Minor Hemoglobin Research Articles

Minor fetal hemoglobins in relation to gestational age

Cord blood samples were obtained at delivery in 44 normal women, at gestational periods ranging from 27 to 41 weeks. All women had a normal glucose screening test and 15 were delivered preterm. With the use of Biorex 70 chromatography, red cell lysates analyzed to determine the percentages of the different hemoglobin (Hb) components, i.e., Hbfla+b, HbFlc, Hbfo, and Hl)A. The percentage of HbFla+b in fetal blood was higher than the corresponding percentage in adult blood. The percentages and ratios of the various Hb components were compared in preterm and term fetuses and their correlation to gestational age was tested. HbF and HbF/ A ratios were higher in preterm fetuses and were negatively correlated with gestational age. HbA and HbA/F ratios were lower in preterm fetuses than in term fetuses and correlated positively with gestational age. The percentages of minor components Hbfla+b, and HbFlc were not significantly different in the preterm and the term fetuses, and they did not have statistically correlation with gestational age. However, the ratios of these minor hemoglobins to HbF, i.e., HbFla+b/HbF, and HbFlc/HbF and HbFl/HbF were found to be significantly higher in term fetuses as compared to preterm fetules and to correlate positively with gestational age. This is probably due to the gradual lengthening of the life span of fetal erythrocytes as term approaches, which results in the erythrocytes increased exposurte to the glycolytic intermediates and possibly to glucose. (Am. J. Obstet. Gynecol. 141:704, 1981.)

14] Preparation of hemoglobin carbamylated at specific NH2-terminal residues

Publisher Summary This chapter focuses on the carbamylation of the amino groups of hemoglobin. The procedure described is only one of three methods that can be employed to prepare gram amounts of hemoglobin carbamylated at specific NH 2 -terminal positions. Hemoglobin obtained from a lysate of fresh erythrocytes in the oxygenated form is the starting material; purification to homogeneity is achieved later by the chromatography of the separate α and β chains after carbamylation. Blood from normal individuals, from patients with sickle cell anemia, or from patients heterozygous for abnormal hemoglobin Providence (Lys- β 82 → Asn or Asp) has been used successfully in the procedure. The chapter describes the use of specifically carbamylated hemoglobin tetramers, removal of minor hemoglobins, preparation and recombination of hemoglobin chains, and conversion of carbon monoxyhemoglobin to oxyhemoglobin. For the isolation of the hemoglobin chains, a modification of the procedure of Bucci and Fronticelli is used.

A murine model for the switch from fetal to adult hemoglobin during ontogeny

In murine erythroleukemia cells, the minor/major hemoglobin (Hb) ratio depends on the cell line and the inducing agent. To determine whether mouse minor hemoglobin is a “fetal” hemoglobin in vivo, globin chain composition and synthesis rates were determined in DBA/2 mice of various ages ranging from 14-day embryos to > 6-mo adults. Globin chains were separated by electrophoresis on polyacrylamide gels containing urea and Triton X-100. This method separates the embryonic (x,y,z) and the adult (alpha, beta ma, beta mi) globin chains. Fourteen day embryos had only 5%-10% adult globins but approximately 30% of the adult beta chains were beta mi. The % beta mi decreased with age and reached 20% in adult mice. Biosynthetic studies led to more pronounced differences: beta mi synthesis was 45% of total beta chain production in 14-day embryos and declined to 22% in adults. Thus beta minor/total beta globin synthesis declines during mouse ontogeny. This resembles qualitatively the human switch from fetal to adult hemoglobin and provides a murine model for studies of hemoglobin regulation.

