Determination of in vivo carbon monoxide production in laboratory animals via exhaled air.
BACKGROUND
In vitro assays play an important role in the understanding of the heme oxygenase (HO)/carbon monoxide (CO) pathway. However, because physiological roles for the products of this pathway are hypothesized, it is becoming increasingly important to perform in vivo studies. Since CO production is primarily mediated by HO and is excreted mainly by the lungs, measurements of total body CO excretion (VeCO) via the breath allow continuous, noninvasive monitoring of heme degradation and CO and bilirubin production. Here, we describe a modified flow-through method for the collection and quantitation of CO from small laboratory animals.


METHODS
Mice and rats were studied in gas-tight chambers supplied with a continuous flow of CO-free air. CO in the exhaust air was measured by gas chromatography with a reduction gas analyzer. After establishing baseline VeCO levels, animals were administered various xenobiotics known to alter HO activity and further monitored for changes in CO production for up to 12 h without observable distress.


RESULTS
Administration of heme (substrate for HO) resulted in reproducible increases in CO production; whereas, prior administration of zinc protoporphyrin (ZnPP, HO inhibitor) or cobalt protoporphyrin (CoPP, HO inducer) resulted in respective dose-dependent decreases and increases in the heme-induced CO production.


CONCLUSIONS
We have demonstrated that this noninvasive method of CO quantitation reliably estimates heme degradation with sensitivity to distinguish between different types of HO-manipulating xenobiotics in a dose-dependant manner in both mouse and rat models. Furthermore, VeCO measurements allow nearly real-time determinations of CO and bilirubin formation, which helps to illustrate the time course of drug action.
Dercho
Ryan A
RA
Department of Pharmacology and Toxicology, Queen's University, Kingston, ON, Canada K7L 3N6.
Nakatsu
Kanji
K
Wong
Ronald J
RJ
Stevenson
David K
DK
Vreman
Hendrik J
HJ
D000818:Animals; D001944:Breath Tests / Q000295:instrumentation / Q000379:methods; D002248:Carbon Monoxide / Q000032:analysis* / Q000378:metabolism; D004867:Equipment Design; D045853:Exhalation; D006418:Heme / Q000494:pharmacology; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D051381:Rats; D017207:Rats, Sprague-Dawley
D006418:Heme; D002248:Carbon Monoxide; D006419:Heme Oxygenase (Decyclizing)