Oxidative and osmotolerant effects in Salvator merianae (Squamata: Teiidae) red blood cells during hibernation / Efeitos de oxidação e osmotolerância dos eritrócitos de Salvator merianae (Squamata: Teiidae) durante a hibernação

Abstract Hibernation is a natural condition of animals that lives in the temperate zone, although some tropical lizards also experience hibernation annually, such as the lizard native from South America, Salvator merianae, or "tegu" lizard. Even though physiological and metabolic characteristic associated with hibernation have been extensively studied, possible alterations in the red blood cells (RBC) integrity during this period remains unclear. Dehydration and fasting are natural consequences of hibernating for several months and it could be related to some cellular modifications. In this study, we investigated if the osmotic tolerance of RBCs of tegu lizard under hibernation is different from the cells obtained from animals while normal activity. Additionally, we indirectly investigated if the RBCs membrane of hibernating tegus could be associated with oxidation by quantifying oxidized biomolecules and the activity of antioxidant enzymes. Our findings suggest that RBCs are more fragile during the hibernation period, although we did not find evidence of an oxidative stress scenario associated with the accentuated fragility. Even though we did not exclude the possibility of oxidative damage during hibernation, we suggested that an increased RBCs volume as a consequence of hypoosmotic blood during hibernation could also affect RBCs integrity as noted.

Resumo A hibernação é uma condição natural dos animais que vivem na zona temperada, embora alguns lagartos tropicais também experenciem hibernação anualmente, como é o caso do lagarto nativo da América do Sul, Salvator merianae ou "teiú". Embora as características fisiológicas e metabólicas associadas à hibernação tenham sido amplamente estudadas, possíveis alterações na integridade das hemácias durante esse período ainda permanecem obscuras. A desidratação e o jejum são consequências naturais da hibernação por vários meses e podem estar relacionadas a algumas modificações celulares. Neste estudo, investigamos se a tolerância osmótica de hemácias do lagarto teiú sob hibernação são diferentes das células obtidas de animais em atividade normal. Além disso, investigamos indiretamente por meio da quantificação de biomoléculas oxidadas e da atividade de enzimas antioxidantes se a membrana das hemácias dos teiús em hibernação poderia estar associada à oxidação. Nossos resultados sugerem que as hemácias possuem maior fragilidade durante o período de hibernação, embora não tenhamos encontrado evidências de um cenário de estresse oxidativo associado à essa fragilidade acentuada. Embora não tenhamos excluído a possibilidade de dano oxidativo durante a hibernação, sugerimos que um aumento no volume das hemácias como consequência de sangue hipoosmótico durante a hibernação também poderia afetar a integridade de hemácias, tal como foi observado.

Oxidative and osmotolerant effects in Salvator merianae (Squamata: Teiidae) red blood cells during hibernation

Abstract Hibernation is a natural condition of animals that lives in the temperate zone, although some tropical lizards also experience hibernation annually, such as the lizard native from South America, Salvator merianae, or tegu lizard. Even though physiological and metabolic characteristic associated with hibernation have been extensively studied, possible alterations in the red blood cells (RBC) integrity during this period remains unclear. Dehydration and fasting are natural consequences of hibernating for several months and it could be related to some cellular modifications. In this study, we investigated if the osmotic tolerance of RBCs of tegu lizard under hibernation is different from the cells obtained from animals while normal activity. Additionally, we indirectly investigated if the RBCs membrane of hibernating tegus could be associated with oxidation by quantifying oxidized biomolecules and the activity of antioxidant enzymes. Our findings suggest that RBCs are more fragile during the hibernation period, although we did not find evidence of an oxidative stress scenario associated with the accentuated fragility. Even though we did not exclude the possibility of oxidative damage during hibernation, we suggested that an increased RBCs volume as a consequence of hypoosmotic blood during hibernation could also affect RBCs integrity as noted.

