Isolation and characterization of hydrophobic polypeptides in human bile.
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Polypeptides were isolated from human bile by extraction with chloroform/methanol, followed by reversed-phase chromatography in methanol/ethylene chloride and gel filtration in chloroform/methanol. Peptides were characterized by SDS/PAGE, sequence analysis and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. This identified haemoglobin alpha chain, ATP synthase lipid-binding protein subunit 9, an N-terminal fragment of mac25/insulin-like growth factor-binding protein 7 and an internal fragment of monocyte differentiation antigen CD14, all not described previously in bile. In addition, alpha1-antitrypsin, known in bile from previous work, was also identified. The hydrophobic character of haemoglobin alpha chain is not apparent from its amino acid sequence, but the other polypeptides all have major hydrophobic segments. These results show that several proteins are removed upon organic solvent extraction used for delipidation during the preparation of samples for proteome analysis. Several of the polypeptides found are unexpectedly present in bile, suggesting that specific excretion mechanisms may be involved. (+info)
Erythroid bone marrow activity and red cell hemoglobinization in iron sufficient beta-thalassemia heterozygotes as reflected by soluble transferrin receptor and reticulocyte hemoglobin in content. Correlation with genotypes and Hb A(2) levels.
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BACKGROUND AND OBJECTIVES: Ferrokinetic studies and erythroid cell ultrastructural studies have indicated some degree of ineffective erythropoiesis in heterozygous beta-thalassemia, although a wide case-to-case variation was observed. In this study we applied rapid biochemical and hematologic measurements to assess erythroid marrow activity (sTfR) and reticulocyte hemoglobin content (CHr) in iron-sufficient individuals with heterozygous beta-thalassemia and investigated the correlation with the degree of globin polypeptide chain imbalance by comparing parameters between beta-thalassemia heterozygotes with genotypes of variable severity. DESIGN AND METHODS: We studied 57 iron-sufficient adults with heterozygous beta-thalassemia, divided into groups according to genotype: group A: beta(silent)-thalassemia heterozygotes, group B: beta(+)-thalassemia heterozygotes and group C: beta(0)-thalassemia heterozygotes. Twenty-one hematologically normal individuals served as controls (group D). We measured hematologic parameters including CHr with a Bayer-Advia 120 hematology analyzer. Hemoglobins were analyzed by high performance liquid chromatography, while biochemical parameters of iron status (iron, ferritin, transferrin and sTfR) were measured with chemical, luminometric and nephelometric methods. RESULTS: We found significant positive correlations between sTfR values for all beta-thalassemia heterozygote groups when plotted against Hb A(2) and Hb F levels (r=0.566, p<0.0001 and r=0.283, p<0.03, respectively) and significantly negative correlation between CHr and Hb A(2) values (r=-0.790, p<0.00001). These data reflect the fine association of globin polypeptide chain imbalance with erythron expansion and the greater degree of ineffective erythropoiesis in beta-thalassemia heterozygotes with more severe genotypes. INTERPRETATION AND CONCLUSIONS: This study is the first demonstration that sTfR and CHr are useful parameters for evaluating the relative severity of different genotypes in heterozygous beta-thalassemia. (+info)
Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of alpha thalassaemia.
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AIMS: To assess the accuracy and precision of measuring haemoglobin A(2) by high performance liquid chromatography (HPLC) in the presence and absence of sickle cell trait, with or without alpha thalassaemia trait. METHODS: The haemoglobin A(2) percentage and the haemoglobin A(2) plus S percentages were determined by HPLC on 82 normal controls and 78 patients with sickle cell trait, respectively; the alpha thalassaemia status of each patient was determined by polymerase chain reaction. Red cell indices and haemoglobin A(2) and S percentages were compared in patients with two, three, or four alpha genes. RESULTS: Of the 78 patients with sickle cell trait, 17 were heterozygous for alpha(+) thalassaemia (-alpha(3.7)/alphaalpha) and 13 were homozygous (-alpha(3.7)/-alpha(3.7)). Microcolumn chromatography showed that the haemoglobin A(2) percentage was slightly, but significantly, higher than normal in sickle cell trait. HPLC determinations of haemoglobin A(2) percentage in patients with sickle cell trait are precise but inaccurate, the percentage being appreciably overestimated. The measured haemoglobin A(2) percentage is stable for one week, but inaccuracy increases by two weeks in most samples. Despite this inaccuracy, there are significant differences in the HPLC "haemoglobin A(2) percentage" between groups of individuals with two, three, and four alpha genes. CONCLUSIONS: Haemoglobin A(2) determinations by HPLC are precise but inaccurate. Nevertheless, there are significant differences in the haemoglobin A(2) percentage in subjects with two, three, and four alpha genes. Although there is some overlap between groups, this can be useful, together with the red cell indices, in predicting the likelihood of coexisting alpha thalassaemia. (+info)
Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography. (+info)
Implications of increased haemoglobin A2 values in HIV positive women in the antenatal clinic.
