On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hydration-dependent conformational states of hemoglobin. Equilibrium and kinetic behavior

The equilibrium and kinetics of methemoglobin conversion to hemichrome induced by dehydration were investigated by visible absorption spectroscopy. Below about 0.20 g water per g hemoglobin only hemichrome was present in the sample; above this value, an increasing proportion of methemoglobin appeared with the increase in hydration. The transition between the two derivatives showed a time-dependent biphasic behavior and was observed to be reversible. The rates obtained for the transition of methemoglobin to hemichrome were 0.31 and 1.93 min-1 and for hemichrome to methemoglobin 0.05 and 0.47 min-1. We suggest that hemichrome is a reversible conformational state of hemoglobin and that the two rates observed for the transition between the two derivatives reflect the α- and β-chains of hemoglobin.

Hemoglobin as AS/alfa talassemia - Importância diagnóstica

Sickle Cell disease is a generic term for a group of genetic disorders characterized by the predominance of hemoglobin S. These disorders include Sickle Cell anemia, the Sickle Cell beta Thalassemia syndromes and Hemoglobinopathies in which hemoglobin S is in association with another abnormal hemoglobin, such as hemoglobin S/C. The Sickle Cell trait (hemoglobin AS) associated with Alpha Thalassemia presents alterations in the red blood cells morphology, usually absent in the heterozygous for this hemoglobin variant. The interaction between hemoglobin Sand alpha Thalassemia has been described as one of the factors responsible for the improvement in the clinical picture of homozygous of hemoglobin S (Sickle Cell Anemia), decreasing the number of episodes of pain. The genetic mechanisms of this influence are evaluated using molecular analyses of the human globin genes. With the objective of verifying the presence of alpha Thalassemia in heterozygous of hemoglobin S, with anemia, sent to the Laboratory of Hemoglobins, Department of Biology, UNESP, São José do Rio Preto, SP, we analyzed 1002 blood samples with Sickle Cell trait, in the period from 1990 to 1998. The samples were picked with EDTA 5% as anticoagulant, after previous authorization of the carriers. Appropriated counseling and management requires definitive diagnosis. For the laboratorial diagnosis the blood samples were submitted to electrophoretic procedures in alkaline and acid pH and cytological evaluation of hemoglobin H. The electrophoretic procedures confirmed the presence of hemoglobin AS. The cytological evaluation evidenced the presence of alpha Thalassemia. Of this total analyzed, 16(1,59%) blood samples presented the association between hemoglobin AS and alpha Thalassemia and two individuals belonged of the same family. Our results addressed us to suggest to the routine laboratories, that is important to accomplish the research of alpha Thalassemia among the Sickle Cell trait, with anemia, to verify the interaction with alpha Thalassemia, supplying to the carriers a important information on its hematological profile, genetic pattern of hemoglobinopathies and the appropriated counseling. Rev.bras.hematol.hemoter.,2000,22(3):388-394.

Effect of fortification of drinking water with iron plus ascorbic acid or with ascorbic acid alone on hemoglobin values and anthropometric indicators in preschool children in day-care centers in Southeast Brazil

Background. Iron-deficiency anemia currently is the most frequently occurring nutritional disorder worldwide. Previous Brazilian studies have demonstrated that drinking water fortified with iron and ascorbic acid is an adequate vehicle for improving the iron supply for children frequenting day-care centers. Objective. The objective of this study was to clarify the role of ascorbic acid as a vehicle for improving iron intake in children in day-care centers in Brazil. Methods. A six-month study was conducted on 150 children frequenting six day-care centers divided into two groups of three day-care centers by drawing lots: the iron-C group (3 day-care centers, n = 74), which used water fortified with 10 mg elemental iron and 100 mg ascorbic acid per liter, and the comparison group (3 day-care centers, n = 76), which used water containing only 100 mg ascorbic acid per liter. Anthropometric measurements and determinations of capillary hemoglobin were performed at the beginning of the study and after six months of intervention. The food offered at the day-care centers was also analyzed. Results. The fo od offered at the day-care center was found to be deficient in ascorbic acid, poor in heme iron, and adequate in non-heme iron. Supplementation with fortified drinking water resulted in a decrease in the prevalence of anemia and an increase in mean hemoglobin levels associated with height gain in both groups. Conclusions. Fortification of drinking water with iron has previously demonstrated effectiveness in increasing iron supplies. This simple strategy was confirmed in the present study. The present study also demonstrated that for populations receiving an abundant supply of non-heme iron, it is possible to control anemia in a simple, safe, and inexpensive manner by adding ascorbic acid to drinking water. © 2005, The United Nations University.

