LEC12

12 Glycolysis

12.1 Introduction

Glycolysis is the decomposition of glucose by hydrolysis. Most of the glucose in muscle is stored as glycogen. Thus , glycolysis is also the decomposition of glycogen. Glycolysis is supremely important in understanding how muscles are converted to meat. Glycolysis normally causes an increase in acidity (= decrease in pH) as muscles are converted to meat. Too much or too little acid - and the meat is ruined.

IF you have not taken a course in biochemistry - concentrate on section 12.2 to 12.5. If you are bored by yet another repetition of glycolysis have a look at sections 12.6 onwards.

12.2 Living muscle

In living muscle, the complete oxidation of glucose to carbon dioxide and water requires oxygen, and it releases a lot of energy. Much of this energy is captured by adding a phosphate group to another molecule that already has two phosphate groups. Thus, adenosine diphosphate (ADP) is converted to adenosine triphosphate (ATP).
The ATP molecule carries this energy within the myofibre, and it may be released to another biochemical system by cleaving off the added phosphate.
Muscle contraction is a primary user of ATP in the living animal, but substantial amounts of ATP are used by the membranes around and within the myofibre for maintaining ionic concentration gradients.
One molecule of glucose may be combined with oxygen to add a phosphate group to each of 36 molecules of ADP.
In addition to 36 molecules of ATP, this produces carbon dioxide and water.
However, if the muscle is without oxygen (anoxic) after slaughter, the most it can normally gain by oxidizing each glucose unit is two molecules of ATP.

12.3 Glycogen

Structure of a glucose unit with its carbon atoms numbered is shown below.

These are joined by straight 1-4 linkages or branched 1-6 linkages to form glycogen.

Glycogen is the primary storage carbohydrate in myofibres and appears in electron micrographs as single granules or clumps of granules located in the sarcoplasm (cytoplasm) between myofibrils and under the cell membrane.
Scanning tunneling microscopy (one of the most powerful types of microscope yet invented) reveals glycogen granules are ellipsoidal with a laminar structure which suggests they grow from one edge rather than a central point.
In longitudinal sections of skeletal muscle, glycogen and its associated enzymes are concentrated at the I bands (one of the transverse bands across the muscle fibre when examined microscopically).
Glycogen is a polysaccharide formed by the linking together of large numbers of glucose units. However, the glycogen from some animal tissues is a proteoglucan (glycoprotein) that may contain other monosaccharides and phosphate ester groups.
The concentration of glycogen in foetal muscle increases steadily towards the end of gestation and peaks at the time of birth. Pigs have very high muscle glycogen levels at birth but they decline rapidly after birth to reach future adult levels within a week.
Glycogenin is a protein acting as a starting point for the formation of new glycogen.

The image below shows a transverse section of pork stained by the periodic acid-Schiff reaction for glycogen. All the myofibres started with glycogen, but most myofibres have depleted their glycogen during the conversion of muscle to meat. Note how some of the myofibres have a central core of glycogen.

12.4 After exsanguination

Once the animal has been exsanguinated, the oxygen within the muscles is rapidly exhausted, and the process of converting muscle to meat really begins.
Glycogenolysis is the enzymatic degradation of glycogen, and is the first step in the release of energy by the oxidation of glucose units.
Phosphorylase erodes straight chains (from their non-reducing ends at carbon 4) and attaches a phosphate group to the carbon atom at position 1 as it removes a glucose unit.
Phosphorylase erodes straight chains until it comes to the fourth glucose unit preceding a branch point.
The three glucose units before the fourth one carrying the branch are removed together, and are added to an adjacent free straight chain so the 1-6 linkage thus exposed at the branch point may be severed.
A second enzyme, debranching enzyme, performs this task.
Instead of being released as glucose-1-phosphate, the glucose unit released from a branch point remains as free glucose.
Thus, glycogenolysis liberates glucose-1-phosphate and glucose in a ratio determined by the mean length of straight chains and the number of branch points.

