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An acetyl group is linked to the sulfhydryl (SH) tail of CoA to form acetyl CoA. CoA is made up of a nucleotide head (adenine + ribose + phosphate), a vitamin B5 body, and a sulfhydryl group-containing beta-mercaptoethylamine tail.

Methyl group-labeled acetyl CoA is created from 14C-labeled pyruvate. It is mixed up between C2 and C3 of succinate and turns into C4 of alpha-ketoglutarate. It is not released during the cycle's initial turn. It is not released in the second turn because only C1 and C4 are released in the first turn. The C2 and C3 of OAA, on the other hand, are changed into the C2 and C3 of alpha-ketoglutarate, which are subsequently distributed across all four carbons of succinate. Half of the 14C (at C1 and C4 of OAA) will therefore be released during the cycle's third rotation. Half of the remaining 14C would release as CO₂ after each rotation.

Hence, three rounds of cycle are required.