Haemoglobin interference in the bichromatic spectrophotometry of NAD(P)H at 340/380 nm

The negative bias observed in the NADPH-based bichromatic measurement of glucose in haemolysates (da Fonseca-Wollheim, F., Heinze, K.-G. & Liss, E. (1992) Temperature-dependent matrix effect in the direct enzymatic measurement of blood glucose, this journal 30, 371-375) is caused by shifts in the UV absorbance of haemoglobin which affect the absorbance difference delta A340/380 nm. In model experiments with haemoglobin solutions, spectral changes resulting in decreases of the absorbance at 340 nm and/or increases at 380 nm were found to occur for the following three reasons: 1. Oxidation of haemoglobin-O2 with formation of Fe(III) derivatives. Methaemoglobin formation is accelerated by lowering the pH, raising the temperature from 25 to 37 degrees C or by adding organic phosphates (inositol hexakisphosphate, ATP). At pH 6, addition of plasma increases the rate of methaemoglobin formation, while at pH values > 7, haemoglobin-O2 is stabilised. The oxidation of haemoglobin-O2 in the presence of sodium lauryl sulphate is also accompanied by a decrease of delta A340/380 nm. The haemichromes formed in this reaction exhibit stable UV light absorptivity. 2. Increase in the temperature of the haemoglobin-O2 solution. It is shown that the temperature-induced shifts in the haemoglobin-O2 absorptivity are reversible and that similar changes occur with the chemically more stable cyanomethaemoglobin. 3. Deoxygenation of haemoglobin-O2 at low pO2. Theoretically, the variation of factors influencing the pO2 (0.5) such as temperature, pH and allosteric effectors can also lead to changes in delta A340/380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)