RISK OF OVERESTIMATION OF FREE MALONDIALDEHYDE IN PERFUSED RAT HEARTSDUE TO HOMOGENIZATION ARTIFACTS

Objectives: The aims of the present study were to determine (1) whethe r free malondialdehyde (MDA) was artifactually formed during homogeniz ation of myocardial tissue and (2) whether free MDA was increased in r eperfused rat hearts. Methods: Groups of isolated buffer-perfused rat hearts were subjected to control perfusion, or 20 min of ischemia, or 20 min of ischemia followed by 5 or 30 min of reperfusion. The hearts were subsequently assayed for free MDA by ion-pairing high-performance liquid chromatography following homogenization in the absence or pres ence of the antioxidant butylated hydroxytoluene (0.01%). Results: Tis sue homogenates prepared in the absence of butylated hydroxytoluene co ntained significantly higher (P < 0.001) free MDA levels than tissue h omogenates from the same hearts prepared in the presence of butylated hydroxytoluene. Free MDA levels of tissue homogenates prepared in the presence of butylated hydroxytoluene were below the detection limit (2 0 pmol/mg protein) in 27 of 30 tissue homogenates, irrespective of the perfusion protocol. Control experiments showed that the presence of b utylated hydroxytoluene did not interfere with the detection of free M DA. Conclusions: These results indicate that free MDA was formed artif actually during tissue homogenization in the absence of butylated hydr oxytoluene. Furthermore, free MDA could not be detected in perfused ra t hearts after control perfusion, or 20 min of ischemia, or 20 min of ischemia followed by 5 or 30 min of reperfusion.