LDL conditioned with heme arginate or hematin.

Heme arginate–conditioned LDL was a weaker inducer for heme oxygenase than hematin-conditioned LDL. (A) For heme oxygenase mRNA analysis, human umbilical vein endothelial cell monolayers were incubated for 1 hour with LDL (50 μg/mL protein) alone (second lane), LDL treated with H₂O₂ (6.25 μmol/L) (third lane), LDL treated with hematin (1.25 μmol/L) plus H₂O₂ (6.25 μmol/L) (fifth lane), and LDL treated with heme arginate (1.25 μmol/L) plus H₂O₂ (sixth lane) in HBSS, and then replaced with medium for 4 hours. For negative control (first lane), HBSS was free of lipoprotein, and for positive control (fourth lane), cells were exposed to hematin alone (10 μmol/L) for 1 hour followed by a 4-hour incubation with culture medium. RNA was isolated, electrophoresed, blotted, and hybridized with a ³²P-labeled heme oxygenase cDNA probe. (B) Autoradiograph was quantified by videodensitometry and expressed as arbitrary OD units. (C) Ethidium bromide–stained nylon membrane with the corresponding 18S and 28S rRNA for the above autoradiogram. (D) Heme oxygenase enzyme activity (picomole of bilirubin formed per milligram of cell protein per 60 minutes) was measured at 8 hours after exposure of endothelium to the same test solutions as for Northern blot in (A). Results represent enzyme activity (mean ± SE) of at least 3 experiments done in duplicate.P < .001 versus bar 5.