidence that in stress, triggered by adrenaline, glycogen is preferred even if there were other energy substrates available, i.e., even if the stress did not involve high physical activity. The lactate formed in the glycolysing fibres will be transported into the blood via monocarboxylate transporters (MCT) that move lactate through the cell membranes from the higher concentration toward the lower. Lactate is destined to be exported to the liver to be used as a substrate for the synthesis of glycogen or glucose-6-phosphate. While on its way, some of it will probably also be aerobically
Encyclopedia of Meat Sciences, Volume 1
doi:10.1016/B978-0-12-384731-7.00245-2
Conversion of Muscle to Meat | Glycogen utilized either by the oxidative muscle fibres or by the heart, if the lactate concentration in them is lower than that in blood.
for allowing a rapid release of glucose from its structure by glycogen phosphorylase (GP) (Figure 1). A 100 mM solution of glucose would create a high osmotic pressure, whereas the equivalent 0.002 mM of glycogen does not, the glycogen molecule being more a particle (granule) than an element in a solution. The maximum sized glycogen molecule/granule of a diameter of 40 nm contains approximately 55 000 glucose units, and 2100 nonreducing chain ends. The total molecular weight is approximately 107 Da. Each glycogen molecule has 40–50 GP dimers (or 20–25 tetramers) bound to it, facilitating the immediate and fast cleavage of the glucosyl units. Glycogen synthase (GS), glycogen branching enzyme (GBE), AMP-activated protein kinase (AMPK), as well as glycogen debranching enzyme (GDE) are also bound to the glycogen complex, and thus, readily available when needed. There are also several other proteins with regulatory roles bound to the complex (Figure 2). In the core of the glycogen molecule is glycogenin (molecular weight (MW) 37 000), an enzyme-acting protein (glucosyltransferase) that serves as a primer for the molecule. In glycogen formation, uridine diphosphate moves the first eight glucosyl units to glycogenin after which GS takes over and extends the chain of glucosyl units via α(1→4) linkages. Glycogen branching enzyme then transfers an A-chain of 6–7 glucosyl units forming an α(1→6) linkage between the original chain and the transferred chain. A new branch point must be at least 4 glucosyl units apart from the previous branch point. Theoretical calculations have shown that the optimal chain length is 13 (12–14) glucose units, and that it is optimal to have two branching points in each B-chain (Figure 1). Each level of chains forms a concentric tier, and all new tiers double the number of chains of the previous tier. After 12 tiers, the structure will turn self-limiting, eventually due to the spatial relations between the glycogen molecule and GS. The distance of four glucose units between the branching points thus leaves tails of 4 or 5 glucosyl units.
Structure of Glycogen Theoretical studies have revealed how the glycogen molecule has taken its form in the biological evolution. The glycogen molecule is optimized for storing a maximum amount of glucose in the smallest possible volume, and at the same time,
A-chain
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Figure 1 Glycogen molecule (5 tiers) according to Meléndez-Hevia and others. Each unbranched A (dotted line) and branched B (solid line) chain has 12–14 glucosyl residues. G ¼glycogenin. Adapted from Immonen, K., 2000. Bovine muscle glycogen concentration in relation to diet, slaughter and ultimate beef quality. Doctoral Thesis, University of Helsinki, EKT Series no 1203, 90 pp. Available at: https://helda. helsinki.fi/bitstream/handle/10138/20879/bovinemu.pdf?sequence ¼2 (accessed 15.08.13).
Malin
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Figure 2 A schematic summary of proteins known to interact with glycogen. GDE, glycogen debranching enzyme; AMPK, AMP-activated protein kinase; GBE, glycogen branching enzyme; GM and PP1R6, regulatory subunits of protein phosphate 1 PP1; GN, glycogenin; GNIP, glycogenininteracting protein; GS, glycogen synthase; GSK, glycogen synthase kinase; GP, glycogen phosphatase; PhK, phosphorylase kinase; PTG, protein targeting to glycogen. Reproduced from Graham, T.E., Yuan, Z., Hill, A.K., Wilson, R.J., 2010. The regulation of muscle glycogen: The granule and its proteins. Review. Acta Physiologica 199, 489–498.
