The TCA cycle (Fig. 2) is a major pathway in the central metabolism, having two main functions: It provides different compounds that are precursors for the cell ana-bolism and generates most of the metabolic energy. Regarding metabolism compart-mentalization, this pathway takes place inside the mitochondria. It is also important to highlight its central role as an internal mechanism to balance the rate of glycolysis and glutaminolysis, as discussed later. The connection between TCA and glycolysis is made through pyruvate, which is first transported into the mitochondria and further transformed in acetyl coenzyme A (CoA). Acetyl CoA is also partially provided by the degradation of some amino acids: tyrosine, lysine, leucine, and isoleu-cine. As previously discussed, most of the pyruvate obtained in the metabolization of glucose is diverted to lactate, and therefore only a small percentage is incorporated into the mitochondria. However, some differences are observed in the reported results, probably due to the different cell lines and growth conditions employed by different authors. Some authors have reported very low values (13,15,16), where the flux from pyruvate to acetyl CoA, through PDHC, accounted for less than 1%
of the rate of glucose consumption, compared with other reported values of 8-14% (17,21), and even up to 40% (14). Acetyl CoA enters the cycle reacting with oxaloa-cetate to form citrate. At this point, an important flux of citrate leaves the TCA cycle and is exported to the cytosol, where it contributes to lipid formation with concomitant production of pyruvate. Lipid formation is a highly demanding biosynthetic pathway in tumoral cell lines. The combination of a low incorporation of acetylCoA into the cycle and the outflow of citrate severely depletes the flux in the TCA cycle; this needs to be replenished in order to fulfill the energy generation function in the cell. This is done at the level of a-ketoglutarate. Glutamine is the precursor molecule for this, transformed to glutamate inside the mitochondria, and further to a-ketoglu-tarate. As a consequence, it is found that the flux from citrate to a-ketoglutarate can be as low as 10% of the flux through citrate synthase (12,14). This phenomenon is called the truncated citric acid cycle (36). The primary role of glutamine is to supply the necessary intermediate to keep the cycle operating, which reveals the importance of this compound as the main source of carbon and energy (37,38). Other possible anaplerotic reactions that could have a similar effect of TCA cycle replenishment at the level of the oxaloacetate molecule seem to be nonfunctional in mammalian cells, since no PEPCK or PC has been detected (13,15,16). Oxaloacetate is used for the biosynthesis of aspartate and asparagine. At the level of succinyl CoA there is a contribution from different amino acids such as isoleucine, valine, threonine, and methionine that can represent up to 10% of the flux from succynil CoA to succinate. At the level of succinate there is a new input, derived from the degradation of tyrosine and leucine, that can represent up to 7% of the flux from fumarate to malate (14,17). At the level of malate there is another important export flow from the cycle, used by the cells as an overflow mechanism to balance the flux through the cycle. Indeed, due to the high rate of glutamine uptake in most mammalian cells and the anaplerotic reaction of a-ketoglutarate incorporated into the cycle, the flux from succinate to malate is higher than the flux from oxaloacetate to citrate. Since the stoi-chiometry of the reaction between oxaloacetate and acetyl CoA is one to one, the cell must relieve any malate in excess from the cycle, in order to avoid accumulation of intermediates. Malate is therefore exported out of the TCA cycle, and further converted to pyruvate, as will be discussed in the next section.
The TCA cycle is directly linked to metabolic energy generation in the form of ATP, based in the respiratory system, located in the internal membranes of the mitochondria. The different molecules of NADH and FADH are oxidized, with oxygen as the final electron acceptor. Normally, a maximum of three ATP molecules per NADH and two ATP molecules per FADH can be reached, although some authors have suggested that mammalian cells can adjust their P/O ratio as a result of the culture conditions, adjusting to a more efficient metabolism when they are exposed to an oxygen limitation.
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