Fully-automated spectrophotometric method for measurement of antioxidant activity of catalase.
OBJECTIVE
Develop fully automated assay of antioxidant catalatic activity of catalase.


METHODS
The assay is based on standard, clinical chemistry automated analyzer methods for measuring hydrogen peroxide by using the Trinder reagent. Catalase competes with 324 U/L horseradish peroxidase (type XII) and Trinder reagent for hydrogen peroxide produced by 46 U/L uricase action on urate. Unit activity is defined as 50% inhibition of maximal color development.


RESULTS
Within-run coefficients of variation (cv) were 2% for standards and samples, whereas between-run cv was 3.1% for standards and 7.3% for samples. Dilutional parallelism and linearity were demonstrated for 8-fold dilutions of samples over the range 0.1 to 1.1 U/mL. Recovery of added catalase was complete. Samples are stable to freezing and storage for 1 week at -80 degrees C. Activities (units/mL) ranged from 0.29 to 0.41 in human and canine plasma, and for erythrocytes from 48 to 70 in man, 17 to 19 in dogs, and 60 to 89 in rats. Rat liver activity (units/g wet weight) was age-dependent and ranged from 17 to 24 at 2 months, and from 19 to 37 at 6 months.


CONCLUSIONS
The first, fully automated assay for the measurement of catalatic activity of catalase in plasma, erythrocytes, and liver is demonstrated for multiple species. The assay is simple, precise, relatively inexpensive, and rapid.
Slaughter
M R
MR
Safety Assessment, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR, UK.
O'Brien
P J
PJ
D000818:Animals; D000975:Antioxidants / Q000032:analysis*; D002374:Catalase / Q000032:analysis* / Q000378:metabolism*; D002624:Chemistry, Clinical / Q000379:methods; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000201:enzymology; D005615:Freezing; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis; D008099:Liver / Q000201:enzymology; D008297:Male; D008954:Models, Biological; D010949:Plasma / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012016:Reference Values; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D013997:Time Factors; D014527:Uric Acid / Q000494:pharmacology
D000975:Antioxidants; D014527:Uric Acid; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D002374:Catalase