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Until recently, glycogen was thought to exist as two separate forms: proglycogen and macroglycogen. Particles with molecular weight of 400 kDa are called proglycogen, which due to its protein content of approximately 10%, is insoluble in acidic solutions. Glycogen molecules of a much higher molecular weight are called macroglycogen, which in turn maintain its water-solubility even in acidic solutions due to the dominant abundance of carbohydrate. It has been postulated that proglycogen is utilised in severe stress as well as postmortem, whereas macroglycogen is used during endurance exercise, and less so postmortem. The existence of different glycogen pools has, however, been challenged. It has been concluded that instead of existing as two separate forms, there is a continuum of glycogen molecules of different size. It has also been claimed that muscle fibres accommodate glycogen in multiple sites; some free in cytosol, and some bound to the sarcoplasmic reticulum. There are, therefore, differences in both solubility and availability for use. Microscopically, the granules are most abundant between the myofibrils, but also inside the fibrils. In humans, the average diameter of a glycogen granule is 25 nm, i.e., tier 8 is mainly in use, and less than 20% of the granules have built all 12 tiers. In conclusion, glycogen particles are not of fixed molecular weight, but instead, are subject to on-going resynthesis or degradation depending on the level of physical activity and/or stress, as well as the nutritional status of the animal.
Metabolism of Glycogen The main function of glycogen is to serve as a source of glucose for anaerobic glycolysis (includes glucose-1-P to pyruvate and, with the help of muscle-type lactate dehydrogenase, from pyruvate to lactate). Anaerobic glycolysis is a very rapid metabolic pathway producing 3 ATP of energy per glucosyl unit, the yield being much lower, however, than if pyruvate was utilised aerobically. The biochemical role of lactate formation is to regenerate NAD+ for glycolysis, and one proton will be simultaneously bound to pyruvate when lactate is formed. There has been an intensive discussion over the source of such protons, and it seems that physiologists, dealing with the pH range of 7.2–6.4 of a dynamic living muscle, and meat and animal scientists, working with a broader range of pH, i.e., from 7.2 all the way down to the pH 5.0 of a more stable postmortem muscle, see the matter quite differently. The protons derive from earlier stages of glycolysis, so that different pKa-values of glycolytic metabolites and phosphates at different postmortem pH-values relate to free proton formation. Irrespective of the sources of protons, and/or the buffering capacity of meat, there is an abundance of data confirming the linear negative correlation between lactate content and pH-value. In the breakdown of glycogen, there are two enzymes directly involved: GP that releases glucose is strongly controlled by various factors, including hormonal, and GDE that disassembles the branch points. The rate of the process is limited by the availability of free HPO42−. The pKa value of H2PO4 is 7.2, and at pH 5, the pool of HPO42− is virtually nonexistent. GP (842 amino acids and a MW of 97.4 kDa) amounts to approximately 2% of the soluble proteins of a muscle, and has
an immense acting capacity. GP is activated both by allosteric interactions as well as reversible phosphorylation. The inactive form b, of GP can be found in resting muscles, and is activated by a cascade involving epinephrine, calcium, cyclic AMP, AMPactivated protein kinase (which activates phosphorylase kinase), and inhibited, however, by glucose-6-P and ATP. GP is biologically active as a dimer of two identical subunits, but can also be found as a tetramer. There are dimeric GP molecules attached to each glycogen molecule/particle. Each GP monomer consists of two domains: the C-terminal domain (the one responsible for catalysing the reaction), and the N-terminal domain (responsible for regulation of the enzyme). The binding of phosphate or AMP at the effector sites leads to changes in the conformation of the protein, and increases the acceptance of glycogen at the active site. One subunit is being active whereas the other, as attached to the molecule, is regulating the activity. When the outermost tier is full of glycosyl units, 50% of all glucose within a glycogen particle lies in the unbranched A-chains, whereas the remaining 50% constructs the B-chains, irrespective of the total number of tiers. GP can only cut 9 out of the 13 consecutive 1–4-linked glucosyl units of a linear A-chain. Thus, according to theoretical calculations, GP can initially cleave 34.6% of the glucosyl units of any glycogen particle. At the fourth glucosyl unit from the 