Modification of Hemoglobin by Acetaldehyde: A Time Course Study by High Pressure Liquid Chromatography

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Abstract
Acetaldehyde forms stable adducts with proteins, and rapidly eluting hemoglobins on cation exchange chromatography have been found to be elevated in persons consuming excess alcohol. Incubation of hemoglobin hemolysate with 5 mM acetaldehyde at 37°C for various time intervals resulted in linear increases in the amounts of hemoglobin (Hb)A1a+b and HbA1c fractions determined by cation exchange high pressure liquid chromatography. The rate of formation of the HbA1c fraction was significantly higher (p < 0.001) than that of the HbA1a+b fraction. No increases in the amounts of minor hemoglobins were observed when hemoglobin was incubated with 0.05 mM acetaldehyde. Incubation of hemoglobin with 5 m acetaldehyde followed by reduction with sodium borohydride (NaBH4.) resulted in a significant increase in both HbA1a+b and HbA1c fractions. The rate of formation of the HbA1c fraction was again significantly faster than that of HbA1a+b. Dialysis of nonreduced acetaldehyde‐ modified hemoglobin had no effect on the amounts of the two minor hemoglobin fractions. Dialysis of NaBH4‐reduced acetaldehyde‐modified hemoglobin resulted in decreased amounts of the HbA1a+b fractions but no changes in the HbAlc fractions. Incubation with sodium cyanoborohydride led to minimal changes in chromatographic properties of hemoglobin. The clinical utility of acetaldehyde‐modified hemoglobin eluting in the HbA1c fraction in the detection of excess alcohol consumption appears to be limited by the high concentration of acetaldehyde required. Furthermore, attempts to stabilize acetaldehyde‐Schiff base adducts of hemoglobin with reducing agents must include appropriate controls, since the reductive step alone may lead to changes in the chromatographic properties of hemoglobin.