The effect of Hb F and alpha-thalassemia on the red cell indices in sickle cell anemia.

This study examines the effect of different levels of fetal hemoglobin (Hb F) and the presence or absence of genes for alpha-thalassemia on the red cell indices and degree of anemia among 102 patients with homozygous sickle cell disease (S/S) between the ages of 15 and 62 years. Patients were divided into those with an average Hb F of less than 10 gm/L ("low" Hb F group) and those with greater than 10 gm/L ("high" Hb F group). alpha-Thalassemia was assessed by restriction enzyme analysis of DNA by the Southern blotting technique. Homozygosity for the beta(s) gene was confirmed by restriction enzyme analysis of DNA using the enzyme Mst II. There were 51 patients with four alpha-globin genes, 28 of whom had "high" and 23 "low" Hb F levels. Fifty-one patients had alpha-thalassemia, 38 of whom were heterozygous and 13 homozygous for the 3.7 kb alpha-thalassemia deletion. Nine had "high" and 31 had "low" Hb F. Irrespective of alpha-globin genotype, patients in the high Hb F group had a higher mean Hb, Hct, MCV, and MCH than those in the low HB F group. In patients without alpha-thalassemia Hb F was positively correlated with MCV and MCH (p less than 0.001), patients with high Hb F levels having macrocytosis confirmed by microhematocrit studies. Patients with alpha-thalassemia had a lower MCHC than patients with four alpha-globin genes and this was not significantly affected by the level of Hb F. The combination of alpha-thalassemia and high levels of Hb F appears to result in a distinctive S/S phenotype that is similar to the type of S/S disease described in Southern India.

Use of biotinylated probes for detecting sickle cell anemia.

Earlier, we reported that the 5' end of the normal beta-globin gene (beta) resides on a 1.14-kilobase DNA fragment, whereas the 5' end of the sickle cell gene (beta s) resides on a 1.34-kilobase fragment. In that blot hybridization analysis, we used genomic DNA digested with restricted endonuclease Mst II, and radioactive probes with short half-life. We demonstrate here that, if a biotinylated probe is used instead in a slightly modified procedure, sickle cell anemia can be quickly and directly detected if there is as much as 5 micrograms of total genomic DNA in the sample. This procedure obviates the special precautions necessary when radioactive materials are used.

Modification of sickle hemoglobin by acetaldehyde and its effect on oxygenation, gelation and sickling.

Erythrocytes from sickle cell anemia patients and chromatographically purified Hb S0 were incubated with 0.25 to 120 mM acetaldehyde for 15 min to 6 h at different temperatures and pH. Several hemoglobin adducts stable to dialysis were separated by Biorex 70 chromatography and the proportions of the adducts formed were dependent on the period of incubation, acetaldehyde concentration, pH and temperature. Acetaldehyde treatment showed an increase in solubility, minimum gelling concentration and oxygen affinity and a decrease in sickling which showed a dependence on Hb S modification. With 0.25 to 1 mM acetaldehyde, significant inhibition of sickling was observed without any effect on the physical characteristics of the hemoglobin molecule. Acetaldehyde may act as a gelation inhibitor as well as a cell sickling inhibitor.

Synthesis of the minor fetal hemoglobin Fic in colonies of erythropoietic precursors isolated from human umbilical cord blood.

The biosynthesis of the minor hemoglobin FIc, which contains acetylated gamma chains, and the major hemoglobin Fo was studied during erythroid cell differentiation and maturation in cultures of erythroid precursors isolated from five human umbilical cord blood samples. A gradual decrease in the synthesis of Hb FIc was observed during the maturation of the erythroid cells when cultured from 7 to 15 days. The synthesis of Hb Fo did not show a consistent change; however, the relative synthesis of Hb FIc was the highest in the 7-day-old colonies and decreased at day 15 to levels observed in the reticulocytes. The addition of sodium butyrate, which is known to promote histone acetylation, significantly increased the synthesis of Hb FIc in 7-day-old colonies.

Non-enzymatic acetylation of human hemoglobins.

Chromatographically purified Hb F0, Hb A0 and Hb S0 fractions were incubated with 70 microM [14C]acetyl-CoA for 3 h in 20 mM Tris-HCl, pH 7.6 and 8.6. The acid-precipitable radioactivity was monitored and the hemoglobins were separated by Biorex 70 chromatography. The pH dependence of acetylation showed an increase in acetylation with an increase in pH from 7.6 to 8.6, whereas an increase in ionic strength decreased acetylation. Incubation of Hb F0 with [14C]acetyl-CoA resulted in modified hemoglobin and gamma chains that co-chromatographed with Hb FIc and gamma 1c chains, respectively. Acetylation of Hb A0 and Hb S0 produced minor hemoglobins whose chromatographic mobilities were slightly faster than those of Hb AIc and Hb SIc, respectively, Radioactivity peaks also appeared at the leading edges of the major hemoglobin zones as well, which indicates that, like non-enzymatic glycosylation, non-enzymatic acetylation of hemoglobins involves both specific and nonspecific reactions.

Oxygen binding of fetal and adult bovine hemoglobin in the presence of organic phosphates and uric acid riboside.

The P50 values of "stripped" fetal and adult bovine hemoglobin were 18.4 and 28.9 respectively. Neither the oxygen-hemoglobin dissociation curve nor the Hill coefficient, n, of fetal or adult bovine hemoglobin was affected by uric acid riboside (UAR), 2,3-diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), or inositol pentaphosphate (IPP). Combinations of UAR and ATP with adult bovine hemoglobin or 2,3-DPG and ATP with fetal hemoglobin also had no effect. It was concluded that neither adult nor fetal bovine red cells contained an identifiable compound which affects the binding of oxygen to hemoglobin.