Quality control material containing hemoglobin for blood gas and pH measurement: Improvement of the stability of stroma-free hemoglobin solution

Abstract

In stroma-free hemoglobin solution (SFHS) formation of methemoglobin (hemiglo-bin; Hi) occurs over a period of some months, due to the fact that Hi reduction stops in hemolysates. SFHS should contain active hemoglobin (Hb), which is able to bind oxygen and should not contain inactive Hb (Hi, carboxyhemoglobin) which does not bind oxygen. Reversible binding of oxygen by Hb is only possible when the molecule is in its reduced (Fe⁺⁺) form. In red blood cells (RBC) Hb is in the reduced form. The formation of Hi, which contains Fe⁺⁺⁺ as a result of Hb oxidation, is the first step in Hb degradation. This step is reversible in RBC.

Previously, we have described the preparation of SFHS containing the methemoglobin reductase (MR) system of RBC. To improve the stability of SFHS, we first investigated the formation of Hi as a function of pH and ionic strength and quantified the MR activity in SFHS. Non-enzymatic Hi reduction was studied with substances as ascor-bate and glutathione. Stimulation of MR by EDTA was tested. Inhibition of Hi formation was studied with nicotinic acid amide in the presence and absence of NADH. It is concluded that ascorbate and glutathione are not effective during extended periods of storage of SFHS, and that EDTA causes formation of large amounts of Hi. Nicotinic acid amide did not inhibit Hi formation. NADH, as a substrate for the MR system, is very effective in keeping Hi low.