The Mechanism of Ascorbic Acid Interference on the Determination of Hydrogen Peroxide with Catalase
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Ascorbic acid (AH<SUB>2</SUB>) is well known to give false positive errors in the determination of hydrogen peroxide (H<SUB>2</SUB>O<SUB>2</SUB>) with the catalase-Hantzsch reaction which involves the formation of formaldehyde from H<SUB>2</SUB>O<SUB>2</SUB> and methanol, followed by the color development based on the condensation reaction of formaldehyde formed and acetylaceton added in the presence of ammonium ion.<BR>Present study was undertaken to elucidate a possible mechanism of the AH<SUB>2</SUB> interference in the H<SUB>2</SUB>O<SUB>2</SUB> determination. Dehydro-ascorbic acid (DA) was prepared by use of immobilized ascorbate oxidase-tube. The oxidation-reduction relationship between AH<SUB>2</SUB> and DA was investigated with spectral analysis and using the Hantzsch reaction and aldehyde dehydrogenase reaction.<BR>1.AH<SUB>2</SUB> continuously increases the color development of the catalase-Hantzsch reaction in the absence of H<SUB>2</SUB>O<SUB>2</SUB>, suggesting the formation of H<SUB>2</SUB>O<SUB>2</SUB> or formaldehyde in the reaction mixture.<BR>2.AH<SUB>2</SUB> is oxidized by a sparing amount of oxygen and Cu<SUP>2+</SUP> to form H<SUB>2</SUB>O<SUB>2</SUB> and DA. <BR>3.DA formed reacts with methanol even in the absence of catalase to regenerate AH<SUB>2</SUB> and formaldehyde. H<SUB>2</SUB>O<SUB>2</SUB> reacts with methanol in the presence of catalase to form formaldehyde. The formaldehyde increases the color development of the Hantzsch reaction.<BR>4.AH<SUB>2</SUB> regenerating system (AH<SUB>2</SUB>, O2 and methanol) continuosly supplies H<SUB>2</SUB>O<SUB>2</SUB> and formaldehyde which are used as substrates of the catalase-Hantzsch reaction and then increase the color development. The coupled reaction of DA reduction (DA to AH<SUB>2</SUB>) and methanol oxidation (methanol to formaldehyde) seems to play a most important role in AH<SUB>2</SUB> interference.
- Japan Society of Clinical Chemistryの論文
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