Resumo A hibernação é uma condição natural dos animais que vivem na zona temperada, embora alguns lagartos tropicais também experenciem hibernação anualmente, como é o caso do lagarto nativo da América do Sul, Salvator merianae ou teiú. Embora as características fisiológicas e metabólicas associadas à hibernação tenham sido amplamente estudadas, possíveis alterações na integridade das hemácias durante esse período ainda permanecem obscuras. A desidratação e o jejum são consequências naturais da hibernação por vários meses e podem estar relacionadas a algumas modificações celulares. Neste estudo, investigamos se a tolerância osmótica de hemácias do lagarto teiú sob hibernação são diferentes das células obtidas de animais em atividade normal. Além disso, investigamos indiretamente por meio da quantificação de biomoléculas oxidadas e da atividade de enzimas antioxidantes se a membrana das hemácias dos teiús em hibernação poderia estar associada à oxidação. Nossos resultados sugerem que as hemácias possuem maior fragilidade durante o período de hibernação, embora não tenhamos encontrado evidências de um cenário de estresse oxidativo associado à essa fragilidade acentuada. Embora não tenhamos excluído a possibilidade de dano oxidativo durante a hibernação, sugerimos que um aumento no volume das hemácias como consequência de sangue hipoosmótico durante a hibernação também poderia afetar a integridade de hemácias, tal como foi observado.

Oxidative and osmotolerant effects in Salvator merianae (Squamata: Teiidae) red blood cells during hibernation.

Hibernation is a natural condition of animals that lives in the temperate zone, although some tropical lizards also experience hibernation annually, such as the lizard native from South America, Salvator merianae, or "tegu" lizard. Even though physiological and metabolic characteristic associated with hibernation have been extensively studied, possible alterations in the red blood cells (RBC) integrity during this period remains unclear. Dehydration and fasting are natural consequences of hibernating for several months and it could be related to some cellular modifications. In this study, we investigated if the osmotic tolerance of RBCs of tegu lizard under hibernation is different from the cells obtained from animals while normal activity. Additionally, we indirectly investigated if the RBCs membrane of hibernating tegus could be associated with oxidation by quantifying oxidized biomolecules and the activity of antioxidant enzymes. Our findings suggest that RBCs are more fragile during the hibernation period, although we did not find evidence of an oxidative stress scenario associated with the accentuated fragility. Even though we did not exclude the possibility of oxidative damage during hibernation, we suggested that an increased RBCs volume as a consequence of hypoosmotic blood during hibernation could also affect RBCs integrity as noted.

Hemoglobin (Hb) Val de Marne (Hb Footscray) in Brazil: the first case report.

Hemoglobin (Hb) variants involving alpha-chains are less common in the global population than Hb variants resulting from beta-chain alterations. Generally, alpha-chain Hb variants are caused by point mutations affecting alpha-1 and/or alpha-2 genes of the alpha-globin cluster (HBA1 and HBA2). In Brazil, the most prevalent alpha-chain Hb variant is Hb Hasharon. In this study, we present the first case of an Hb Val de Marne variant in the Americas, specifically in Brazil.

Cytochemical characteristics of blood cells from Brazilian tortoises (Testudines: Testudinidae).

The hematology of wild and captive animals is essential for obtaining details about species and represents a simple method of diagnosing disease and determining prognosis. Few studies have described the morphology of chelonian blood cells, which are more common in sea and freshwater turtle species. Thus, in order to further our understanding and recognition of different chelonian cells types, the present study aimed to describe blood cells from the two species of Brazilian tortoises, Chelonoidis carbonarius and C. denticulatus. Cytochemical analysis of tortoise blood tissue with Panótico®, made it possible to describe all the of the chelonian cell types (with the exception of thrombocytes): erythrocytes, agranular leukocytes (monocytes and lymphocytes), and granular leukocytes (eosinophils, heterophils, basophils, and azurophils). These data are of high importance for establishing hematological profiles of Brazilian tortoises and reptiles. Therefore, based on our results and on comparative analyses with data from the literature for other reptile species, we can conclude that the blood cells described for Brazilian tortoises are found in all species of reptiles that have been analyzed thus far, and may be characterized and used as a comparative parameter between different groups to evaluate the health status of these animals.

Thesis Abstract Morphological and phylogeographic analysis of Brazilian tortoises (Testudinidae).

