Red cell aspartate aminotransferase saturation with oral pyridoxine intake.

CONTEXT AND OBJECTIVE: The coenzyme of aspartate aminotransferase is pyridoxal phosphate, generated from fresh vegetables containing pyridoxine. Vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronies syndrome respond to high pyridoxine doses. The objective was to investigate the oral pyridoxine oral dose that would lead to maximized pyridoxal phosphate saturation of red cell aspartate aminotransferase. DESIGN AND SETTING: Controlled trial, in Hematology Division of Instituto Adolfo Lutz. METHODS: Red cell aspartate aminotransferase activity was assayed (before and after) in normal volunteers who were given oral pyridoxine for 15-18 days (30 mg, 100 mg and 200 mg daily). In vitro study of blood from seven normal volunteers was also performed, with before and after assaying of aspartate aminotransferase activity. RESULTS: The in vivo study showed increasing aspartate aminotransferase saturation with increasing pyridoxine doses. 83% saturation was reached with 30 mg daily, 88% with 100 mg, and 93% with 200 mg after 20 days of oral supplementation. The in vitro study did not reach 100% saturation. CONCLUSIONS: Neither in vivo nor in vitro study demonstrated thorough aspartate aminotransferase saturation with its coenzyme pyridoxal phosphate in red cells, from increasing pyridoxine supplementation. However, the 200-mg dose could be employed safely in vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronies syndrome treatment. Although maximum saturation in circulating red cells is not achieved, erythroblasts and other nucleated and cytoplasmic organelles containing cells certainly will reach thorough saturation, which possibly explains the results obtained in these diseases.

Red cell aspartate aminotransferase saturation with oral pyridoxine intake

CONTEXTO E OBJETIVO: A enzima aspartato aminotransferase apresenta o piridoxal fosfato como coenzima, oriunda da piridoxina existente em alimentos vegetais frescos. A anemia sideroblástica responsiva à vitamina B6, mielofibrose e síndrome de Peyronie respondem a altas doses de piridoxina. O objetivo foi investigar a máxima resposta da aspartato aminotransferase à suplementação oral com piridoxina. TIPO DE ESTUDO E LOCAL: Experimento controlado, na Seção de Hematologia, Instituto Adolfo Lutz. MÉTODOS: A atividade da aspartato aminotransferase eritrocitária foi determinada (antes e após) em voluntários que receberam suplementação por 15-18 dias (30 mg, 100 mg e 200 mg diariamente). Estudo in vitro também foi realizado, com sangue de sete indivíduos. As atividades enzimáticas antes e após a incubação foram determinadas, seguindo o mesmo protocolo do estudo in vivo. RESULTADOS: O estudo in vivo revelou um aumento gradativo da saturação da aspartato aminotransferase com doses crescentes de piridoxina. 83% de saturação foi alcançada com 30 mg diariamente, 88% com 100 mg e 93% com 200 mg. O estudo in vitro não revelou saturação de 100%.CONCLUSÕES: Tanto in vivo quanto in vitro, não se revelou saturação completa da aspartato aminotransferase por sua coenzima piridoxal-5-fosfato nos eritrócitos. Entretanto, a dose de 200 mg diariamente poderia ser empregada com segurança no tratamento da anemia sideroblástica, mielofibrose e síndrome de Peyronie. Embora a saturação máxima nos eritrócitos não seja atingida, os eritroblastos e outras células nucleadas que contenham as organelas citoplasmáticas certamente atingirão a saturação completa, possivelmente à razão dos resultados obtidos nas doenças citadas.

The absolute recommendation of chamber Neubauer method for platelets counting instead of indirect methods in severe thrombocytopenic patients

Accurate and precise platelet counting is crucial for recommending platelets transfusion for thrombocytopenic patients, principally when platelet counts are bellow 30,000/µl. As most laboratories still use the indirect methods for confirming low automated platelet counts, this work compared two indirect methods used in practice (Fonio and Nosanchunk et al.) with the International Committee for Standardization in Hematology recommended direct method (Brecher and Cronkite). The obtained data show that the indirect methods present low precision and accuracy, and that the direct method should always be employed in severe thrombocytopenic samples thanks to its high precision

Is automated platelet counting still a problem in thrombocytopenic blood?

