Method for quantification of myocardial infarction and inflammatory cell infiltration in rat cardiac tissue.
A method to quantitate both creatine phosphokinase (CPK) and myeloperoxidase (MPO) activity from the same cardiac tissue homogenate preparation is described. Depletion of CPK specific activity is used to quantitate myocardial infarct size, while MPO activity is utilized as a marker for polymorphonuclear leukocyte and monocyte infiltration into inflammatory sites. However, the standard assay systems are not compatible, necessitating the use of different groups of animals for these two parameters. This leads to an increase in cost, effort, and variability. The described method utilized a standard CPK methodology. It was found that interference in the MPO assay was likely caused by 2-mercaptoethanol present in the homogenate buffer (IC50 = 90 microM). Washing of the 30 K X g pellet followed by rehomogenization restored the MPO activity. Negligible MPO activity was found in the original supernatant or washes. Through the use of this technique, MPO activity was measured in the hearts of myocardial infarcted animals. The results indicated that MPO activity generated from CPK homogenate pellets compared favorably to the activity seen using standard methodology homogenates. The procedure described thus allowed the simultaneous determination of myocardial CPK specific activity and MPO activity, resulting in decreased animal usage and potentially less variability.
Griswold
D E
DE
Department of Immunology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406.
Hillegass
L M
LM
Hill
D E
DE
Egan
J W
JW
Smith
E F
EF
D000818:Animals; D003402:Creatine Kinase / Q000378:metabolism; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D009203:Myocardial Infarction / Q000473:pathology*; D009206:Myocardium / Q000201:enzymology / Q000473:pathology*; D009195:Peroxidase / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D008623:Mercaptoethanol; D009195:Peroxidase; D003402:Creatine Kinase