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Technical Report #3 – Cellular Energy Production: The Krebs Cycle, Electron Transport, and Oxidative Phosphorylation

Humans and other animals obtain energy to support life, growth, and activity from food. The basic ques-tion is how is the energy contained in food extracted and transformed into a form which can be directly used as fuel by the body. The source of energy used by the body is the potential energy contained in the chemical bonds of food. Energy is either released or consumed during chemical reactions, depending on the relative energies of the reactants versus the products. The main foods used as energy substrates by the body are carbohydrates and fats. Carbohydrates and fats are different chemically, but have in common that they both contain carbon-hydrogen bonds. In this state, carbon is said to be reduced. In aerobic metabolism the carbon and hydrogen react with oxygen, forming CO2 and H2O.

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The reaction between hydrogen and oxygen to make water is extremely exergonic (this is the reaction that turned the Hindenburg into a fireball). This reaction releases energy because hydrogen and oxygen are more stable (i.e., have less energy) when they are joined together as water than when they exist separately. Most of the energy derived from the aero-bic metabolism of foods is from this reaction. Fats provide twice the caloric density of carbohydrates 9 calories per gram for fat as compared to 4 calories per gram for carbohydrate. The reason fat contains more energy than carbohydrate is that in fat the carbon is in a more reduced form (Zubay, 1983, p. 482) more hydrogen is packed in per carbon atom. In aerobic metabolism the carbon and hydrogen in foods react with oxygen to produce CO2 and H2O. This reaction releases energy because carbon diox-ide and water molecules contain less energy than the original food molecules and oxygen.

The same reac-tion occurs when a piece of food burns in the camp fire. In that situation the energy released by the reac-tion is simply liberated as heat to the surroundings. In the body the reaction is broken down into many small steps and the energy which is released is captured in a molecule called adenosine triphosphate, or ATP. About 67% of the energy obtained in glucose (the body’s chief fuel molecule) is captured by ATP and the rest is liberated as heat (Zubay, 1983, p. 395). This is a very impressive efficiency level compared to other machines. ATP is the immediate source of energy used to fuel nearly all cellular processes, including muscular contraction. The role of ATP is not to store energy (that is the role of body fat and glycogen) but rather to transfer energy from a food molecule to some other cellular molecule which is going to perform work (Vander, Sherman, Luciano, 1980, p. 80). Ener-gy is the ability to do work. Potential energy is energy which is stored and has the potential to perform work if it is released. The energy contained in a chemi-cal bond is a form of potential energy. The potential energy contained in the chemical bonds of food mol-ecules is released during oxidation, and this energy is transferred via ATP to other molecules which perform cellular work everything from muscular contraction to protein synthesis .

Conceptually, it is convenient to break up this process into four stages, although in fact these stages are inti-mately linked in the cell. The first stage of carbohy-drate metabolism is glycolysis and the first stage of fat metabolism is beta-oxidation. The following stages of energy production are common to both fats and carbo-hydrates, and are the Krebs cycle, electron transport, and oxidative phosphorylation. The Krebs CycleThe central energy producing pathway in the body is the Krebs cycle (figure 1), named for the German chemist Hans Krebs. Krebs originally postulated this process, also known as the TCA cycle, in 1937 and was later awarded the Nobel Prize in 1953 for this work. Energy substrates derived from carbohydrates or fatty acids enter the Krebs cycle as the intermedi-ate acetyl-CoA. The ultimate end of the process is to convert the chemical energy contained in foods into ATP.

Adenosine triphosphate is an unstable molecule containing a high energy phosphate bond. When ATP is split, the energy contained in this phosphate bond is released and is available to perform work inside the cell. ATP is the immediate source of energy for nearly all cellular processes, and thus has earned its reputa-tion as “the energy currency of the cell.”Carbohydrates are initially metabolized via an anaerobic process known as glycolysis. Glu-cose enters the glycolytic pathway and is con-verted into two molecules of pyruvate, generat-ing a net yield of two ATP molecules. Under anaerobic conditions, as may be temporarily experienced in muscular tissue during weight training, pyruvate is reduced to lactate, or lactic acid, which causes a burning sensation in the muscle. Glycolysis is a relatively inefficient process, yielding only two ATPs per glucose molecule. The two lactate molecules account for roughly 93% of the energy present in the original glucose molecule, so only about 7% of the energy embodied in glucose is made avail-able for use. Of this, about 50% is captured in ATP (Zubay, 1983, p. 305).