CONTEXT: Reliable platelet counting is crucial for indicating prophylactic platelet transfusion in thrombocytopenic patients. OBJECTIVE: To evaluate the precision and accuracy of platelet counting for thrombocytopenic patients, using four different automated counters in comparison with the Brecher & Cronkite reference method recommended by the International Committee for Standardization in Hematology (ICSH). TYPE OF STUDY: Automated platelet counting assessment in thrombocytopenic patients. SETTING: Hematology Laboratory, Hospital do Servidor Público Estadual de São Paulo, and the Hematology Division of Instituto Adolfo Lutz, São Paulo, SP, Brazil. MAIN MEASUREMENTS: Brecher & Cronkite reference method and four different automated platelet counters. PARTICIPANTS: 43 thrombocytopenic patients with platelet counts of less than 30,000/microliter. RESULTS: The ADVIA-120 (Bayer), Coulter STKS, H1 System (Technicom-Bayer) and Coulter T-890 automatic instruments presented great precision and accuracy in relation to laboratory thrombocytopenic samples obtained by diluting blood from normal donors. However, when thrombocytopenic patients were investigated, all the counters except ADVIA (which is based on volume and refraction index) showed low accuracy when compared to the Brecher & Cronkite reference method (ICSH). The ADVIA counter showed high correlation (r = 0.974). However, all counters showed flags in thrombocytopenic samples. CONCLUSION: The Brecher & Cronkite reference method should always be indicated in thrombocytopenic patients for platelet counts below 30,000 plt/microliter obtained in one dimensional counters.

Is automated platelet counting still a problem in thrombocytopenic blood?

CONTEXT: Reliable platelet counting is crucial for indicating prophylactic platelet transfusion in thrombocytopenic patients. OBJECTIVE: To evaluate the precision and accuracy of platelet counting for thrombocytopenic patients, using four different automated counters in comparison with the Brecher & Cronkite reference method recommended by the International Committee for Standardization in Hematology (ICSH). TYPE OF STUDY: Automated platelet counting assessment in thrombocytopenic patients. SETTING: Hematology Laboratory, Hospital do Servidor Público Estadual de Säo Paulo, and the Hematology Division of Instituto Adolfo Lutz, Säo Paulo, SP, Brazil. MAIN MEASUREMENTS: Brecher & Cronkite reference method and four different automated platelet counters. PARTICIPANTS: 43 thrombocytopenic patients with platelet counts of less than 30,000/æl RESULTS: The ADVIA-120 (Bayer), Coulter STKS, H1 System (Technicom-Bayer) and Coulter T-890 automatic instruments presented great precision and accuracy in relation to laboratory thrombocytopenic samples obtained by diluting blood from normal donors. However, when thrombocytopenic patients were investigated, all the counters except ADVIA (which is based on volume and refraction index) showed low accuracy when compared to the Brecher & Cronkite reference method (ICSH). The ADVIA counter showed high correlation (r = 0.947). However, all counters showed flags in thrombocytopenic samples. CONCLUSION: The Brecher & Cronkite reference method should always be indicated in thrombocytopenic patients for platelet counts below 30,000 plt /µl obtained in one dimensional counters

Red cell glutathione reductase saturation obtained with oral riboflavin supplementation

The erythrocyte glutathione reductase (E-GR) saturation with its coenzyme flavin adenine dinucleotide (FAD) affords the nutritional status regarding the vitamin B2, as FAD proceeds from riboflavin intake. E-GR normally exhibits physiological partial saturation with its coenzyme. OBJECTIVE: To study the maximum in vivo saturation which could be reached by increasingly oral vitamin supplemetantion. METHODS: Three groups of normal volunteers were given 3 mg (group A), 20 mg ( group B) and 50 mg (group C) riboflavin daily during twenty days. The enzyme activities were assayed before and after supplementation, with and without coenzyme addition to the reagent system. RESULTS: 77 per cent, 83 per cent and 87 per cent saturation were observed in the groups A, B and C respectively. CONCLUSION: Increasing in vivo saturation was observed with increasing oral doses of riboflavin supplementation, but total saturation was not observed even with the highest oral 50 mg roboflavin supplementation

