The collection of human blood from both patients and healthy volunteers is necessary for the development of laboratory assays required for studies of the role of nitric oxide, inflammatory mediators and endothelial function in inflammatory diseases that involve the blood vessels. ? ? This protocol continues to be used for the purposes stated, to collect blood from patients and volunteers for the development of laboratory assays. To date we have enrolled 210 individuals from the NIH site and collected blood numerous times to be used in experimental assays. Some of these samples have been used for gene expression studies and nitrite bioavailability studies. ? ? Glutathione peroxidase-1 (GPX-1) is the most significant catalytic antioxidant in the red cell. Congenital deficiency of GPX1 has been reported in association with hemolytic anemia due to oxidant stress. Hemolytic rate has been implicated in the development of sickle vasculopathy.? ? In another substudy, red cell pellets were collected from 32 subjects with sickle cell disease and 17 healthy African-American control subjects, and were stored frozen until assayed. A novel microplate immunoaffinity capture assay was used that measures both immunoreactive PGX1 protein and enzyme activity.? ? Subjects with SCD had higher levels of red cell GPX1 activity than controls (2.42 plus or minus 0.12 vs. 1.86 plus or minus 0.15 units/mg red cell protein (mean plus or minus SEM, p=0.006). PGX1 activity correlated with low reticulocyte count (r = minus 0.50, p = 0.004), low indirect bilirubin (r = minus0.45, p = 0.01) and high fetal hemoglobin expression (r = 0.39, p = 0.03). GPX1 activity in SCD subjects on hydroxyurea was much higher than those not on hydroxyurea (2.75 plus or minus 0.12, n = 12 vs. 1.79 plus or minus 0.13, n = 11, p less than 0.001), suggesting that hydroxyurea might induce GPX1 expression. In the SCD subjects off hydroxyurea, low GPX1 activity closely correlated with high serum lactate dehydrogenase level, a marker of high hemolytic rate (r = minus 0.85, p = 0.002), suggesting that low GPX1 levels might contribute to rapid hemolytic rate. In a multivariate model, GPX1 was independently associated with reticulocyte count even when adjusted for fetal hemoglobin level.? ? In addition to fetal hemoglobin, hydroxyurea may also induce expression ofGPX1, a potent antioxidant that might have a role in decreasing hemolysis associated with the robust oxidant stress of SCD. This finding is consistent with previously published evidence of GPX1 induction by hydroxyurea n cancer cell lines, with increased antioxidant function. Additional studies are needed to confirm this phenomenon in a larger cohort of patients with SCD and to further evaluate the combined effect of fetal hemoglobin co-induction, but this finding suggests a potential mechanism by which hydroxyurea may produce clinical benefit in SCD even in the absence of significant fetal hemoglobin induction.? ? In an additional substudy, we enrolled 15 subjects from Children's National Medical Center. We tested a number of compounds, present in BCAA metabolism, for their ability to up-regulate embryonic globin gene expression in mouse erythroleukemias cells, an adult murine erythroid cell.? ? We are interested in whether children with inherited disorders of branched-chain amino acid (BCAA) metabolism have an associated elevation in fetal hemoglobin. This hypothesis arose from the observation that children with a deficiency in a pathway-distal enzyme for BCAA metabolism resulting in propionic acidemia (PA) had levels of fetal hemoglobin that were higher for longer than was seen in metabolically normal children.? ? In this study, we have investigated children with a wider range of disorders in BCAA metabolism. The BCAAs are isoleucine, leucine and valine. The metabolic disorders investigated here included maple syrup urine disease (MSUD), which involves the first metabolic step after deamination for all 3 BCAAs, isovaleric acidemia (IVA), which involves the second step of deamination for leucine, and methylmalonic acidemia (MMA) and propionic academia which involve, respectively, the penultimate and antepenultimate enzymes for isoleucine and valine metabolism to succinyl-CoA.? ? In 10 subjects, 3 to 24 years of age, with disorders of intermediary BCAA metabolism, there was a modest, but statistically significant, elevation in HbF in patients with IVA, PA, or MMA than was seen in patients with MSUD, at 0.9 % vs. 0.25% (p= 0.017, figure 1), when analyzed by HPLC. ? ? Further, we found that the predominant gamma globin chain in patients with BCCA-abnormalities was gamma-2, gG at 8-12% of total betaglobin chains (gamma + beta, figure 2), although less meaningful in a single patient with Hb Sb-thalassemia. However, no gamma-2 globin was detectable in patients with MSUD.? ? It is likely that metabolic stability (that is, level of control and therefore level of intermediary metabolites) may be relevant to the more mild induction of HbF seen here than in earlier studies.? ? These studies have not been associated with any morbidity to date. We plan to continue these investigations over the next year, so that we might extend these measurements to additional subjects, with a particular emphasis those with MSUD. It is expected that this will complete the investigation.
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