Determination of Digitoxin-induced Elevated Hydrogen Peroxide Levels in SK-Hep-1 Cells Using a Chemiluminescent Method
JENG-SHU WEI1,2,4*, HUI-LING HUANG1, CHUN-YI KAO2, CHANG-HUI LIAO3 AND KUO-WEI CHANG4
1. Department of Medical Biotechnology and Laboratory Sciences,
2. Graduate Institute of Basic Medical Sciences,
3. Graduate Institute of Natural Products,
4. Graduate Institute of Biochemical and Biomedical Engineering,
Chang Gung University, 259 Wenhwa 1st Rd., Kweishan, Taoyuan 333, Taiwan (R.O.C.)
(Received: January 10, 2007; Accepted: June 3, 2007)
ABSTRACT
Drug-induced intracellular hydrogen peroxide overproduction is proposed as an upstream event in apoptosis signaling. It is therefore important to develop a sensitive method to quantify hydrogen peroxide in biological samples undergoing cell death. We report herein a luminol-based chemiluminescent assay with a suitable detection range of best linearity from 1 to 0.1 μM hydrogen peroxide with a CV range of 5.7% to 21.7% for intraday measurement at the detection range. We hypothesized that digitoxin-treated human hepatoma SK-Hep-1 cells produce hydrogen peroxide to cause cytotoxicity, which was verified by measuring the spent media by our chemiluminescent method in a dose-dependent manner. Taken together, our sensitive luminol-based chemiluminescent assay has potential use in the quantification of trace amounts of digitoxin overdose-induced hydrogen peroxide in biological samples.
Key words: digitoxin, SK-Hep-1 cells, hydrogen peroxide, chemiluminescence