Risk of overestimation of free malondialdehyde in perfused rat hearts due to homogenization artifacts.
OBJECTIVE
The aims of the present study were to determine (1) whether free malondialdehyde (MDA) was artifactually formed during homogenization of myocardial tissue and (2) whether free MDA was increased in reperfused 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 presence of the antioxidant butylated hydroxytoluene (0.01%).


RESULTS
Tissue homogenates prepared in the absence of butylated hydroxytoluene contained significantly higher (P < 0.001) free MDA levels than tissue homogenates 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 (20 pmol/mg protein) in 27 of 30 tissue homogenates, irrespective of the perfusion protocol. Control experiments showed that the presence of butylated hydroxytoluene did not interfere with the detection of free MDA.


CONCLUSIONS
These results indicate that free MDA was formed artifactually during tissue homogenization in the absence of butylated hydroxytoluene. Furthermore, free MDA could not be detected in perfused rat hearts after control perfusion, or 20 min of ischemia, or 20 min of ischemia followed by 5 or 30 min of reperfusion.
Verbunt
R J
RJ
Department of Cardiology, University Hospital Leiden, Netherlands.
Egas
J M
JM
Van der Laarse
A
A
D000818:Animals; D000975:Antioxidants; D016477:Artifacts; D002084:Butylated Hydroxytoluene; D002851:Chromatography, High Pressure Liquid; D005189:False Positive Reactions; D008297:Male; D008315:Malondialdehyde / Q000032:analysis*; D017202:Myocardial Ischemia / Q000378:metabolism*; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar
D000975:Antioxidants; D002084:Butylated Hydroxytoluene; D008315:Malondialdehyde