Description of electrophoretic and chromatographic hemoglobin profile of Rhinoclemmys punctularia

Studies of the hemoglobin pattern in Brazilian reptiles are important for determining ecological and phylogenetic relationships, but they are scarce. Peripheral blood samples were obtained from 7 males and 18 females of Rhinoclemmys punctularia. The hematological profile was based on the total hemoglobin and hematocrit values. The hemoglobin profile was obtained using electrophoretic procedures at different pH, isoelectric focusing, globin chain electrophoresis, and HPLC. The hematocrit (31 ± 2%) and total hemoglobin (7.5 ± 0.2 g/dL) values did not indicate gender variations. Alkaline pH electrophoresis of the total blood samples treated with 1% saponin demonstrated the presence of four well-defined hemoglobin fractions, one major component (fraction I), showing cathodic migration and three others faster than fraction I with anodic migration. When the samples were precipitated with chloroform, only two hemoglobin fractions were observed, similar to fractions I and III from the first procedure. Isoelectric focusing and HPLC showed the same pattern. With acid and neutral pH electrophoresis, two fractions with anodic migration were observed. The globin chain identification at alkaline pH showed two fractions, but four fractions were observed at acidic pH, suggesting that different polypeptide chains are involved in the hemoglobin molecule. The chromatographic separation of the total blood sample demonstrated that the major fraction comprised 81.9% and the minor 18.1%. The results obtained demonstrated a similarity between these hemoglobin components and those of some Chelidae reported in the literature for both land and aquatic animals, reflecting the adaptation to environmental conditions. ©FUNPEC-RP.

Three-dimensional visualization of human hemoglobin phenotypes with HPLC

Hemoglobinopathies were included in the Brazilian Neonatal Screening Program on June 6, 2001. Automated high-performance liquid chromatography (HPLC) was indicated as one of the diagnostic methods. The amount of information generated by these systems is immense, and the behavior of groups cannot always be observed in individual analyses. Three-dimensional (3-D) visualization techniques can be applied to extract this information, for extracting patterns, trends or relations from the results stored in databases. We applied the 3-D visualization tool to analyze patterns in the results of hemoglobinopathy based on neonatal diagnosis by HPLC. The laboratory results of 2520 newborn analyses carried out in 2001 and 2002 were used. The Fast, F1, F and A peaks, which were detected by the analytical system, were chosen as attributes for mapping. To establish a behavior pattern, the results were classified into groups according to hemoglobin phenotype: normal (N = 2169), variant (N = 73) and thalassemia (N = 279). 3-D visualization was made with the FastMap DB tool; there were two distribution patterns in the normal group, due to variation in the amplitude of the values obtained by HPLC for the F1 window. It allowed separation of the samples with normal Hb from those with alpha thalassemia, based on a significant difference (P > 0.05) between the mean values of the Fast and A peaks, demonstrating the need for better evaluation of chromatograms; this method could be used to help diagnose alpha thalassemia in newborns.

Abnormal hemoglobin phenotypes in carriers of mild anemia in Latin America.

We looked for abnormal hemoglobins in blood samples sent for diagnosis of anemia. Identification of the hemoglobins was made using electrophoretic, chromatographic and molecular procedures. The 2020 blood samples were of patients from various regions of Brazil and from some other Latin American countries. Among the abnormal hemoglobins that we found, 3.5% are known to be rare, while 51% had an electrophoretic profile similar to that of Hb S at alkaline pH. Differentiation was possible only by combining electrophoretic and chromatographic methods. Hb Hasharon, an alpha globin chain mutant, was the most frequently found variant hemoglobin; it accounted for 14.3% of the abnormal DNA samples. The other abnormal hemoglobin phenotypes displayed distinct electrophoretic profiles; most of them migrated faster than Hb A. The frequencies of the different abnormal hemoglobin profiles that we found reflect the miscegenation of the Latin American population and indicate the importance of hemoglobin studies using various methods in combination for accurate diagnosis and appropriate counseling of carriers and their families.