Porphobilmogen deaminase gene mutations in Brazilian acute intermittent porphyria patients.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder resulting from porphobilmogen deaminase (PBGD) deficiency. Seven unrelated Brazilian patients were investigated regarding PBGD gene mutations by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) analysis followed by direct DNA sequencing. The PBG gene screening disclosed abnormal SSCP patterns in exons 7, 9, 12, 13, and 15, as well as in introns 3 and 10. Direct DNA sequencing revealed the occurrence of three nonsense mutations (R149X, R225X, and R325X) in exons 9, 12, and 15, respectively, and one missense mutation G111R in exon 7. The G111R mutation was detected in two unrelated patients. Intragenic polymorphisms (3119G/T in intron 2, 3581G/A in intron 3, 7052A/G and 7064C/A in intron 10, and -65C/T in exon 1) were also observed. In addition, two silent mutations (V202V in exon 10 and A266A in exon 13) were found. The latter has not heretofore been reported. Thus, this study revealed the mutations involved in Brazilian symptomatic AIP patients, as well as the intragenic polymorphisms found in the patients.

Normal erythrocyte calpain I activity on membrane proteins under near-physiological conditions in patients with essential hypertension.

CONTEXT: It has been reported that the equilibrium between the erythrocyte protease calpain I and its physiological inhibitor calpastatin is disrupted in patients with essential hypertension. OBJECTIVE: To investigate the activity of non-purified calpain I in hemolysates against the erythrocytic membrane proteins, rather than against other substrates. DESIGN: Evaluation of calpain I red cell activity upon its own physiological substrates in hypertensive patients, in a near-physiological environment. SETTING: LIM-23 and LIM-40 of Hospital das Clinicas of the Faculty of Medicine of USP. SAMPLE: Patients with moderate primary hypertension over 21 years of age who were given amlodipine (n:10) and captopril (n:10) for 8 weeks, plus normal controls (n:10). MAIN MEASUREMENTS: Red cell membrane proteins were incubated with and without protease inhibitors and with and without calcium chloride and underwent polyacrylamide gel electrophoresis. RESULTS: Digestion of bands 2.1 and 4.1 was observed, indicating calpain I activity. No statistical differences regarding bands 2.1 and 4.1 were observed before treatment, between the controls and the hypertensive patients, either in ghosts prepared without calcium or with increasing concentrations of calcium. Nor were statistical differences observed after treatment, between the controls and the patients treated with amlodipine and captopril, or between the patients before and after treatment with both drugs. CONCLUSION: The final activity of non-purified calpain I upon its own physiological substrate, which was the approach utilized in this study, may more adequately reflect what happens in red cells. Under such conditions no imbalance favoring calpain I activity increase was observed. The protective factor provided by calpastatin against calpain I activity may diminish under hypertension.

Normal erythrocyte calpain I activity on membrane proteins under near-physiological conditions in patients with essential hypertension

CONTEXT: It has been reported that the equilibrium between the erythrocyte protease calpain I and its physiological inhibitor calpastatin is disrupted in patients with essential hypertension. OBJECTIVE: To investigate the activity of non-purified calpain I in hemolysates against the erythrocytic membrane proteins, rather than against other substrates. DESIGN: Evaluation of calpain I red cell activity upon its own physiological substrates in hypertensive patients, in a near-physiological environment. SETTING: LIM-23 and LIM-40 of Hospital das Clinicas of the Faculty of Medicine of USP. SAMPLE: Patients with moderate primary hypertension over 21 years of age who were given amlodipine (n:10) and captopril (n:10) for 8 weeks, plus normal controls (n:10). MAIN MEASUREMENTS: Red cell membrane proteins were incubated with and without protease inhibitors and with and without calcium chloride and underwent polyacrylamide gel electrophoresis. RESULTS: Digestion of bands 2.1 and 4.1 was observed, indicating calpain I acitivity. No statistical differences regarding bands 2.1 and 4.1 were observed before treatment, between the controls and the hypertensive patients, either in ghosts prepared without calcium or with increasing concentrations of calcium. Nor were statistical differences observed after treatment, between the controls and the patients treated with amlodipine and captopril, or between the patients before and after treatment with both drugs. CONCLUSION: The final activity of non-purified calpain I upon its own physiological substrate, which was the approach utilized in this study, may more adequately reflect what happens in red cells. Under such conditions no imbalance favoring calpain I activity increase was observed. The protective factor provided by calpastatin against calpain I activity may diminish under hypertension

