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What are the assumptions made during the calculation of net gain of ATP?

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It is possible to make calculations of the net gain of ATP for every glucose molecule oxidised, but in reality this can remain only a theoretical exercise. 

These calculations can be made only on certain assumptions that: 

1. There is a acquential, orderly pathway functioning, with one substrate forming the next and with glycolysis, TCA cycle and ETS pathway following one after another. 

2. The NADH synthesised in glycolysis is transferred into the mitochondria and undergoes oxidative phosphorylation. 

3. None of the intermediates in the pathway are utilised to synthesise any other compound.

4. Only glucose is being respired – no other alternative substrates are entering in the pathway at any of the intermediary stages.

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