A Murine Model for the Switch From Fetal to Adult Hemoglobin During Ontogeny

In murine erythroleukemia cells, the minor/major hemoglobin (Hb) ratio depends on the cell line and the inducing agent. To determine whether mouse minor hemoglobin is a "fetal" hemoglobin in vivo, globin chain composition and synthesis rates were determined in DBA/2 mice of various ages ranging from 14-day embryos to > 6-mo adults. Globin chains were separated by electrophoresis on polyacrylamide gels containing urea and Triton X-100. This method separates the embryonic (x,y,z) and the adult (alpha, beta ma, beta mi) globin chains. Fourteen day embryos had only 5%-10% adult globins but approximately 30% of the adult beta chains were beta mi. The % beta mi decreased with age and reached 20% in adult mice. Biosynthetic studies led to more pronounced differences: beta mi synthesis was 45% of total beta chain production in 14-day embryos and declined to 22% in adults. Thus beta minor/total beta globin synthesis declines during mouse ontogeny. This resembles qualitatively the human switch from fetal to adult hemoglobin and provides a murine model for studies of hemoglobin regulation.

Minor hemoglobins in sickle-cell heterozygotes and homozygotes with and without diabetes

Minor hemoglobins in the red cell lysates of Hb S heterozygotes (AS) and homozygotes (SS) with and without juvenile-onset diabetes were separated by Biorex 70 column chromatography. These minor hemoglobins were identified as Hb A Ia+b, Hb A Ic, Hb S Ia+b, and Hb S Ic in AS persons and Hb F I, Hb F 0, Hb S Ia+b, and Hb S Ic in SS patients. The levels of all four components were elevated 2 to 3 fold in AS persons with diabetes. Likewise, the percentages of Hb S Ia+b and Hb S Ic were significantly increased in a diabetic SS patient. The proportions of the Hb A I and Hb S I components in the AS persons were nearly the same, indicaing that Hb A and Hb S are glycosylated in vivo to about the same extent. In vitro studies confirm these findings. It was also confirmed by in vitro studies that the glycosylation of hemoglobin involves both specific and nonspecific binding of carbohydrate to α and β chains.

Identification of hemoglobin E by the isopropanol solubility test

Hemoglobin (Hb) E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid βE-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of βE globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/β (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.

Functional properties of the glycosylated minor hemoglobins A 1a-1, A 1a-2 and A 1b. EPR evidence for increased stability of the low affinity quaternary structure and decreased susceptibility to organic phosphate

Electron paramagnetic resonance (EPR) spectra of the glycosylated minor hemoglobins A 1a-1, A 1a-2, A 1b and A 1c and the major hemoglobin A 0 in the nitrosyl form have been obtained in the absence and presence of inositol hexaphosphate. In the absence of inositol hexaphosphate, nitrosyl hemoglobins A 1a-1, A 1a-2 and A 1b exhibited a triplet hyperfine structure centered at g = 2.009 which has been shown to be diagnostic of the low affinity (T) quaternary structure. Addition of inositol hexaphosphate to nitrosyl hemoglobins A 0, A 1c, A 1b and A 1a-2 developed a triplet hyperfine structure of the EPR spectra but the magnitude of the hyperfine was decreased in the order of hemoglobins A 0, A 1c, A 1b and A 1a-2. However, inositol hexaphosphate had essentially no effect on the EPR spectrum of nitrosyl hemoglobin A 1a-1. The present results account qualitatively for the oxygen binding properties of these glycosylated minor hemoglobins in the framework of a two-state allosteric model.

Structural analysis of the minor human hemoglobin components: Hb AIa1, Hb AIa2 and Hb AIb.

Human hemolysate contains several minor hemoglobin components, including Hb AIa1, Hb AIa2, Hb AIb and Hb AIc which are post-translational modifications of the major component, Hb A0. Hb AIc is known to contain glucose attached to the N terminus of the beta chains by a ketoamine linkage. We separated the alpha and beta globin chains from purified Hb AIa1, Hb AIa2 and Hb AIb by ion-exchange chromatography. The beta chains were reducible by sodium borohydride and gave a positive thiobarbituric acid test. These results indicated that they are modified by ketoamine-linked carbohydrate. In addition, phosphate analysis revealed 1.5 phosphate residue associated with each beta AIa1 chain and 1 phosphate residue with each beta AIa2 chain. Hb AIa1, Hb AIa2 and Hb AIb were all found to be contaminated by non-globin proteins. Protein-sequencing approaches demonstrated that the N termini of beta AIa1, beta AIa2 and beta AIb were blocked. In support of this conclusion, analysis of tryptic digests of beta AIa2 and B AIb revealed modified N-terminal peptides. We conclude that, like Hb AIc, components Hb AIa1, Hb AIa2 and Hb AIb also contain a sugar moiety linked to the N terminus of the beta chain.