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Universal antenatal haemoglobinopathy screening in this hospital has identified several women with increased haemoglobin A(2) values, but without hypochromic microcytic red cell indices. This report describes two cases where there is evidence that the raised haemoglobin A(2) value is not caused by heterozygous beta thalassaemia, but rather results from these patients being human immunodeficiency virus (HIV) positive and on antiretroviral therapy. This will have important implications as universal antenatal haemoglobinopathy screening becomes more widespread, and as the number of HIV positive women of childbearing age increases. (+info)
Genetic modifiers of beta-thalassemia.
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As the defective genes for more and more genetic disorders become unravelled, it is clear that patients with apparently identical genotypes can have many different clinical conditions even in simple monogenic disorders. Beta thalassemia occurs when there is a deficiency in the synthesis of beta globin chains. The clinical manifestations of beta thalassemia are extremely diverse, spanning a broad spectrum from severe anemia and transfusion-dependency to the asymptomatic state of thalassemia trait. The remarkable phenotypic diversity of the beta thalassemias is prototypical of how a wide spectrum of disease severity can be generated in single gene disorders. The most reliable and predictive factor of disease phenotype is the nature of the mutation at the beta globin locus itself. However, relating phenotype to genotype is complicated by the complex interaction of the environment and other genetic factors at the secondary and tertiary levels, some implicated from family studies, and others, as yet unidentified. This article reviews the clinical and hematologic diversity encountered in beta thalassemia with an overview of the modifier genes that moderate their disease expression. (+info)
The detection and diagnosis of hemoglobin A2' by high-performance liquid chromatography.
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Hemoglobin (Hb) A2' is a hematologically silent variant of HbA2 that is detected easily by high-performance liquid chromatography (HPLC), where it elutes in the S window. Our purposes were to define diagnostic criteria for the HbA2' trait using the Variant II (Bio-Rad, Hercules, CA) and to determine the prevalence of HbA2' in a metropolitan patient population. All Hb screens (N = 5,862) performed during a 26-month period were reviewed for new hemoglobinopathies. We identified 57 cases of HbA2' trait, making it the fourth most prevalent Hb variant detected in this population after HbS, HbC, and beta-thalassemia minor For HbA2' trait cases, the mean HbA2 level was 1.7% (SD, 0.17%), and the mean HbA2' level was 1.3% (SD, 0.18%). Six possible HbA2'/beta-thalassemia double heterozygotes were identified, for whom the sum of the HbA2 and HbA2' exceeded 4% of total Hb. Hb variants that might interfere with detection of HbA2' include HbS, glycosylated HbC, and HbG2. Diagnostic criteria proposed for the HbA2' trait by HPLC are HbA2 of 2% or less, S window peak of 1% to 2%, no previous diagnosis of HbS, and absence of HbG and HbC. (+info)
Known and new delta globin gene mutations and their diagnostic significance.
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Mutations in the delta-globin gene (HBD, MIM# 142000) are not pathologically relevant. However, since high HbA2 levels are diagnostic for beta-thalassemia trait and a lowered level for an alpha- or delta-mutation, co-inheritance of delta- and beta-gene defects may lead to misinterpretation of diagnostic results. We examined 29 cases with low HbA2 level diagnosed in our laboratory, in the presence or absence of a second HbA2 fraction. We found a delta globin gene mutation in 20 cases. In total four different known mutations were found, three structural and one expressional. Moreover, two new defects were observed, one causing a structural abnormality and one a beta-thalassemia. The structural abnormality HBD c.431A->G (p.His144Arg)(dcd 143 CAC->CGC) was homologous to the beta-globin gene variant called Hb-Abruzzo and we have named this mutation HbA2 -Abruzzo. The new delta-thalassemia defect HBD c.-118C->T (d -68 C->T) has no homology on the beta-globin gene (HBB, MIM# 141900). All mutations caused a low HbA2 level and through this could lead to misdiagnosis when inherited together with a beta-thalassemia. (+info)