Hemoglobin polymorphism and hematological profile of Geoffroy's side-necked turtle (Phrynops geoffroanus, Testudines) in the northwestern region of São Paulo State, Brazil

Complete blood counts and hemoglobin isoform data were gathered from 36 specimens of the turtle species Phrynops geoffroanus from the northwestern region of São Paulo State, Brazil. They were collected in an urban area. The hemoglobin profiles were obtained after red blood cell lysis and by electrophoretic migration in alkaline pH, acid pH, and neutral pH buffer. The hemoglobin components were confirmed using high-performance liquid chromatography (HPLC). Erythrogram analysis included hematocrit, total hemoglobin concentration, total red blood cell count, and red blood cell indices. The leukogram included a total white blood cell count and a calculation of the percent values of neutrophils, lymphocytes, monocytes, basophils, eosinophils, heterophils, and azurophils. HPLC analysis revealed three hemoglobin components; the first with a concentration of 5.5%, the second was a major component with an average concentration of 67.1%, and the third with a concentration of 28.5%. The hematological profile obtained for these specimens allowed us to establish a pattern for P. geoffroanus in São Paulo State Northwestern region. The average hematocrit values were 22.5% for females and 24.0% for males. For total hemoglobin, we found average values of 6.66 g/dL in females and 7.22 g/dL in males. The number of white blood cells was 2725 x 103/μL for females and 2775 x 103/μL for males. There was a predominance of heterophils, eosinophils, and monocytes in both sexes. No significant differences were found between males and females for hematological profile. The hematological results were compared to literature data for other Chelonia. They were similar to what is known for fresh water turtles. © FUNPEC-RP.

Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS

Further characterization of hemoglobin of Glossoscolex paulistus (HbGp) subunits was performed based on SDS-PAGE, size exclusion chromatography (SEC) and MALDI-TOF-MS analysis. SDS-PAGE has shown a total of four linker chains, two quite intense and two of lower intensity. HbGp fractions (I-VI), obtained by size exclusion chromatography (SEC), from oligomeric dissociation at alkaline pH 9.6, were monitored. Fraction I is identical to the whole protein. The monomeric chains c, obtained from the trimer abc reduction, present four isoforms with MM 17,336 Da, 17,414 Da, 17,546 Da and 17,620 Da. Furthermore, the trimer subunit presents two isoforms, T 1 and T 2, with MM 51,200 ± 60 and 51,985 ± 50 Da, respectively. Based on SDS-PAGE, the linker chains seem to be distributed along the different fractions of the SEC chromatogram, appearing along the peaks corresponding to fractions I-V. The fraction IV contains, predominantly, trimers with some linkers contamination. The strong interaction of linker chains L with the trimers abc, makes it difficult to obtain these subunits in pure form. The monomer d in fraction VI appears to be quite pure, in agreement with previous studies. © 2011 Elsevier Ltd. All rights reserved.

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations. © 2012 Elsevier B.V.

Use of hemoglobin as alternative to peroxidases in cholesterol amperometric biosensors

Sophisticated molecular architectures can be produced with the layer-by-layer (LbL) method, which may combine distinct materials on the same film. In this study, we take advantage of this capability to produce cholesterol amperometric biosensors from LbL films containing hemoglobin (Hb) and cholesterol oxidase in addition to the polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(ethylene imine) (PEI). Following an optimization procedure, we found that an LbL film deposited onto ITO substrates, with the architecture ITO(PEI/Hb)5(PEI/COx)10, yielded a sensitivity of 93.4 μA μmol L-1 cm-2 for cholesterol incorporated into phospholipid liposomes, comparable to state-of-the-art biosensors. Hb acted as efficient electron mediator and did not suffer interference from phospholipids. Significantly, cholesterol could also be detected in real samples from chicken egg yolk, with no effects from potential interferents, including phospholipids. Taken together these results demonstrate the possible fabrication of low cost, easy-to-use cholesterol amperometric biosensors, whose sensitivity can be enhanced by further optimizing the molecular architectures of the LbL films. © 2012 Elsevier B.V.

Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media

The thermal denaturation and aggregation of the HbGp, in the oxy- and cyanomet-forms, was investigated by DSC, AUC, DLS, optical absorption and CD, in the pH range from 5.0 to 7.0. Oxy-HbGp has a denaturation process partially reversible and dependent on the temperature. DSC melting curve is characterized by a single peak with Tc value of 333.4±0.2K for oxy-HbGp, while two peaks with Tc values of 332.2±0.1 and 338.4±0.2K are observed for cyanomet-HbGp, at pH 7.0. In acidic pH oxy- and cyanomet-HbGp are more stable showing higher Tc values and aggregation. AUC data show that, HbGp, at pH 7.0, upon denaturation, remains undissociated at 323K, presenting oligomeric dissociation at 333 (12±3% of tetramer and 88±5% of whole HbGp) and 343K (70±5% of monomer and 30±2% of trimer). DLS data show that the lag period before aggregation is dependent on the temperature and HbGp concentration. Optical absorption and CD results show that the increase of temperature leads to the oxy-HbGp oxidation and aggregation, above 331K, in acidic pH. CD data, for HbGp, present a greater thermal stability in acid medium than at neutral pH, with similar Tc values for both oxidation forms. Our data are consistent with previous studies and represents an advance in understanding the thermal stability of oligomeric HbGp structure. © 2012 Elsevier B.V.