In the presence of oxygen a different metabolic fate is available to pyruvate. Instead of being converted into lactate, pyruvate is decarboxylated to generate acetyl-CoA. Acetyl-CoA is also produced by beta-oxidation of fatty acids, as discussed Tech-nical Report #2.The basic point of the Krebs cycle is to provide the chemical means of completely oxidizing the carbon of glucose or fatty acids to CO2 and the hydrogen to H2O. This allows much more of the energy contained in the food molecule to be extracted and used by the cell, as compared to anaerobic metabolism. In each turn of the Krebs cycle two carbons enter as acetate and two carbons exit as CO2. The cycle involves eight intermediates, each of which is converted into the next by an enzyme specific for that step (figure 1). These reactions are localized in the mitochondria, the site of aerobic energy production within the cell. The first stage of carbohydrate metabolism, glycolysis, occurs in the cytoplasm and does not require oxygen.

The end-product of glycolysis, pyruvate, enters the mitochondria to be further metabolized. In the mi-tochondria pyruvate is converted into acetyl-CoA by pyruvate dehydrogenase. Fats are oxidized to produce acetyl-CoA within the mitochondria. Long chain fatty acids must be ferried across the mitochondrial mem-brane by the carnitine shuttle, while medium chain fatty acids can transverse the membrane by passive diffusion.Acetyl-CoA donates the two-carbon compound acetate to a four-carbon acceptor oxaloacetate thereby gener-ating citrate, a six-carbon compound. During one turn of the cycle two molecules of carbon dioxide are liber-ated, ultimately regenerating oxaloacetate. The Krebs cycle intermediates are not consumed in the cycle and there is no net loss of carbon in the process, ignoring any side reactions which may occur. The cycle can thus be viewed as catalytic, since a relatively small amount of oxaloacetate can be used to metabolize an arbitrary amount of acetyl-CoA.

The activity of this pathway is controlled by the levels of its substrates and products, so that its level of energy production matches the energy needs of the cell. As the concentration of substrates increases, or the concentration of end products decreases, the activ-ity of the cycle increases. The most sensitive factors which directly regulate the cycle’s activity are the NAD/NADH ratio and the ATP/ADP ratio. The activ-ity of the first step in the pathway is also sensitive to the concentration of oxaloacetate.Under normal conditions the concentration of interme-diates such as oxaloacetate is not limiting. Medium chain triglycerides enter mitochondria independent of the carnitine shuttle, and thus bypass an important regulatory step in fatty acid oxidation (refer to Techni-cal Report #2). Medium chain triglycerides are oxi-dized so rapidly that the acetyl-CoA which is produced can overwhelm the amount of oxaloacetate available to accept it (Bach and Babayan, 1982). Some portion of the acetyl-CoA is then diverted to another metabolic fate ketogenesis. In ketogenesis two molecules of acetyl-CoA combine to form ketone bodies, primar-ily acetoacetic acid and beta-hydroxybutarate (refer to Technical Report #2). This process is diminished if oxaloacetate precursors, such as aspartate and pyru-vate, are co-administered with the MCTs (Bach and Babayan, 1982; Crozier, 1988).

This suggests that the ketogenic properties of MCTs are due, in fact, to their ability to overwhelm the capacity of the Krebs cycle at the level of oxaloacetate.Only one ATP molecule is produced directly by each turn of the Krebs cycle. This is referred to as “sub-strate level phosphorylation” since the generation of ATP is directly coupled to a specific chemical reaction. In other words, ADP participates as a substrate in the reaction. Most of the energy derived from aerobic metabolism comes from subsequent oxidation of the NADH and FADH2 produced by the cycle. This is referred to as “oxidative phosphorylation” since here ATP synthesis is coupled to the oxidation of NADH and FADH2. Aerobic metabolism can the be thought of as having two phases: the oxidative phase in which electrons (in the form of hydrogen atoms) are removed from organic substrates and transferred to coenzyme carriers (FAD and NAD), followed by the reoxidation of the reduced coenzymes (FADH and NADH2) by the transfer of electrons (again in the form of hydrogen) to oxygen, generating H2O (Zubay, 1983, p. 325).