Enzymes and membrane proteins of ADSOL-preserved red blood cells

CONTEXT: The preservative solution ADSOL (adenine, dextrose, sorbitol, sodium chloride and mannitol) maintains red cell viability for blood trans-fusion for 6 weeks. It would be useful to know about its preservation qualities over longer periods. OBJECTIVE: To determine some red cell biochemical parameters for peri-ods of up to 14 weeks in order to determine whether the red cell metabo-lism integrity would justify further studies aiming at increasing red cell preservation and viability. DESIGN: Biochemical evaluation designed to study red cell preservation. SETTING: Sao Paulo University erythrocyte metabolism referral center. SAMPLE: Six normal blood donors from the University Hospital of the Universidade Federal do Paranß, Curitiba, Brazil. MAIN MEASUREMENTS: Weekly assay of erythrocyte adenosine-5Ý-triphosphate (ATP), 2,3-diphosphoglycerate (2,3DPG), hexokinase (HX), phosphofructokinase (PFK), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconic dehydrogenase (6-PGD), glyceraldehyde-3-phosphate dehydrogenase (GAPD), glutathione reduc-tase (GR), glutathione peroxidase (GSHPx), plasma sodium and potas-sium, blood pH, and membrane proteins of red cells preserved in ADSOL were studied during storage for 14 weeks storage. RESULTS: During ADSOL preservation, erythrocyte ATP concentration decreased 60 percent after 5 weeks, and 90 percent after 10 weeks; the pH fell from 6.8 to 6.4 by the 14th week. 2,3-DPG concentration was stable during the first week, but fell 90 percent after 3 weeks and was exhausted after 5 weeks. By the end of the 5th week, an activity decrease of 16-30 percent for Hx, GAPD, GR, G-6-PD and 6-PGD, 35 percent for PFK and GSHPx, and 45 percent for PK were observed. Thereafter, a uniform 10 percent decay was observed for all enzymes up to the 14th week. The red blood cell membrane pro-teins did not show significant alterations in polyacrylamide gel electro-phoresis (SDS-PAGE) during the 14 weeks. CONCLUSION: Although the blood viability was shown to be poor from the 6th week up to the 14th week of storage due to ATP and 2,3-DPG depletion, the other biochemical parameters remained in fairly good condition for longer storage. As there is a gradual and uniform decay in activity throughout these 14 weeks, it seems that ADSOL-preserved red cells may be used as red cell enzyme standards and membrane proteins as well

Hematological and biochemical profiles of rats (Rattus norvegicus) kept under microenvironmental ventilation system

Previous studies reported that rats (Rattus norvegicus) kept under microenvironmental ventilation systems (MEV) present better productive and health parameters when compared to animals kept under general diluting ventilation (GDV). The objective of the present research trial was to evaluate hematological and biochemical profiles of rats kept under MVS. In order to achieve this objective, two different trials were designed: Trail 1 (E1), in which it was evaluated the reproductive performance of males and females submitted to two different air speed limits - FV1, from 0.03 to 0.26 m / sec and FV2, from 0.27 to 0.80 m / sec. In Trial 2 (E2) it was evaluated different bed change intervals (3, 5, 7 and 9 days), for males kept under constant air speed (0.5 m / sec). Values for hemogram and biochemical patterns of these animals were compared to those of rats kept under GDV. Results show statistical differences in some of the studied parameters not only for the comparison between GVD and E1 and GVD and E2, but also between both groups submitted to MEV (E1 and E2). However, values found for all studied parameters are inside the normal range reported for this species, what indicates that MEV does not induce important changes in the physiological parameters evaluated

