NADH-MetHb reductase activity was measured using cyanogenic methemoglobin as a substrate, or dichlorophenol-indophenol as a substrate to determine yellow delivery enzyme activity, or cytochrome b5 as a substrate to determine b5R activity. It should be emphasized that the results in the test tube (at high substrate concentrations) do not exactly reflect the degree of reduction in catalytic efficiency in living cells (at low substrate concentrations) because of the different mechanisms of action of the different genetic variants. Because, if the enzyme molecular structure is changed so that its affinity with the substrate NADH is reduced (Km value increases), under physiological conditions, the concentration of NADH in the cell is very low and the enzyme activity is almost completely lost; however, when the enzyme activity is measured in the test tube, the NADH added is equivalent to tens or even hundreds of times of the physiological concentration, and the enzyme activity can be measured completely. If the enzyme activity is measured at low substrate concentrations, its instantaneous initial velocity must be recorded with a time scan, otherwise the error is too large. Other genetic enzyme deficiency diseases have similar problems. Enzyme activity is stable in normal humans, and changes in enzyme activity indicate that certain enzymes have been released into the blood, urine, or body fluids following damage or disease in certain organs and tissues of the body. Therefore, the measurement of enzyme activity in blood, urine or body fluids can be used to understand or determine the onset and progression of certain diseases.