Glycosylation and acetylation of hemoglobin in infants of normal and diabetic mothers.

The minor hemoglobins, which are formed post-translationally by acetylation or glycosylation, were studied in cord blood from 34 newborn infants. There were 9 normal infants, 8 infants of gestational (chemical) diabetics, and 17 infants of insulin-dependent diabetics. Among these groups, no significant differences in the acetylated fetal (FI) or other minor hemoglobin fractions were found by either isoelectric focusing in polyacrylamide gels or chromatography on DEAE-cellulose columns. These studies emphasize the technical limitations of detecting small amounts of glycosylated hemoglobin in newborns. The biological implications of the acetylated fetal hemoglobin fraction remain obscure.

Frequency and distribution of structural variants of hemoglobin and thalassemic states in Western Japan.

Hemolysates from 100,000 people who visited the Kyushu University Hospital and affiliated hospitals during the past 15 years were screened for hemoglobinopathies using electrophoresis on thin-layer starch gel; those exhibiting an abnormality were characterized further on clinical, biochemical, and genetic grounds. Of about 97,000 adult and 3,140 cord blood samples, 29 contained electrophoretically detectable abnormalities in the heterozygous condition. Another 17 samples had quantitative changes in the levels of the minor hemoglobin components. Of the thalassemic conditions, 12 involved beta-thalassemia, 3 alpha-thalassemia, 1 delta beta-thalassemia, and 1 delta-thalassemia. Among 45 carriers of beta-thalassemia from 12 families, 5 were noted to have thalassemia intermedia since they exhibited much more severe hemolytic syndromes than those with typical beta-thalassemia minor. The frequency with which we could detect a structural variant of Hb A in the adults by electrophoresis was one in 3,800 samples. About one in 8,000 carried a beta-thalassemia gene.

Hemoglobin A1 and Diabetic Retinopathy

The levels of the minor hemoglobin A1 components were measured in a consecutive series of 102 diabetic patients who were extensively studied for signs of diabetic retinopathy. We found a statistically significant relationship between metabolic control, as reflected by the hemoglobin A1 level, and the severity of diabetic retinopathy in patients with diabetes diagnosed before 30 years of age (P less than or equal to .001). We did not demonstrate a significant correlation between metabolic control and the severity of retinopathy in patients with diabetes diagnosed after the age of 30 years. We found significantly more severe retinopathy among patients with longer duration of the disease, in men, in whites, in diabetics diagnosted before 30 years of age who were treated with lower insulin doses, and in obses patients with the onset of diabetes after the age of 30 years.

Use of the minor hemoglobin ratio for the determination of gestational age

Maternal and cord blood hemoglobin A1c were quantitated at the time of delivery by radioimmunoassay. Other cord blood hemoglobins were quantitated by column chromatography using Bio-Rex 70. The minor hemoglobin ratio : formula: (see text),showed an excellent linear correlation (r2 = 0.88) with gestational age. Therefore, the ratio of : formula: (see text), could be a useful test to determine gestational age in the third trimester.

Synthesis of hemoglobin Aic and related minor hemoglobin by erythrocytes. In vitro study of regulation.