Inherited hemoglobin disorders in an Afro-Amazonian community: Saracura

ABSTRACT: The most common hemoglobinopathies, viz, hemoglobins S and C, and α-and β-thalassemias, were investigated through the molecular screening of 116 subjects from the community of Saracura, comprising fugitive African slaves from farms of the municipality of Santarém, in the west of Pará State, Brazilian Amazon. The observed frequency of the HBB*S gene (0.9%) was significantly lower than that encountered in other Afro-derived communities in the region. Concomitantly, the absence of the HBB*C allele has been reported for most of the Afro-Amazonian communities thus far studied. As remnant populations of quilombos are generally small, the heterogeneous distribution of HBB*S and HBB*C alleles among them is probably due to genetic drift and/or founder effect. The observed frequency of 3.7 kb deletion in Saracura (8.5%) was consistent with the African origin of the population, with a certain degree of local differentiation and admixture with individuals of Caucasian ancestry, placed in evidence by the occurrence of - -(MED) deletion (1.2%), a common mutation in Mediterranean regions. As regards f-thalassemia, among the seven different mutations found in Saracura, three βºand two β+ mutations were of Mediterranean origin, and two β+ of African. Thus, only 28% of the local β-thalassemia mutations found in Saracura were of African origin.

Origin of the hemoglobin S gene in a northern Brazilian population: the combined effects of slave trade and internal migrations

Com o objetivo de investigar a origem da mutação bS na população da região norte do Brasil, foram analisados polimorfismos de DNA no complexo de genes b da hemoglobina em 30 pacientes com anemia falciforme na população de Belém, a capital do Estado do Pará. Sessenta e sete por cento dos cromossomos bS analisados apresentaram o haplótipo Bantu, 30% o haplótipo Benin e 3% o haplótipo Senegal. A origem da mutação bS na população de Belém, estimada de acordo com a distribuição de haplótipos, não está de acordo com a esperada com base em dados históricos sobre o tráfico de escravos para a região norte, os quais indicam uma reduzida contribuição de escravos da região do Benin. Essas diferenças podem ser atribuídas ao tráfico interno de escravos, bem como ao posterior fluxo de populações imigrantes, particularmente de nordestinos. A distribuição de haplótipos em Belém não difere significativamente da observada em outras regiões brasileiras, muito embora os dados históricos sugiram que a maioria dos escravos procedentes da região do Atlântico-Oeste africano, onde predomina o haplótipo Senegal, foi trazida para o norte do Brasil, enquanto que o nordeste (Bahia, Pernambuco e Maranhão) recebeu o maior contingente de escravos oriundos da região centro-oeste africana, onde o haplótipo Benin é o mais comum. Nós sugerimos que as diferenças regionais quanto à procedência dos escravos africanos também foram modificadas pelo tráfico de escravos estabelecido entre as diferentes regiões brasileiras e posteriormente pelos movimentos migratórios.

Live High-Train High increases hemoglobin mass in Olympic swimmers

This study tested whether 3-4 weeks of classical Live High-Train High (LHTH) altitude training increases swim-specific VO2max through increased hemoglobin mass (Hb(mass)).Ten swimmers lived and trained for more than 3 weeks between 2,130 and 3,094 m of altitude, and a control group of ten swimmers followed the same training at sea-level (SL). Body composition was examined using dual X-ray absorptiometry. Hb(mass) was determined by carbon monoxide rebreathing. Swimming VO2peak was determined and swimming trials of 4 x 50, 200 and 3,000 m were performed before and after the intervention.Hb(mass) (n = 10) was increased (P < 0.05)after altitude training by 6.2 +/- A 3.9 % in the LHTH group, whereas no changes were apparent in the SL group (n = 10). Swimming VO2peak was similar before and after training camps in both groups (LHTH: n = 7, SL: n = 6). Performance of 4 x 50 m at race pace was improved to a similar degree in both groups (LHTH: n = 10, SL: n = 10). Maximal speed reached in an incremental swimming step test (P = 0.051), and time to complete 3,000 m tended (P = 0.09) to be more improved after LHTH (n = 10) than SL training (n = 10).In conclusion, 3-4 weeks of classical LHTH is sufficient to increase Hb(mass) but exerts no effect on swimming-specific VO2peak. LHTH may improve performance more than SL training.