Clinical Chemistry, Vol 33, 493-497, Copyright © 1987 by American Association for Clinical Chemistry

Fast kinetic method for methemoglobin developed and adapted for quantification of hemoglobin in whole blood

RH Cummings and HL Pardue

We studied the kinetics of the reaction of cyanide with methemoglobin (mHb), used the information to develop a fast kinetic method for quantifying methemoglobin, then used that method to quantify hemoglobin (Hb) in whole blood based on the reaction with ferricyanide to produce mHb. Reaction conditions for mHb and cyanide are adjusted to give pseudo-first-order behavior with an apparent rate constant that increases linearly with cyanide concentration, decreases linearly with pH, and follows an Arrhenius-type relationship with temperature. Absorbance change, computed from kinetic data with a curve-fitting method, varies linearly with mHb concentration. Although the reactions are monitored most reliably with stopped-flow mixing, cyanide concentration was used to decrease reaction velocities to be compatible with centrifugal mixing. Kinetic results (y) for Hb in blood by both mixing methods correlate well with equilibrium results (x): y = 1.000x + 0.02 mmol/L, Syx = 0.06 mmol/L with stopped-flow mixing; y = 1.03x + 0.12 mmol/L, Syx = 0.07 mmol/L with centrifugal mixing; r = 0.96 and n = 21 in each case.