The discriminative potentials of biogeography, vocalization, morphology, cytogenetics, hemoglobin, and molecular profiling of cytochrome b as taxonomic techniques for differentiating Brazilian tortoises were evaluated in this study. In Brazil, two species of tortoises are described, Chelonoidis carbonarius and Chelonoidis denticulatus. However, in the present study, some animals that were initially recognized based on morphological characters and coloring did not correspond to the typical pattern of C. carbonarius; these animals were classified as morphotypes 1 and 2. It was proposed that these morphotypes are differentiated species, and they should not be considered as a single taxonomic unit with C. carbonarius. Tortoises analyzed were provided by the National Institute for Amazonian Research (INPA); the Emilio Goeldi Museum, PA; municipal zoos in São José do Rio Preto, SP, and Araçatuba, SP; and the Reginaldo Uvo Leone breeding farm for Wild and Exotic Animals, Tabapuã, SP. Based on the data obtained using biogeographic evaluation of specimens in the literature, it was found that C. carbonarius is distributed in the Northeast Region of Brazil, and no animal of this pattern was observed in the investigated collections. On the other hand, C. denticulatus is found in all the states of the Legal Amazonia. In addition, isolated individual records of this species exist in the Atlantic Forest in Espírito Santo and Rio de Janeiro and in the Midwest Region composed of the states of Goiás, Mato Grosso, and Mato Grosso do Sul. In the Northeast Region, C. denticulatus occurs in the State of Bahia. Morphotype 1 has a wider geographical distribution than C. carbonarius, possibly because of several distribution reports associated with C. carbonarius, indicating erroneous association of morphotype 1 as a single taxonomic unit with C. carbonarius. Morphotype 2 is found only in the states of Pará, Maranhão, and Piauí. These biogeographic data indicate that the distribution of C. carbonarius can partially be explained by the fact that all the morphotypes are considered as a single taxonomic unit. Behavioral aspects such as intraspecific communication may be as reliable as morphological or molecular data for inferring evolutionary relationships. Analysis of the physical characteristics of vocalization [fundamental frequency (Hz), interval between notes (s), duration of each note (s), and number of notes from each vocalization] between C. carbonarius and morphotype 1 revealed statistically significant differences in the interval between notes (s) (P = 0.0000); duration of each note (s) (P = 0.0000); frequency of notes (Hz) (P = 0.0009); and number of notes (P = 0.0002). The results of preference experiments using sound stimulus were inconclusive with respect to species-specific vocalization preference; only females of C. carbonarius showed intraspecific vocalization preference, indicating possible reproductive isolation mechanisms. To explore the presence of sexual dimorphism and morphological differences between C. denticulatus, C. carbonarius, and morphotype 1, descriptive statistics to analyze the data obtained for the investigated measures were used. Two sets of analysis were conducted - the first for each group, to compare the sexes; and the second for each sex, to compare the groups. To examine the interspecific variation in size and shape, a correlation matrix inprincipal component analysis was used. Next, I used factor analysis to rank the features showing >0.75 correlation in the differentiation between the sexes. The results were consistent with the hypothesis that morphotype 1 corresponds to a new species, because it differs from the species pattern in terms of morphology, coloring, and sexual dimorphism. The results of classical cytogenetic analysis - to differentiate C. denticulatus, C. carbonarius, and morphotype 1 - revealed no consistent data that would enable its use as a taxonomic parameter. Conventional Giemsa staining revealed a diploid chromosome number of 2n = 52 for all the evaluated groups. The patterns obtained using chromosomal banding techniques showed high similarity and low reproducibility. Moreover, the sensitivity and resolution were insufficient to enable differentiation between the three groups, implying the existence of conserved characteristics of the karyotype in Testudinidae. To establish a hemoglobin profile for C. denticulatus, C. carbonarius, and morphotype 1, and to visualize the hemoglobin fractions of each group, I conducted acid and alkaline electrophoreses. The results of high-performance liquid chromatography revealed percentage differences in the hemoglobin fractions. In addition, electrophoresis of the polypeptide chains under acid and alkaline pH conditions showed the globin composition of each fraction. The observed differences in the chromatographic profile between C. carbonarius and morphotype 1 with respect to C. denticulatus validated the technique as an additional method for elucidating taxonomic issues in Testudinidae. The similarities observed in the hemoglobin profiles of C. carbonarius and morphotype 1 suggest recent separation between these groups. Alignment of mitochondrial DNA cytochrome b fragments revealed a degree of homogeneity among the C. denticulatus samples and the sequences published in the literature, indicating low genetic variability of the mitochondrial DNA cytochrome b fragment for this species. In contrast, the sequences of the mitochondrial DNA cytochrome b fragments from C. carbonarius and morphotypes 1 and 2 differed from those available in the published literature databases, indicating a variable genetic structure. This may be because it does not consider taxonomic divisions within C. carbonarius. Phylogenetic analysis did not reveal an appropriate phylogenetic signal for differentiating C. carbonarius and morphotypes 1 and 2. However, this analysis did differentiate C. denticulatus, indicating that the separation of C. carbonarius and morphotypes 1 and 2 is more recent than the separation of C. denticulatus and C. carbonarius. Owing to the problems associated with the use of cytochrome b in phylogenetic analysis, the polytomy observed for C. carbonarius and morphotypes 1 and 2 does not exclude the hypothesis that these samples represent different species. It is possible that the inclusion of morphological, behavioral, and hemoglobin profile data in a mixed matrix with the molecular data would enable the separation of morphotypes 1 and 2 as monophyletic species. This analysis would require data regarding external groups, to facilitate a more robust phylogenetic analysis, thereby enabling greater taxonomic resolution. In view of the differences in the biogeographical pattern, vocalization, specific-sound preference, and morphology found in the present study, I propose that morphotype 1 should be considered a new species. Data regarding the conservation status of natural tortoise populations in Brazil should be reviewed, because of the intensive human pressure on this species. There are no private or public plans for the conservation or recovery of natural tortoise populations. Hence, it is very likely that these animals have a more endangered status than that reported in the literature.