While the structure of glycogen and the mechanism of glycogenolysis are important in understanding the conversion of muscles to meat, the remainder of the pathway by which glycogen is anaerobically oxidized to lactate need only be covered in general principle. The most important question to be answered is why lactate is produced under anaerobic conditions, yet hardly at all under aerobic conditions.

After a series of steps in the glycolytic pathway, molecules with six carbon atoms derived from the glucose units of glycogen are split to produce two molecules of pyruvate, each with three carbon atoms.
All the glycolytic enzymes (except for hexokinase) are concentrated in the I band.
If aerobic conditions prevail, pyruvate formed in the cytosol of the myofibre now enters a mitochondrion (the metabolic furnace of the cell).
After entering a mitochondrion, pyruvate is converted to acetyl-CoA which becomes fused to oxaloacetate to form citrate.
The citrate then is oxidized in the Krebs' cycle, which is completed by the regeneration of oxaloacetate.
Continuous activity of the Krebs' cycle is fueled by a range of carbohydrates, fatty acids and amino acids, and is the primary system for the aerobic generation of energy.
Large numbers of molecules of ATP are produced from ADP by a series of reactions, oxidative phosphorylation, in the mitochondrial membrane.
Under aerobic conditions, the production of two pyruvate molecules from a glucose-1-phosphate molecule results in the reduction of 2NAD+ .
Thus, somewhere else in the myofibre, NADH must be re-oxidized for glycolysis to continue.
Aerobically, this occurs as a consequence of mitochondrial Krebs' cycle activity, although NADH and NAD+ do not actually cross the mitochondrial membrane.
In anaerobic living muscles and in meat, the Krebs' cycle is halted, and NADH is re-oxidized in the cytosol by lactate dehydrogenase (LDH) during the conversion of pyruvate to lactate.
Pyruvate is of no immediate use anaerobically since mitochondrial oxidation has ceased, but its conversion to lactate ensures a continued supply of NAD+ for the continuation of glycogenolysis and anaerobic glycolysis in the cytosol.
However, since these events form only the initial stages of complete carbohydrate oxidation, they do not regenerate much ATP.

LDH adds hydrogen to pyruvate to produce lactic acid and exists in a number of isoenzymes separable electrophoretically by their net electrical charge. If LDH-1 is prevalent, it facilitates aerobic metabolism, where possible, since it is inhibited by pyruvate and lactate. LDH-5 is not inhibited by high levels of lactate and pyruvate, and it facilitates anaerobic metabolism. LDH-1 is typical of cardiac muscle while LDH-5 is typical of skeletal muscles, particularly those adapted for anaerobic conditions during contraction. In skeletal muscles, the ratio of LDH-1 to LDH-5 corresponds to the dominant activity pattern of a muscle. For example, muscles capable of sustained activity and which only use aerobic metabolism have high LDH-1. LDH-5 is the dominant isoenzyme in skeletal muscles from slaughter-weight pigs.

12.5 Regulation of glycogenolysis

The regulation of glycolysis in a myofibre of a live animal is integrated with the metabolic state of the myofibre and its immediate energy needs.
The metabolic state of the myofibre is profoundly affected by hormones, particularly adrenaline, and by the extent of recent contractile activity of the myofibre.
Phosphorylase is particularly important in the conversion of muscle to meat since it may be a primary control site for post mortem glycolysis.
Phosphorylase in muscle is most active when it is, itself, phosphorylated (a-form). When dephosphorylated, it is less active (b-form).
In general terms, therefore, phosphorylase is switched on and off by the addition or removal of its phosphate, with on and off states being relative rather than absolute.
The activity of phosphorylase b is dependent on the presence of AMP but the activity of phosphorylase a is not.