The reduction of oxygen to water to extremely exergonic and most of the ATP is generated during this process. The oxidation of acetyl-CoA involves removal of electrons (as hydrogen) from the Krebs cycle inter-mediates and transfer of hydrogen to the coenzymes FAD and NAD. In the process, these coenzymes are reduced to FADH and NADH2. (In chemistry, “oxi-dation” is the removal of electrons and “reduction” is the addition of electrons.) Subsequently, the reduced coenzymes are reoxidized by transfer of the hydrogens to oxygen in the “electron transport chain.” Ultimate-ly, ATP is synthesized by oxidative phosphorylation of ADP, which is driven by a proton gradient generated in the process of electron transport (Zubay, 1983, p. 325).The reactions of the Krebs cycle can be summarized by the following equation (Zubay, 1983, p. 335):acetyl-CoA + 2 H2O + 3 NAD+ + FAD + ADP + Pi 2 CO2 + 3 NADH + 3 H+ + FADH2 + CoA + ATP Two carbons enter as acetate and exit as CO2, pro-ducing ATP, NADH, and FADH2 as byproducts. The FADH and NADH2 in turn enter the electron transport chain to be further metabolized.Electron Transport and Oxidative PhosphorylationAlthough some ATP is directly generated by the Krebs cycle, more significant products of the cycle are the reduced coenzymes NADH and FADH2. Most of the energy contained in the starting material is still pres-ent in these coenzymes. The primarily energy yield of aerobic metabolism occurs when NADH and FADH2 are re-oxidized to NAD and FAD.

This process is known as electron transport because electrons from NADH and FADH2 are transported via a chain of electron carriers and are ultimately transferred to mo-lecular oxygen.  Oxygen is a very electronegative element, meaning that it has a strong affinity for electrons. In essence, oxygen and hydrogen combine to form water because oxygen has a high affinity for electrons, and hydrogen represents an easy source. Hydrogen does not have a strong affinity for electrons and basically gets trapped into sharing its electrons with oxygen. The overall reactions can be summarized as (Zubay, 1983, p. 364): NADH + H+ + 1/2O2 NAD+ + H20 G = -52.6 kcal/mol FADH2 + 1/2O2 FAD + H20 G = -43.4 kcal/molThe reduced coenzymes NADH and FADH2 serve as donors of electrons (as hydrogen) which combine with oxygen to form water. The delta-G expression indicates that the reaction will proceed spontaneously with the release of energy. Enough energy is released to drive the synthesis of several ATPs. Therefore, rather than wasting energy, the above reaction is di-vided up into several small steps.

The energy release is thus parcelled out in small packets to allow ATP to be generated more efficiently (Zubay, 1983, p. 365).  To achieve this, electrons are transported from the reduced coenzymes to oxygen via a series of carri-ers, arranged in the order of increasing electron affin-ity (figure 2). These electron carriers are molecules (some of them proteins) capable of undergoing revers-ible oxidation-reduction reactions.These electron transporters are embedded within the mitochondrial inner membrane (mitochondria are double-membraned structures). The energy which is released as electrons are transported down the chain to acceptors of ever increasing electron affinity is not directly used to synthesize ATP. Instead, the energy is used to generate a proton gradient across the inner mitochondrial membrane. This results in an electric field across the membrane (about 0.14 V) as well as a pH gradient (about 1.4 units). Protons are actively pumped across the inner mitochondrial membrane us-ing the energy derived from electron transport. In or-der to establish a proton concentration gradient across the membrane, the membrane must be impermeable to passive diffusion of protons.

Protons re-enter the mitochondrial matrix (driven by the concentration gra-dient) through a protein structure embedded within the membrane known as F0. F0 is physically attached to another protein structure known as F1-ATPase, which directly synthesizes ATP from ADP and phosphate. The precise mechanism by which energy is transferred from F0 to F1 and subsequently used to drive ATP synthesis is still under investigation, but may involve protein conformational changes or channeling of pro-tons through the enzyme active site (Zubay, 1983, p. 393). In summary, ATP is a molecule used to transfer energy from fuel substrates to cellular machinery performing work. The specific way in which this is accomplished is simple in principle but complicated in its actual ex-ecution. In principle, energy is derived from foods by their reaction with oxygen, just as when food is burned in a fire. Instead of being released as heat to the sur-roundings, some of the energy is captured as ATP. To achieve this efficiently, the process is broken down into several small steps. The first stage is to convert food molecules into a two-carbon compound, acetyl-CoA. For carbohydrates this is achieved by glycolysis followed by decarboxylation of pyruvate; fatty acids are converted to acetyl-CoA by beta-oxidation.