Influence of ßS allele in the lipid peroxidation and antioxidant capacity parameters.

INTRODUCTION: The oxidative process plays a fundamental role in the pathophysiology of sickle cell anemia (SCA), and population and environmental characteristics may influence redox balance. The aim of this study was to evaluate lipid peroxidation and antioxidant capacity in Brazilian Hb S carriers undergoing different therapies. METHODS: Blood samples from 270 individuals were analyzed (Hb SS, n = 68; Hb AS, n = 53, and Hb AA, n = 149). Hemoglobin genotypes were assessed through cytological, electrophoretic, chromatographic, and molecular methods. Plasma lipid peroxidation and antioxidant capacity were measured by spectrophotometric methods. RESULTS: Patients with SCA who used iron-chelating drugs combined with hydroxyurea, associated with regular transfusions, showed lower levels of TBARS (P ≤ 0.05), higher levels of TEAC (P ≤ 0.01), and lower TBARS/TEAC ratio (R = 255.8). The redox profile of Hb AS subjects was not statistically different (P > 0.05) from that of Hb AA subjects. CONCLUSION: The data suggest that oxidative stress is lower in the patients with SCA who received regular blood transfusions associated with the combined use of HU and iron chelators than the group received only HU. The redox system of the Hb AS carriers is compatible with the control group.

Frequencies of -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms in sickle cell anemia patients from Brazil.

Sickle cell anemia is an affection that causes chronic inflammation, with consequences for vaso-occlusion, oxidative stress and cytokine production. Genetic polymorphisms in markers involved in this process can modulate the inflammatory response, including polymorphisms -308G/A of TNFA (tumor necrosis factor alpha) and -509C/T of TGFB1 (transforming growth factor beta 1), reported to increase TNF-α and TGF-ß1 production, respectively. Changes in the cytokine balance are important risk factors for clinical events; consequently, we examined the frequencies of these polymorphisms in 240 Brazilian sickle cell anemia patients from southeast Brazil. PCR-RFLP was used to detect these polymorphisms. The -509C/T (TGFB1) polymorphism was more frequent than -308G/A (TNFA), with allelic frequency of 0.3 for the mutant allele T (TGFB) agaist 0.1 for the mutant allele A (TNFA). These allelic frequencies are similar to those known from populations with ethnicity similar to the Brazilian population. Inheritance of these polymorphisms does not seem to be associated with that of the Hb S mutation; however, this information could be useful in analyses of specific clinical characteristics of sickle cell anemia.

Geoffroy's side-necked turtle [Phrynops geoffroanus (Schweigger, 1812), Testudines: Chelidae] as a model for evolutionary ecotoxicology: relationship between environmental contamination, conditions and genetic variability.

The objective of this study was to evaluate the role of human activity factors, such as environmental contamination and habitat changes, as drivers for changing the physiological, biochemical, and genetic diversity of Geoffroy's side-necked turtle populations in one of the most impacted watersheds in southeastern Brazil. The impact of chemical and organic contamination was determined by ecotoxicological analyses to assess the action of some of the major components involved in protection against oxidative stress, phase I and II detoxification metabolism, and antioxidant capacity. The results indicated the influence of domestic and industrial effluents on detoxification metabolism and oxidative stress. However, in spite of increased activity and effect of EROD (CYP1A1) and glutathione S-transferase (GST) activity, GST average values in the urban area agreed with those expected for hypoxic conditions according to the literature. This observation suggests that increased GST in response to ROS production due to the presence of pollutants increases the antioxidant defense network, controlling the oxidative damage caused by hypoxia and reperfusion. To determine the conditions that are reflected in individual ability (fitness), we evaluated the mathematical relationship between weight and length, and found that changes in body shape and weight increase, allowing inferences about animal health and welfare. The data obtained indicate differences in conditions that are associated with the area, but also with sex and reproductive period, and contamination gradient, indicating a strong influence of environmental stressors on the physiology of the specimens. The evaluation of genetic structure among populations of Preto River and Felicidade Stream, based on microsatellites, demonstrated that there was no genetic differentiation, due to extensive gene flow between the areas and high genetic diversity. However, after analysis of intrapopulation structure, we observed the existence of five genetic groups that reflected changes in habitat created by damming and siltation, which initiate separation processes (barriers) between sub-populations. The relationship between the data obtained for biochemical parameters, condition factors and genetic diversity was analyzed by heterozygosity-fitness correlation. The negative relationship observed may be explained by the profile of structural and ecological changes in the populations studied, indicating the important influence of humans on the biology of natural populations. Therefore, Phrynops geoffroanus shows adaptation to environmental contamination, and ecological changes and possible loss of habitat are altering the genetic diversity of the populations studied. This is the first study evaluating all these aspects of P. geoffroanus simultaneously in natural populations in Brazil, using this species as a model.

