Quantitative determination of carbon monoxide in cell culture supernatants by spectrophotometric analysis

Abstract

A simple, sensitive, nonradioactive method for measuring carbon monoxide (CO) in cell culture supernatant is described. Dissolved CO reacts with hemoglobin (Hb) to carboxyhemoglobin (HbCO) in a modified Conway cell. HbCO is quantified by spectrophotometric analysis, and total concentration of CO given in μg CO/l cell culture supernatant is mathematically calculated. Furthermore, we compared our newly developed method with a recently published method. Confluent human umbilical vein epithelial cells (HUVEC) were incubated with 10 −6 M hydrocortisone known to induce heme oxygenase-2 protein and transcript expression for 4 h and CO production was measured. Levels following hydrocortisone treatment were significantly enhanced compared to controls when using our newly developed technique ( p<0.05), whereas only a nonsignificant trend could be observed using the recently published method. We conclude that this nonradioactive technique to quantify CO is more sensitive than previous ones, thereby allowing to measure even physiologic quantities of CO in aqueous solutions.