Fig. 4. Estimating D by fitting simple equations to complicated biological situations. When O₂ is consumed by metabolic processes, estimating D in biological structures is non-trivial, as metabolism draws down interior O₂ levels and alters the time course of change after external step changes. Here we simulated step change traces under both first-order and Michaelis–Menten reaction kinetics and then fitted a simple equation (Eqn. 9) to the simulated traces. Under first-order kinetics (A), estimated D was good when initial drawdown (or ) was small. At higher k, giving greater initial drawdown, fitted D increasingly overestimated the known D used in the simulations (3x10^–6). Under Michaelis–Menten kinetics (B), estimated D was again good when initial drawdown was small. At higher V_max, giving greater initial drawdown, the down- and up-traces were highly asymmetrical. In this case, fitted D from the up-trace always gave values closer to the known, simulated value.