Evaluation of HPFH and 뫧-thalassemia mutations in a Brazilian group with high Hb F levels.

Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (뫧-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in 뫧-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish 뫧-thalassemia and none were heterozygous for Sicilian 뫧- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the 뫧-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.

Comparison of oxidative stress and the frequency of polymorphisms in the HFE gene between hemoglobin S trait blood donors and sickle cell disease patients.

It is well documented that Hb S and iron affect blood cells, and trigger oxidative processes and generation of free radicals with potential for lipid peroxidation. We evaluated the frequency of polymorphisms in the HFE gene in Hb AS blood donors and how these polymorphisms influenced lipid peroxidation and antioxidant capacity. Blood samples were collected from 211 Hb AS blood donors, 119 Hb AA blood donors as a control group, and 28 sickle cell disease patients (Hb SS). The H63D allele was found at a frequency of 10.5% in the Hb AS samples, and the C282Y allele frequency was 0.7%. In the control group, the frequencies of the H63D and C282Y alleles were 13.4 and 2.1%, respectively. In the sickle-cell disease patients, the H63D and the C282Y allele frequencies were 10.7 and 3.5%, respectively. The frequencies of the C282Y and H63D polymorphisms in Hb AS blood donors are similar to those reported for the Brazilian population. Serum malondialdehyde values, indicative of lipid peroxidation, were highest in sickle cell patients, independent of the polymorphisms in the HFE gene, with significant differences, showing the influence of Hb S allele in the levels of lipid peroxidation. However, the trolox equivalent antioxidant capacity average levels, indicative of the antioxidant capacity, were reduced with significant differences, indicating that in spite of a lipid peroxidation raise, this is not followed by the increased of the antioxidant capacity, leading to oxidative stress.

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Serum ferritin and transferrin saturation levels in ß° and ß(+) thalassemia patients.

There have been few studies on the mutations that cause heterozygous beta-thalassemia and how they affect the iron profile. One hundred and thirty-eight individuals were analyzed, 90 thalasemic ß° and 48 thalasemic ß(+), identified by classical and molecular methods. Mutations in the hemochromatosis (HFE) gene, detected using PCR-RFLP, were found in 30.4% of these beta-thalassemic patients; heterozygosity for H63D (20.3%) was the most frequent. Ferritin levels and transferrin saturation were similar in beta-thalassemics with and without mutations in the HFE gene. Ferritin concentrations were significantly higher in men and in individuals over 40 years of age. Transferrin saturation also was significantly higher in men, but only in those without HFE gene mutations. There was no significant difference in the iron profile among the ß° and ß(+) thalassemics, with and without HFE gene mutations. The frequency of ferritin values above 200 ng/mL in women and 300 ng/mL in men was also similar in ß° and ß(+) thalassemics (P > 0.72). Our conclusion is that ferritin levels are variable in the beta-thalassemia, trait regardless of the type of beta-globin mutation. Furthermore, HFE gene polymorphisms do not change the iron profile in these individuals.

Frequency of ABO blood group system polymorphisms in Plasmodium falciparum malaria patients and blood donors from the Brazilian Amazon region.