1–6-linked branch, the activity of GP ceases. The cascade for the activation of glycogenolysis is very effective. One epinephrine molecule bound to a receptor on a muscle fibre results in 400 000 glucose-1-P units cleaved by GP per second. This means that one molecule of GP can disassemble glycogen at a speed of 30 000 degradations per particle per second. Blood epinephrine concentration of 10−10 M generates an intercellular cyclic adenosine monophosphate (cAMP) concentration of 10−6 M. The relative concentrations of the three successive enzymes of the cascade; cAMPdependent protein kinase, phosphorylase kinase and GP, occur as molar ratios of 1:10:240 including adrenalin at a ratio of 0.0001, indicating an effective amplification of the signal. At the onset of muscle contraction, GP is activated from form b to form a by the increasing content of free cytosolic Ca++, as well as by norepinephrine secreted from the motorend-plates at the sarcolemma. GP then remains active in high concentrations of AMP and phosphate, and will be inhibited again when ATP and glucose-6-P levels have increased. Decreasing levels of ATP and glucose-6-P will start glycogenolysis. In case of nonintensive exercise, the increased blood supply of nutrients and oxygen quickly take over, and GP a is phosphorylated back to b. In a postmortem muscle of the pig, for example, GP stays in the form b for the first 10 min. It should be noted, however, that in a stressed animal, when creatine phosphate has been utilised, glycolysis starts earlier, even before slaughter. In this case, the reaction CP2−+ADP3−+H+ → C+ATP4− will not generate the initial buffering effect. For the continuation of glycogenolysis, GDE must remove the 1–6 linkages. The rate of GDE is not more than 10% of that of GP. The biological significance for this may be that a controlling system is needed to stop excessive breakdown which would cause damages in the fibre. Also GDE is bound to the glycogen particle, and thus, always available. It has a
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Figure 3 The relationship between muscle glycogen concentration and the rate of resynthesis in young bulls. Reproduced with permission from Tarrant, P.V., 1988. Animal behaviour and environment in the dark-cutting condition. In: Fabiansson, S.U., Shorthose, W.H., Warner, R.D. (Eds.), Dark-Cutting in Cattle and Sheep, pp. 8−18. Australian Meat and Live-stock Research & Development Corporation.
double function. In one end of the molecule, there is a transferase removing three glucosyl units and placing them at the end of another chain, most probably B, whereas in the other end of the enzyme complex there is 1,6-glucosidase activity cleaving the last remaining glucosyl unit as free glucose, which is then immediately phosphorylated. Finally, GDE will move further away from the A-chain thus allowing GP to function again. Very little is known about the control of GDE, except that it can only process A-chains of a length of four glucosyl units. It seems that glycogenolysis is independent on GDE at high glycogen concentrations, but at lower glycogen contents, GDE is of more relevance. GDE works optimally at a temperature range of 39–42 °C, and is not particularly sensitive to a lowering of pH. GDE is, however, very sensitive to low temperatures, and has hardly any activity at below 10 °C. It is, therefore, possible that postmortem, at low levels of glycogen, and in cold meat, GDE is rate-limiting. GS is allosterically activated by glucose-6-phosphate. Contrary to GP, GS is thus active when high blood glucose concentrations lead to elevated intracellular glucose-6phosphate. GS is inactivated by an on-going glycogenolysis, as the cAMP cascade ceases glycogen synthesis. GS is phosphorylated by protein kinase A as well as by phosphorylase kinase. Phosphorylation of GS promotes the ‘b’ (less active) conformation. The cAMP cascade with AMPK having a central role thus controls the levels of glycogen. Also the intake of glucose into a fibre is controlled by AMPK. In the liver, glucose-1-phosphate may also, instead of being converted to glycogen, be converted into glucose-6-phosphate, which is then dephosphorylated for the release into the blood stream (from muscle fibres the charged glucose phosphates do not escape). Once degraded, it takes a relatively long time to replenish the glycogen stores. There are, however, marked differences between species. In beef animals, the resynthesis is particularly slow, 1–2 mmol kg−1 h−1, increasing only at very low glycogen stores (Figure 3). In race horses, during three consecutive bouts within 2 h, the glycogen content first decreases approximately 30%, and interestingly, an additional 20% during 4 h at rest. The recovery takes 3 days indicating that the
consumption of carbohydrates is high after repetitive intensive exercises.
