Kinetic methods for quantitation of hemoglobin and methemoglobin
Abstract
The development of two kinetic methods for quantitation of hemoglobin and methemoglobin is described. The kinetics of the hemoglobin-methemoglobin-cyanmethemoglobin chemical system are characterized and this information is used to establish conditions for the methods. In the first method, which is based on the reaction of cyanide ion with methemoglobin, methemoglobin and total hemoglobin concentrations are determined separately. Reaction conditions were adjusted so that pseudo-first order conditions were maintained and a curve-fitting method utilizing a first-order model was used to fit the progress curves. Both species can be quantified in a single experiment by the second method which is based on two parallel first-order reactions, the oxidation of hemoglobin and complexation of methemoglobin with cyanide ion. Both methods were developed using a stopped-flow mixing system and the first method was adapted to a slower, centrifugal-mixing instrument. Regression of determined concentrations of total hemoglobin obtained with the kinetic method for individual quantitation vs those obtained with an equilibrium method yields: slope = $1.00\pm 0.065$, intercept = 0.020 $\pm$ 0.014 mmol/L, s$\sb{\rm yx}$ = 0.06 mmol/L and r = 0.96 when stopped-flow mixing is used and slope = 1.03 $\pm$ 0.05, intercept = 0.12 $\pm$ 0.12 mmol/L, s$\sb{\rm yx}$ = 0.068 mmol/L and r = 0.96 with centrifugal mixing. The simultaneous kinetic method was compared to a multiwavelength spectral-fitting method developed, by this author, for determined concentrations of mHb, total hemoglobin and percent methemoglobin. Regression of determined concentrations of mHb yields, slope = 1.00 $\pm$ 0.04, intercept = $-$2.7 $\pm$ 1.5, r = 0.996 and s$\sb{\rm yx}$ = 0.043 mmol/L and regression of determined percentages of methemoglobin yields: slope = 0.961 $\pm$ 0.043, intercept = $-$2.5 $\pm$ 1.4, r = 0.996 and s$\sb{\rm yx}$ = 2.61%. The results for total hemoglobin cannot be compared by linear regression and are presented in tables in Chapter 3. The results for determined methemoglobin concentrations are better than those for percent methemoglobin because estimates of total hemoglobin concentration, used in the calculation of percentage, are poorer for the kinetic method. This is because less than optimal conditions for the oxidation reaction were used.
Degree
Ph.D.
Advisors
Pardue, Purdue University.
Subject Area
Analytical chemistry
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