There are two conflicting requirements complicating the activation of phosphorylase. Firstly, since phosphorylase initiates the release of considerable amounts of chemical energy, there must be safeguards to prevent its uncontrolled activity. In stress-susceptible pigs, for example, the uncontrolled activity of anaerobic glycolysis may lead to excessive heat production and to a level of acidity that may soon prove fatal. The conflicting requirement is vast amounts of phosphorylase spread through the muscle mass must be rapidly activated by relatively small amounts of adrenaline. The adrenaline activation of severely frightened animals often is called the "fight or flight" response: neither of these responses is likely to be of much survival value if the anaerobic energy supply to body muscles is delayed.

The conflicting demands for fail-safe but rapid activation are satisfied by two particular features of the activation system.

The conversion of phosphorylase b to phosphorylase a is inhibited locally in each myofibre by high concentrations of ATP and glucose-6-phosphate (an intermediate in the conversion of glycogen to lactate). Thus, if the energy released by phosphorylase is not rapidly consumed, the energy release system shuts down. If the energy is used, however, AMP and phosphate further enhance the activation of phosphorylase.
To enable the rapid activation of phosphorylase throughout the musculature, the relatively small amounts of adrenaline arriving at the muscle initiates a series of biochemical changes functioning as an amplifier. A small input leads to a large output.

Adrenaline causes adenyl cyclase to increase its formation, from ATP, of cyclic AMP.
Then cyclic AMP activates protein kinase.
With ATP and magnesium ions present, protein kinase then phosphorylates another enzyme, phosphorylase b kinase b.
The active form, phosphorylase b kinase a, in the presence of magnesium ions, finally activates phosphorylase b to phosphorylase a.
As a final safety factor, if the supply of inorganic phosphate is inadequate, even phosphorylase a will be relatively inactive but the system will be primed for rapid energy production once muscle contraction is initiated.

Remember the sarcoplasmic reticulum?

When animals require energy anaerobically during normal activity, phosphorylase b kinase is activated by calcium ions released from the sarcoplasmic reticulum - normally the trigger for muscle contraction in living muscle. Glycogen granules are closely related to the sarcoplasmic reticulum and to glycogenolytic enzymes as part of a structural complex. The activation system linking muscle contraction to glycogenolysis is short lived to avoid the continuous use and depletion of glycogen reserves. Glycogen is a rapidly available energy source for both brief muscle activity and the early stages of sustained activity. Phosphorylase activity is curtailed by phosphatase, which dephosphorylates phosphorylase a and phosphorylase b kinase a, when muscle activity ceases or an animal recovers from fright. Many features of the system for activating phosphorylase are shared with the activation system for glycogen synthesis, but the shared features are opposite in effect. Thus, the myofibre does not attempt to synthesize new glycogen at the same time that it is breaking it down, and vice versa.

Newborn pigs often have difficulty maintaining their blood glucose levels, and may die of hypoglycaemia after even short periods of starvation. Given they have high glycogen levels at birth, this is not easy to explain, although stored glycogen may simply be inadequate in amount if milk is not available. Despite an adult predisposition to the excessive accumulation of adipose tissue, newborn pigs have very little adipose tissue and liberation of free fatty acids is severely limited. Most phosphorylase is in the relatively inactive b form just after birth. In adult pigs, hyperglycaemia readily occurs in response to exercise and adrenaline secretion.

12. 6 Fate of lactate in living animals

Meat animals may exhibit a considerable range in their general muscular and cardiovascular fitness. At one extreme, sheep and cattle may roam in search of feed and water for part of the year, coping with adverse conditions much like wild animals. At the other extreme, animals may be reared in close confinement, partly to prevent them wasting feed energy by converting it to muscular work. Pigs rarely exercised may have a low oxygen transport capacity whereas regular exercise reduces lactate production.

In living animals, the lactate produced by contracting muscles is removed by the circulatory system, but first it must travel from the myofibre into the interstitial space. Lactate release may be retarded in situations such as exhaustive exercise. A fraction of the lactate leaving a myofibre may be in the form of undissociated lactic acid, and this fraction may increase with a rise in external pH. In living animals, blood flow is increased in active muscles (hyperaemia). When hyperaemia occurs as a secondary response to hypoxia, it may be mediated by the release of adenosine (from AMP A + P) into the interstitial fluid between myofibres.