Factors that influence hemoglobin (Hb)A(Ic) synthesis by intact erythrocytes were studied in vitro. After incubation cells were lysed, and hemoglobins were separated by isoelectric focusing on polyacrylamide slab gels and quantitated by microdensitometry. HbA(Ic) increased with time, glucose concentrations (5-500 mM), and incubation temperature (4 degrees -37 degrees C). Low temperatures allowed prolonged incubations with minimal hemolysis. At 4 degrees C HbA(Ic) increased linearly with time for 6 wk; after incubation at the highest glucose concentration, HbA(Ic) comprised 50% of total hemoglobin. Insulin (1 and 0.1 mU/ml) did not affect HbA(Ic) synthesis in vitro. In addition to glucose, galactose and mannose, but not fructose, served as precursors to HbA(Ic). A good substrate for hexokinase (2-deoxyglucose) and a poor hexokinase substrate (3-O-methylglucose), were better precursors for HbA(Ic) synthesis than glucose, suggesting that enzymatic phosphorylation of glucose is not required for HbA(Ic) synthesis. Autoradiography after erythrocyte incubation with (32)P-phosphate showed incorporation of radioactivity into HbA(Ia1) and A(Ia2), but not HbA(Ib), A(Ic), or A. Acetylated HbA, generated during incubation with acetylsalicylate, migrated anodal to HbA(Ic) and clearly separated from it. Erythrocytes from patients with insulinopenic diabetes mellitus synthesized HbA(Ic) at the same rate as controls when incubated with identical glucose concentrations. Likewise, the rate of HbA(Ic) synthesis by erythrocytes from patients with cystic fibrosis and congenital spherocytosis paralleled controls. When erythrocytes from cord blood and from HbC and sickle cell anemia patients were incubated with elevated concentrations of glucose, fetal Hb, HbC, and sickle Hb decreased, whereas hemoglobins focusing at isoelectric points near those expected for the corresponding glycosylated derivatives appeared in proportionately increased amounts.

5-aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced friend erythroleukemic cells

Porphyrin synthesis and iron accumulation was stimulated by exogenous 5-aminolevulinic acid (ALA) in uninduced Friend erythroleukemic cells (FELC). Uroporphyrin and protoporphyrin were the major intermediate precursors produced. All porphyrin types were conjugated to protein insoluble cellular components and could be extracted only by methanol sulfuric acid esterification. Heme content of the uninduced FELC was increased 6-fold in the presence of 5 × 10 −4 M ALA. As a consequence, the synthesis of the minor murine hemoglobin component was preferentially induced, an effect similar to that expressed by exogenous hemin. Addition of exogenous ALA to 0.5% DMSO-induced cells increased total hemoglobin synthesis with a higher efficiency of the minor hemoglobin. The endogenous synthesis of porphyrin from exogenous ALA was markedly reduced by hemin. Uroporphyrin, coproporphyrin, protoporphyrin and heme were equally repressed, indicating an inhibitory effect of hemin on ALA dehydrase and urosynthetase activities. In addition, hemin repressed [ 3H]leucine incorporation into protein by uninduced cells. Incubation of uninduced cells in culture medium without serum in the presence of hemin blocked their protein synthesis activity, whereas addition of serum exerted a protective effect on living FELC.

Role of nonenzymatic glycosylation in the development of the sequelae of diabetes mellitus

The increased level of the glycosylated hemoglobin (hemoglobin A Ic) in the diabetic patient has proved to be an interesting clue to understanding the biochemical basis of the sequelae of diabetes. This minor hemoglobin, which arises as nonenzymatic postsynthetic addition of glucose to hemoglobin A, actas as an indicator molecule for the glucose environment over a 3–5-wk period prior to measurement. Reasoning that a similar glycosylation reaction could be occurring with other body proteins, we have studied the ocular lens. The lens, like the erythrocyte, is not dependent on insulin for glucose concentration in the extracellular milieu that would be elevated in the diabetic state. These studies have revealed that a high glucose in vivo or an increased glucose or glucose-6-phosphate concentration in vitro leads to the glycosylation of ε-amino groups of lysine residues in bovine and rat lens crystallins. This glycosylation imparts an increased susceptibility of the crystallins to sulfhydryl oxidation. Disulfide crosslinks result in the formation of high molecular weight aggregates and an opalescence of the cyrstallin solutions.

Nonenzymatic glycosylation of human hemoglobin at multiple sites

The most abundant minor hemoglobin component of human hemolysate is Hb A 1c, which has glucose bound to the N-terminus of the β chain by a ketoamine linkage. Hb A 1c is formed slowly and continuously throughout the 120 day lifespan of the red cell. It can be synthesized in vitro by incubating purified hemoglobin with 14C-glucose. Other minor components, Hb A 1a1 and Hb A 1a2 are adducts of sugar phosphates at the N-terminus of the β chain. Hb A 1b contains an unidentified nonphosphorylated sugar at the β N-terminus. In addition, a significant portion of the major hemoglobin component (Hb A 0) is also glycosylated by a glucose ketoamine linkage at other sites on the molecule, including the N-terminus of the α chain and the ϵ-amino group of several lysine residues on both the α and the β chains. The results indicate that the interaction of glucose and hemoglobin is rather nonspecific and suggests that other proteins are modified in a similar fashion.

