Validation of gene expression data for the HO-1 pathway and p21. Gene expression levels are reflected as arbitrary units of fold-change of expression relative to mean control levels. Black lines in B, C, and D are regression lines. (A) Sickle cell disease patients (red bars) have increased mean gene expression (left y-axis) of all enzymes in the heme catabolism pathway and have higher mean serum total bilirubin (right y-axis), the end product of heme breakdown, compared with healthy volunteers (gray bars). Error bars reflect SEM. (B) Carbon monoxide production (determined by carboxy hemoglobin levels measured by co-oximetry) was measured in 24 patients with sickle cell disease (13 HbSS not on hydroxyurea therapy [▴], 8 HbSS on hydroxyurea [▪], 2 HbSC and 1 HbSβ-thalassemia phenotype [○]). Carbon monoxide production correlates with plasma heme levels measured by benzidine assay. (C) Carbon monoxide production correlates with HO-1 gene expression measured by microarray in these same 24 sickle cell disease patients. (D) Serum total bilirubin levels were measured in 27 sickle cell disease patients (14 HbSS not on hydroxyurea therapy [▴], 10 HbSS on hydroxyurea [▪], 2 HbSC and 1 HbSβ-thalassemia phenotype [○], and 13 controls [•]). Serum total bilirubin correlates with biliverdin reductase gene expression measured by microarray. Gene expression levels for biliverdin reductase also correlate with CO production (r = 0.55, P < .005, data not shown). (E) HO-1 and p21 gene expression measured by microarray and real-time PCR show increased expression in 27 sickle cell disease patients. Error bars reflect SEM. (F) Patients with sickle cell disease (n = 4) have increased cellular HO-1 protein levels in their peripheral blood mononuclear cells compared with healthy volunteers (n = 4) as measured by Western blot. Lane 1 represents an HO-1–positive control.