Metabolism
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Experiments with isolated liver cells:
the citric acid cycle and warming
up post-operative patients
Key points from this exercise:
Addition of citrate leads to a considerably greater increase in oxygen consumption than is accounted for by the amount of citrate added, because it has a catalytic effect, increasing the rate at which what we now know is a cyclic pathway can operate. Addition of any of the other intermediates of the cycle has a similar catalytic effect.
When radioactive palmitate is provided as the substrate, it undergoes beta-oxidation to radioactive acetyl CoA, followed by complete oxidation in the citric acid cycle to yield radioactive carbon dioxide.
However, if malonate is added as an inhibitor of succinate dehydrogenase,and oxaloacetate is provided to permit continued uptake of acetyl CoA into citrate, there is no formation of radioactive carbon dioxide, and radioactive succinate, a water-soluble compound, accumulates. This is because the two carbon atoms that are lost from citrate in the first turn of the cycle are not the two that have been added from acetyl CoA.
Citrate is a symmetrical molecule, but in the cycle it behaves asymmetrically. This means that it must be passed directly from citrate synthase onto aconitase, without going into free solution. This metabolic chanelling provides regulation of the metabolism of citrate. When more citrate is being formed than is required for cycle activity and ATP formation, it can leave citrate synthase and go into free solution (because aconitase is saturated) and can be exported from the mitochondrion for fatty acid synthesis in the cytosol.
Lactate and glutamate can undergo complete oxidation in the citric acid cycle, or can be used for synthesis of fatty acids and glucose. Palmitate, which yields acetyl CoA, cannot be used for gluconeogenesis (because the reaction of pyruvate dehydrogenase is irreversible, and for each two carbon atoms added as acetyl CoA, two are lost in the cycle, so there is no net increase in the amount of oxaloacetate in the cell. If lactate and palmitate are provided, all of the lactate is used for gluconeogenesis, and there is greater oxidation of palmitate than when only palmitate is provided, since there is a need to produce the ATP needed for gluconeogenesis from lactate.
When non-radioactive glutamate and radioactive palmitate are added, there is radioactive labelling of the glucose formed from glutamate, because all of the intracellular pool of oxaloacetate has become labelled from the radioactive palmitate. Palmitate (or any other fatty acid or ketone body) is not, and cannot be, a substrate for gluconeogenesis.
When amino acids that yield citric acid cycle intermediates are metabolised they lead to an increase in oxaloacetate, but are not completely oxidised unless the oxaloacetate first undergoes carboxylation to phosphoenolpyruvate, then dephosphorylation to pyruvate and oxidation to acetyl CoA. This involves synthesis and utilisation of ATP, and hence is a thermogenic process, leading to an increase in body temperature. Similarly, if the oxaloacetate is used for gluconeogenesis, there must be increased formation of ATP, which is used in gluconeogenesis, so again this is thermogenic. For more on this see D.A.Bender (2012) British Journal of Nutrition 108: S113-121. Metabolism of “surplus” amino acids. (see also the exercise breathless after sprinting).