On arriving in the liver, lactate may be converted to glucose-6-phosphate, and then stored as glycogen or released as glucose back into the blood. Circulating glucose is available to muscles to be stored as glycogen or used directly as energy for contraction (Cori cycle). After exhaustive exercise, an animal may continue to consume oxygen at an increased rate for some time (oxygen debt). During this recovery period, some lactate may be completely oxidized to release enough energy for the remaining lactate to be converted to glucose (gluconeogenesis) in the liver. When an animal is exsanguinated, the blood can no longer perform its transport function in the Cori cycle, and lactate accumulates in the musculature. There are, however, a number of other possible fates for lactate in living animals.

Heart muscle receives its own blood supply from the coronary artery, straight from the aorta, and cardiac muscle may use circulating lactate as an energy source. The high oxygen concentration in the aorta usually allows complete oxidation of lactate by cardiac muscle. Any lactate remaining in the aorta may not all get pumped to the liver or kidneys to participate in the Cori cycle, because the aorta supplies other major arteries in addition to the hepatic and renal arteries. Much of the circulating lactate may pass through non-contracting muscles oxidizing lactate as an energy substrate, particularly if circulating fatty acids are unavailable. Resting muscles also may take up circulating lactate and reconvert it directly to glycogen, probably via a pathway which is independent of the mitochondrial Krebs' cycle (the evidence is in research papers even if not in your biochemistry book). Finally, also it is possible for all the lactate consumed during an oxygen debt period to be oxidized to carbon dioxide.

Contraction causes an initial drop in myofibre pH because of the hydrolysis of ATP, but this may be followed by an increase in pH from the breakdown of creatine phosphate (CP). CP acts as a short term store of energy since it has a phosphate transferable to ADP by the creatine phosphokinase (CPK). CP is the dominant carrier of energy from mitochondria to myofibrils. CPK and creatine are normally contained within myofibres, but may leak into the blood from damaged or diseased myofibres. In healthy muscle, creatine is slowly but continuously converted to creatinine. Creatinine is lost in the urine in approximate proportion to the muscle mass of the body. The production of lactate following muscle contraction may cause another drop in pH. The interstitial pH between muscle fibres and the pH at the muscle surface are profoundly affected by overall pH levels in the blood.

12.7 Effect of pH on water-holding

Now we can see why the pH of meat generally declines after slaughter - the lactate accumulates as a byproduct of the process that releases energy in an attempt to keep the cell alive. And now we can explain how this affects the quality of the meat, firstly, its water-holding capacity.
If dried meat protein is rehydrated by exposure to increasingly damp air, three water compartments may be detected by the way in which water is taken up: 4% of the water becomes firmly bound as a monolayer round muscle proteins, another 4% is taken up as looser second layer, and 10% of the water accumulates loosely between protein molecules.
Water binding capacity is the ability of meat to bind its own water or, under the influence of external forces such as pressure and heat, to bind added water.
Water absorption or gelling capacity is the ability of meat to absorb water spontaneously from an aqueous environment.
Water binding capacity and water absorption are closely related.
Water absorption may be found from the increase in weight and volume of meat samples placed in an aqueous solution, and water binding capacity may be determined by centrifugation or by pressing the fluid from the meat and measuring it.
Water binding capacity is modified by pH and drops from a high around pH 10 to a low at the isoelectric point of meat proteins between pH 5.0 and 5.1.
At its isoelectric point a protein bears no net charge and its solubility is minimal.
Below pH 5, a value only attained if the pH of a processed meat product is deliberately lowered , water binding capacity starts to increase again.
Thus, as the pH of pork declines post mortem, its water binding capacity decreases, and much of the water associated with muscle proteins is free to leave the myofibre.

Further information

Structure and Development of Meat Animals and Poultry. Pages 495-548.