Which statement is true about the LDH activity assay depicted in the graph over 8 minutes?

• A. Zero-order kinetics between 5 and 8 minutes
• B. The reaction proceeds from lactate to pyruvate
• C. Nonlinearity after 6 minutes is due to substrate exhaustion
• D. The change in absorbance is due to reduction of NAD to NADH

Answer: (C)

The main concept here is how enzyme reaction curves behave when substrate is consumed. In an LDH assay, lactate is converted to pyruvate with NAD+ being reduced to NADH, and the amount of NADH is tracked by increased absorbance at 340 nm. At the start, when lactate is plentiful, the rate is roughly constant and the plot of absorbance versus time is nearly linear. As lactate gets used up, the reaction slows and the slope decreases, so the curve becomes nonlinear toward the later minutes.

So, observing nonlinearity after about 6 minutes is best explained by substrate exhaustion rather than a constant zero-order rate or a misinterpretation of the reaction direction. The rise in absorbance reflects NADH production, which is consistent with lactate being converted to pyruvate, but the key reason the graph bends is that the substrate is running out.