A gas-liquid system for enzyme kinetic studies of volatile organic chemicals. Determination of enzyme kinetic constants and partition coefficients of trichloroethylene.
A gas-liquid system was developed for enzyme kinetic study with volatile organic chemicals (VOCs) by modification of the gas uptake method for the in vivo physiologically based pharmacokinetic experiment. This gas-liquid system, designed in our laboratory, is composed of: 1) a diffusion chamber for adjusting initial vapor concentration by mixing ambient air and the VOCs; 2) a condenser for maintaining the liquid level in the incubation chamber; 3) a stainless-steel metal bellows pump for recirculating vapor in this system; 4) a gas chromatograph equipped with an autosampler and a flame ionization detector; and 5) a computer for controlling automation and data processing. Trichloroethylene (TCE) was used as a model chemical, and enzyme kinetics were studied by measuring the depletion of TCE in the gas phase of the system. TCE-at initial concentrations of 56, 620, and 1240 ppm-was incubated with rat liver microsomes and a NADPH regenerating system in a 100-ml round-bottom flask. Based on parallel enzyme assays using p-nitrophenol as a substrate, cytochrome P450IIE1, activity remained stable up to 3 hr under the incubation conditions (37 degrees C and pH 7.4) whereas addition of glutathione into the incubation mixture did not affect TCE metabolism. Kinetic constants were analyzed using a two-compartment pharmacokinetic model and the computer software SimuSolv. Statistical optimization using the maximum-likelihood method produced apparent in vitro Vmax and KM values of 0.55 nmol/mg protein/min and 0.9 microM, respectively. In addition, this newly developed methodology has a number of advantages over those reported in the literature, including the potential utility of determining tissue partition coefficients of VOCs for physiologically based pharmacokinetic modeling. We conclude that this gas-liquid system is suitable for determination of kinetic constants near realistic environmental concentrations of VOCs including TCE.
Hwang
I Y
IY
Department of Environmental Health, Colorado State University, Fort Collins 80523-1680, USA.
Reardon
K F
KF
Tessari
J D
JD
Yang
R S
RS
D000818:Animals; D046508:Culture Techniques / Q000379:methods; D004798:Enzymes / Q000378:metabolism*; D005740:Gases / Q000032:analysis; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008956:Models, Chemical; D051381:Rats; D011916:Rats, Inbred F344; D014241:Trichloroethylene / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*
D004798:Enzymes; D005740:Gases; D014241:Trichloroethylene