Effect of Glycogen Content on Meat Quality The glycogen contents of animals at rest or just after slaughter have mostly been estimated with the glycolytic potential, i.e., the sum of glycogen, glucose, glucose-6-P and lactate, expressed as lactate equivalents. This is, in principle, a good indicator for antemortem glycogen levels. However, as one connects the relative contents of the above variables with the time postmortem, and especially, with the pH and buffering capacity, it soon becomes evident that in many cases it has not been all that significant of an indicator for meat quality due to complexity of the events. An alternative simple procedure has not, however, at least to date, been introduced for the retrospective determination of preslaughter glycogen contents. The analyses are indeed challenging, especially because glycogenolysis and glycolysis can proceed very rapidly during sample preparation and analysis. It is, therefore, very important to immediately freeze the fresh muscle samples in liquid nitrogen. A resting muscle biopsy taken before any measures of transportation, or just before slaughter, would make the most representative sample, provided that sampling and freezing are, again, carried out promptly. The glycogen contents of resting muscles are usually at the levels of 80–100 mmol kg−1, given as glucose, yet lower levels are frequently found at slaughter due to ante-mortem stress. In pigs, higher levels (of 120 or even 150 mmol kg−1) are found, especially in the Hampshire, and Hampshire crossbreeds. Also in cattle, high resting levels (110– 120 mmol kg−1) can be found in animals that have first been depleted of glycogen, and then allowed to recover on a high energy diet. Pre-slaughter stress, such as transportation, invariably reduces glycogen contents. Although the glycogen levels of mammals are controlled by AMPK through PRKAG3 gene, the mechanism in poultry may be different. In chicken, the glycogen contents of the light Pectoralis major muscles are lower (below 100 mmol kg−1) than in the light
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6.8 6.6 A Model B Model C Model D Model
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Figure 4 Ultimate pH and glycogen content immediately before slaughter in pig M. longissimus dorsi. A–D, various exercise intensities before slaughter. Reproduced from Henckel, P., Karlsson, A., Jensen, M.T., Oksbjerg, N., Søholm Petersen, J., 2002. Metabolic conditions in Porcine longissimus muscle immediately pre-slaughter and its influence on peri- and post mortem energy metabolism. Meat Science 62 (2), 145–155.
mammalian muscles, the buffering capacity, however, being higher. This may be because chicken use the Pectoralis muscles mainly for single intensive bursts for flight, followed then by an aerobic recovery. The threshold glycogen content resulting in ultimate pH-values above the normal of 5.5 is 53 mmol kg−1 in pigs (Figure 4) and 57 in cattle. This means that any induced stress can last for quite some time without affecting the ultimate pH value, even with significant glycogen depletion. Especially in beef, the pyruvate resulting from glycolysis may also be immediately used aerobically by the fibre in question, or by another fibre of better aerobic status. In a young bull, the average rate of glycogen breakdown is 0.18 mmol kg−1 min−1, the maximum being 0.39. The corresponding hourly rates would therefore be 11 and 23 mmol kg−1, should the exercise truly last for hours. There is an abundance of literature on the effects of animal stress on meat quality, and especially on the incidence of the pale, soft and exudative (PSE) meat, as well as the dark, firm and dry (DFD) meat. These aspects will be dealt elsewhere in this book (preslaughter treatment), and are therefore not discussed here in detail. However, the quality effects of preslaughter stress manifest themselves through glycogenolysis, and consequently, through the rate of pH fall, as well as the ultimate pH. When the muscles of pigs and poultry, and more seldom cattle, coming to slaughter have a high content of glycogen accompanied with low levels of creatine phosphate, low oxygen saturation, as well as high body temperature, glycogenolysis and glycolysis will start premortally, and remain rapid for the first few hours postmortem. This will result in a low pH at temperature still being high, and usually in a very low ultimate pH, causing PSE meat. On the contrary, when glycogen content is low already at rest, or has been consumed in a long-term (hours) stress, the consequent low level at slaughter results in a lower rate of pH fall and an elevated ultimate pH. High pH lowers the keeping quality of meat, as well as causes a nontypical taste to it, while
increasing the water-holding capacity and tenderness. Glycogen reserves are hardly ever totally exhausted; approximately 10–20 mmol kg−1 always being left over, even in severely stressed cattle. The glycogenolysis may also cease at a point where, at least in theory, a sufficient amount is left yet to accommodate an additional pH fall of substantial size (Figure 5). It is not known whether the lower keeping quality is related to pH exclusively, or if also the low content of carbohydrate contributes to the alteration of the bacterial flora and/or its metabolism. The liver can store 10% or more of its weight as glycogen, and release it into the blood as glucose. In a slaughter-weight pig, this equates to 150 g of glucose, or 2.4 MJ of energy, and corresponds to approximately 20% of the needs of basic daily metabolism.
Concluding Remarks Livestock live normally a peaceful life without having to deal with frequent, long-lasting physical stress. Stress, namely, the secretion of epinephrine, increases glycogenolysis, and if psychological stress/excitement is accompanied with physical strain, it results in a very rapid use of glycogen. Various preslaughter treatments (collecting, regrouping with fighting and withdrawal of feed, marking, transport, slaughterhouse lairage, and finally, driving to stunning) involve potential physical and psychological stress factors that an animal may not have ever previously experienced. Furthermore, exposure to these treatments may continue from hours to several days, and thus, depending on species as well as the length and type of the logistic channel of each individual case, seriously jeopardize both animal welfare as well as meat quality. The quantity of research in carbohydrate metabolism is overwhelming, but the majority concerns humans and/or laboratory animals, and has been focused on living organisms, i.e., at pH 7.0 and temperature 37 °C. The decreasing pH and
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Figure 5 A suggestion (A) for beef quality categories based on the residual (48 h postmortem) glycogen+glucose content and ultimate pH (B). A I: low-stress, unproblematic; A II: medium-stress, potentially problematic; A III: high-stress, problematic. B shows that substantial amounts of glycogen may be found in chilled beef. Reproduced from Immonen, K., Puolanne, E., 2000. Variation of residual glycogen-glucose concentration at ultimate pH values below 5.75. Meat Science 55, 279–283.