We investigated the ABO genotypes and heterogeneity of the O alleles in Plasmodium falciparum-infected and non-infected individuals from the Brazilian Amazon region. Sample collection took place from May 2003 to August 2005, from P. falciparum malaria patients from four endemic regions of the Brazilian Amazon. The control group consisted of donors from four blood banks in the same areas. DNA was extracted using the Easy-DNA(TM) extraction kit. ABO genotyping was performed using PCR/RFLP. There was a high frequency of ABO*O01O01. ABO*AO01 was the second most frequent genotype, and the third most frequent genotype was ABO*BO01. There were low frequencies of the ABO*O01O02, ABO*AA, ABO*AB, ABO*BB, and ABO*O02O02 genotypes. We analyzed the alleles of the O phenotype; the O(1variant) allele was the most frequent, both in malaria and non-malaria groups; consequently, the homozygous genotype O(1)(v)O(1)(v) was the most frequently observed. There was no evidence of the homozygous O(2) allele. Significant differences were not detected in the frequency of individuals with the various alleles in the comparison of the malaria patients and the general population (blood donors).

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 10(3)/microL for females and 2775 x 10(3)/microL 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.

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.

Lipid peroxidation and antioxidant capacity of G6PD-deficient patients with A-(202G>A) mutation.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an enzymopathy in which reduced NADPH concentrations are not maintained, resulting in oxidative damage. We evaluated G6PD activity, oxidative stress levels and Trolox equivalent antioxidant capacity in individuals with the A-(202G>A) mutation for G6PD deficiency. Five hundred and forty-four peripheral blood samples were screened for G6PD deficiency; we also analyzed lipid peroxidation products measured as thiobarbituric acid reactive species and Trolox equivalent antioxidant capacity. Men with the A-(202G>A) mutation had lower G6PD activity than women with the same mutation. Individuals with the A-(202G>A) mutation also differed in mean Trolox equivalent antioxidant capacity values but not for thiobarbituric acid reactive species values. We concluded that A-(202G>A) mutation is associated with reduced G6PD activity and increased Trolox equivalent antioxidant capacity.

Characterization and oxygen binding properties of des-Arg human hemoglobin

The role of chloride in the stabilization of the deoxy conformation of hemoglobin (Hb), the low oxygen affinity state, has been studied in order to identify the nature of this binding. Previous studies have shown that arginines 141α could be involved in the binding of this ion to the protein. Thus, des-Arg Hb, human hemoglobin modified by removal of the α-chain C-terminal residue Arg141α, is a possible model for studies of these interactions. The loss of Arg141α and all the salt bridges in which it participates is associated with subtle structural perturbations of the α-chains, which include an increase in the conformational flexibility and further shift to the oxy state, increasing oxygen affinity. Thus, this Hb has been the target of many studies of structural and functional behavior along with medical applications. In the present study, we describe the biochemical characterization of des-Arg Hb by electrophoresis, high-performance liquid chromatography and mass spectroscopy. The effects of chloride binding on the oxygen affinity and on the cooperativity to des-Arg Hb and to native human hemoglobin, HbA, were measured and compared. We confirm that des-Arg Hb presents high oxygen affinity and low cooperativity in the presence of bound chloride and show that the binding of chloride to des-Arg does not change its functional characteristics as observed with HbA. These results indicate that Arg141α may be involved in the chloride effect on Hb oxygenation. Moreover, they show that these residues contribute to lower Hb oxygen affinity to a level compatible with its biological function.

Characterization and oxygen binding properties of des-Arg human hemoglobin.

The role of chloride in the stabilization of the deoxy conformation of hemoglobin (Hb), the low oxygen affinity state, has been studied in order to identify the nature of this binding. Previous studies have shown that arginines 141alpha could be involved in the binding of this ion to the protein. Thus, des-Arg Hb, human hemoglobin modified by removal of the alpha-chain C-terminal residue Arg141alpha, is a possible model for studies of these interactions. The loss of Arg141alpha and all the salt bridges in which it participates is associated with subtle structural perturbations of the alpha-chains, which include an increase in the conformational flexibility and further shift to the oxy state, increasing oxygen affinity. Thus, this Hb has been the target of many studies of structural and functional behavior along with medical applications. In the present study, we describe the biochemical characterization of des-Arg Hb by electrophoresis, high-performance liquid chromatography and mass spectroscopy. The effects of chloride binding on the oxygen affinity and on the cooperativity to des-Arg Hb and to native human hemoglobin, HbA, were measured and compared. We confirm that des-Arg Hb presents high oxygen affinity and low cooperativity in the presence of bound chloride and show that the binding of chloride to des-Arg does not change its functional characteristics as observed with HbA. These results indicate that Arg141alpha may be involved in the chloride effect on Hb oxygenation. Moreover, they show that these residues contribute to lower Hb oxygen affinity to a level compatible with its biological function.