The acetyl-CoA, whether derived from carbohydrate or fat, is next metabolized in the Krebs cycle. One ATP molecule is generated per acetyl-CoA directly in the Krebs cycle, by “substrate level phosphorylation.” The carbon entering the Krebs cycle is released as CO2. Hydrogen present in the original food is now in the form of the reduced coenzymes NADH or FADH2. Most of the energy from aerobic metabolism is de-rived from oxidation of these reduced coenzymes in the electron transport chain . The summary reactions of the electron transport chain suggest that the hydro-gen from NADH and FADH2 combine with oxygen to form water, a well known exergonic reaction. How-ever, this does not happen directly. Instead, the energy released as electrons are transported down the chain (to electron acceptors of increasing electron affinity) is used to generate a proton gradient across the mito-chondrial membrane. The movement of protons back inside the membrane through the F0-F1 complex pro-vides the driving force for ATP synthesis by F1. This is referred to as “oxidative phosphorylation” because the phosphorylation of ADP to form ATP is coupled to the oxidative events occurring in the electron transport chain .

Energy Production and the AthleteAthletes experience increased energy need as com-pared to sedentary people . Bicycle racers and other endurance athletes can require as much as 10,000 calories per day to support their activity level. Body-builders commonly consume in excess of 8,000 calories daily to fuel their training and support gains in body weight. The body draws on three different types of food as energy substrates: fats, carbohydrates, and protein. Of these, carbohydrate is the most preferred. Carbohydrates are easily digested and rapidly enter the bloodstream as glucose. Glucose is immediately used as fuel by the cell. A byproduct of glucose metabo-lism is malonyl-CoA, which inhibits carnitine acyl-transferase I. Since long chain fatty acids require the carnitine shuttle in order to be transported inside the mitochondria, they are not used as fuel to a significant extent until the carbohydrates are depleted. Similarly, amino acids can be also oxidized to produce energy but are not used as fuel to a significant extent until carbohydrate is depleted. Of course, one of the primary goals of bodybuilders is to increase muscle mass.

Therefore amino acids are more valuable to use as protein rather than as fuel. Conventional fats are not a good energy source for bodybuilders either since they cannot be metabolized anaerobically and are not burned rapidly enough to meet the energy demands of high intensity exercise such as weight lifting (Coleman, 1991). Medium chain triglycerides are absorbed and metabolized much more rapidly than conventional fats and are immediately available for energy (Bach and Babayan, 1982). MCTs are an excellent quick energy source, harnessing the caloric density of fat but being metabo-lized as rapidly as glucose (Bach and Babayan, 1982).  Furthermore, MCTs and the ketone bodies they pro-duce decrease glucose uptake and utilization (Lavau and Hashim, 1978) and this seems to result in a glu-cose-sparing effect (Cotter et al, 1987). MCTs also have a protein-sparing effect and may reduce skeletal muscle protein catabolism, leaving amino acids avail-able for use as protein instead of being oxidized as fuel (Babayan, 1987; Haymond, Nissen, and Miles, 1983). Medium chain triglycerides are an excellent energy source for anyone experiencing increased en-ergy needs (Bach and Babayan, 1982) and are ideally suited to the special needs of athletes.

References

1. Babayan. Medium chain triglycerides and structured lipids. Lipids 22: 417-420 (1987).

2. Bach and Babayan. Medium chain triglycer-ides: an update. Am. J. Clin. Nutr. 36:950-962 (1982).

3. Coleman. Carbohydrates: the master fuel. In: Sports Nutrition for the 90s, eds.Berning and Steen. Aspen Publishers, 1991.

4. Cotter, Taylor, Johnson, and Rowe, A meta-bolic comparison of pure long chain triglyceride lipid emulsion (LCT) and various medium chain tri-glyceride (MCT)-LCT combination emulsions in dogs Am. J. Clin. Nutr. 45: 927-939 (1987).

5. Crozier. Medium chain triglyceride feeding over the long term: the metabolic fate of C-14 octano-ate and C-14 oleate in isolated rat hepato-cytes. J. Nutr. 118: 297-304 (1988).

6. Guyton. Textbook of Medical Physiology. Published by W.B. Saunders, 1976.

7. Haymond, Nissen, and Miles, Effects of ketone bodies on leucine and alanine metabolism in normal man. In: Amino Acids Metabolism and Medical Applications, Eds. Blackburn, Grant, and Young. Published by John Wright PSG Inc., pages 89-95 (1983).

8. Lavau and Hashim, Effect of medium chain triglyc-eride on lipogenesis and body fat in the rat. J. Nutr. 108: 613-620 (1978).

9. Vander, Sherman, and Luciano. Human Physiology The Mechanisms of Body Function. Published by McGraw-Hill Book Company, 1980.

10. Zubay. Biochemistry. Addison-Wesley Publishing Company, 1983 .

2018-03-13T11:10:22-04:00 August 27th, 2009|Medium Chain Triglyceride Technical Reports|

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