temperature, however, induce changes in enzyme activities, and in the net charges of, for example, phosphates and glycolytic intermediates, thus greatly influencing the regulation of glycogenolysis and glycolysis. As examples, the scientific community does not fully agree from where protons are derived from postmortem, and what it is that stops glycolysis with more or less glycogen still left in the muscle. More research is needed to focus on postmortem biochemistry where pH and temperature are ‘one-way downwards’ variables, and the metabolic profile (glycogen, lipid and protein metabolisms in relation to energy supply and recovery) resulting from the various forms of animal stress needs to be studied in more detail.
See also: Conversion of Muscle to Meat: Color and Texture Deviations; Glycolysis; Rigor Mortis, Cold, and Rigor Shortening. Electrical Stimulation. Growth of Meat Animals: Physiology. Modeling in Meat Science: Meat Quality. Muscle Fiber Types and Meat Quality. Preslaughter Handling: Preslaughter Handling; Welfare including Housing Conditions; Welfare of Animals
Further Reading Fabiansson, S.U., Shorthose, W.R., Warner, R.D. (Eds.), 1989. Dark-cutting in cattle and sheep. Sydney: Australian Meat & Live-stock Research & Development Corporation. Fernandez, X., 1991. A review of the causes of variation in muscle glycogen content and ultimate pH in pigs. Journal of Muscle Foods 2, 209–235. Graham, T.E., Yuan, Z., Hill, A.K., Wilson, R.J., 2010. The regulation of muscle glycogen: the granule and its proteins. Review. Acta Physiologica 199, 489–498. Hamm, R., 1977. Postmortem breakdown of ATP and glycogen in ground muscle: A review. Meat Science 1, 15–39. Henckel, P., Karlsson, A., Jensen, M.T., Oksbjerg, N., Søholm Petersen, J., 2002. Metabolic conditions in Porcinelongissimus muscle immediately pre-slaughter and its influence on peri- and postmortem energy metabolism. Meat Science 62 (2), 145–155. Hocquette, J.F., Ortigues-Marty, I., Pethick, D., Herpin, P., Fernandez, X., 1998. Nutritional and hormonal regulation of energy metabolism in skeletal muscles of meat-producing animals. Livestock Production Science 56, 115–143. Immonen, K., 2000. Bovine muscle glycogen concentration in relation to diet, slaughter and ultimate beef quality. Doctoral Thesis, University of Helsinki, EKT Series no 1203, 90 pp. Available at: https://helda.helsinki.fi/bitstream/handle/ 10138/20879/bovinemu.pdf?sequence=2 (accessed 15.08.13). Immonen, K., Puolanne, E., 2000. Variation of residual glycogen-glucose concentration at ultimate pH values below 5.75. Meat Science 55, 279–283.
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Johnson, L.N., Barford, D., 1990. Glycogen phosphorylase. Minireview. The Journal of Biological Chemistry 265, 2409–2412. Meléndez-Hevia, E., Waddelm, T.G., Shelton, E.D., 1993. Optimization of molecular design in the evolution of metabolism: the glycogen molecule. Biochemical Journal 295, 477–483. Scheffler, T.L., Park, S., Gerard, D.E., 2011. Lessons to learn about post-mortem metabolism using the AMPKy3R200Q mutation in pig. Meat Science 89, 244–250.
Tarrant, P.V., 1988. Animal behaviour and environment in the dark-cutting condition. In: Fabiansson, S.U., Shorthose, W.H., Warner, R.D. (Eds.), Dark-Cutting in Cattle and Sheep, pp. 8−18. Austratian Meat and Live-stock Research & Development Corporation. Ylä-Ajos, M., 2006. Glycogen debranching enzyme activity in the muscles of meat producing animals. Doctoral Thesis, University of Helsinki, EKT Series no 1363, 64 pp. Available at: https://helda.helsinki.fi/bitstream/handle/10138/20823/ glycogen.pdf?sequence=2 (accessed 15.08.13).