On the chromatographic heterogeneity of human fetal hemoglobin

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F I, other minor hemoglobin zones eluting before F I, pre-F I, and after F I, post-F I, have been observed. The relative amounts of the two pre-F I zones and F I are higher in the red cells of adults with 97–100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous δβ-thalassemia and homozygous β 0-thalassemia) than in the red cells of an adult with homozygous β +-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [ 14C]glucose or [ 14C]glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [ 14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F I zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F I zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [ 14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F I synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.

EFFECT OF NORMAL PREGNANCY UPON THE GLYCOSYLATED HAEMOGLOBINS

In this cross-sectional study total glycosylated haemoglobin (Hb AIa+b+c) has been measured in 20 healthy non-pregnant women and in five groups of 20 healthy women at various times during pregnancy. A statistically significant decrease occurs in these minor haemoglobin fractions by about 20 weeks gestation and this reduced level is maintained throughout the rest of pregnancy. If diabetic patients are to have their control assessed during pregnancy by determination of these fractions this fact might be relevant.

'Minor hemoglobins': an alternative approach for evaluating glucose control in pregnancy.

'Minor hemoglobins' (Hb AI) were measured in 178 women during pregnancy and immediately postpartum by a thiobarbituric acid spectrophotometric method. The patients were classified according to an oral glucose tolerance test (GTT) and an assessment of risk factors for diabetes in normal (n = 75), probably normal (n = 72) and chemical diabetics (n = 26). 5 pregnant insulin-dependent diabetics were also studied. Hb AI was unchanged during the second and third trimesters of pregnancy and immediately postpartum (less than 6 days) in women with normal glucose tolerance. Moreover, no difference of Hb AI level was found when compared with a separate group of nonpregnant individuals (n = 15). Chemical diabetics had significantly higher mean third trimester and postpartum Hb AI than normals (p less than 0.001). The highest Hb AI levels during pregnancy were found in the insulin-dependent diabetics, although these were lower compared to a separate group of nonpregnant insulin-dependent diabetics (n = 10; p less than 0.05). During pregnancy, a significant correlation was found between Hb AI and the 2-hour value of the GTT (p less than 0.001). A positive correlation was also found between third trimester maternal Hb AI and infant birth weight (p less than 0.001). Hb AI may complement other parameters in evaluating maternal glucose tolerance during pregnancy.

A simple microchromatographic column for determination of hemoglobins A1a + b and A1c.

A simple microchromatographic homemade column was devised for the measurement of glycosylated minor hemoglobin fractions. The mean and one standard deviation of hemoglobins A1a + b, A1c and A1a + b + c determined by the homemade column in 150 nondiabetic controls and 56 juvenile onset insulin-dependent diabetics were 2.6 +/- 0.5%, 6.0 +/- 1.0%, 8.6 +/- 1.1%, 3.3 +/- 0.9%, 13.7 +/- 2.2% and 16.9 +/- 2.8% respectively. A twofold increase in hemoglobins A1c and A1a + b + c levels was observed in the diabetics as compared to the non-diabetic controls suggesting that the homemade column is valid for the measurement of all three glycosylated hemoglobin fractions and assessment of blood glucose control in diabetic patients. The homemade column procedure yields accurate and reproducible results, is simple to perform, inexpensive, relatively rapid and may be used in the routine clinical laboratory. Hemoglobin A1a + b + c levels measured by a commercial column in 35 non-diabetic controls and in 56 diabetics showed good correlation (R = 0.91) with hemoglobin A1a + b + c levels determined by the homemade column. The commercial column is valid for the measurement of the combined glycosylated hemoglobin A1a + b + c fraction and may be used in the routine clinical laboratory to assess diabetic control.