Hematological and biochemical profiles of rats (Rattus norvegicus) kept under microenvironmental ventilation system / Perfis hematológico e bioquímico de ratos (Rattus norvegicus) mantidos sob sistema de ventilação microambiental

Previous studies reported that rats (Rattus norvegicus) kept under microenvironmental ventilation systems (MEV) present better productive and health parameters when compared to animals kept under general diluting ventilation (GDV). The objective of the present research trial was to evaluate hematological and biochemical profiles of rats kept under MVS. In order to achieve this objective, two different trials were designed: Trail 1 (E1), in which it was evaluated the reproductive performance of males and females submitted to two different air speed limits - FV1, from 0.03 to 0.26 m / sec and FV2, from 0.27 to 0.80 m / sec. In Trial 2 (E2) it was evaluated different bed change intervals (3, 5, 7 and 9 days), for males kept under constant air speed (0.5 m / sec). Values for hemogram and biochemical patterns of these animals were compared to those of rats kept under GDV. Results show statistical differences in some of the studied parameters not only for the comparison between GVD and E1 and GVD and E2, but also between both groups submitted to MEV (E1 and E2). However, values found for all studied parameters are inside the normal range reported for this species, what indicates that MEV does not induce important changes in the physiological parameters evaluated.

Em estudos anteriores, demonstrou-se que ratos mantidos em sistema de Ventilação Microambiental (VMA) apresentaram parâmetros de produtividade e padrão sanitário melhores do que aqueles mantidos em sistema de Ventilação Geral Diluidora (VGD). Outra etapa dos experimentos foi determinar os parâmetros fisiológicos destes animais. O presente estudo foi realizado para avaliar os perfis hematológico e bioquímico de ratos mantidos sob o sistema de VMA. Para tanto, foram realizados dois experimentos diferentes, com ratos mantidos em VMA, quais sejam: Experimento 1 (E1), no qual foi avaliado o desempenho reprodutivo de machos e fêmeas, sob duas faixas de velocidade de ar (FV1 - de 0,03 a 0,26 m/s, e FV2 - de 0,27 a 0,80 m/s); Experimento 2 (E2), no qual foram avaliados diferentes intervalos de troca de cama (3, 5, 7 e 9 dias), para ratos machos mantidos a uma velocidade de ar constante de 0,5 m/s. Os valores do hemograma e de parâmetros bioquímicos destes animais foram comparados com os valores encontrados em ratos mantidos sob VGD. Os resultados obtidos demonstraram diferenças estatísticas em alguns dos parâmetros observados, tanto entre os sistemas VGD e VMA, como entre os diferentes grupos de VMA. Contudo, os valores encontrados em todos os parâmetros avaliados encontram-se dentro de faixas de variação normal para a espécie estudada, como é descrito na literatura. Isto indica que o

Avaliaçäo do quociente das proteínas da membrana eritrocitária e da proteína 3 nas anemias esferocíticas / Red cell membrane proteins ratios in relation to band 3 in spherocytosis

As proteínas da membrana eritrocitária foram estudadas por meio de eletroforese de poliacrilamida (SDS-PAGE) em nove pacientes, utilizando-se o quociente de todas as bandas protéicas em relaçäo à banda 3; um grupo controle normal foi utilizado. O quociente espectrina/banda 3 dos pacientes com esferocitose foi baixo (p=0,0001), o que indica deficiência de espectrina. Foi também observado um aumento relativo geral das demais proteínas, em especial das bandas 4.1 e 4.5 (p=0,046 e p=0,0001, respectivamente), o que pode ser imputado à diminuiçäo da concentraçäo das espectrinas

Search of intravascular hemolysis in patients with the cutaneous form of loxoscelism

Dezenove pacientes que apresentaram a forma clinica cutanea do loxoscelismo foram investigados com o proposito de pesquisar hemolise intravascular sub-clinica. lancando mao da dosagem de haptoglobina, um metodo altamente sensivel que permite detectar discreta presenca de hemolise intravascular. Nao fois encontrada diminuicao do veneno da Loxosceles nestes pacientes.

Padröes eritrocitários obtidos em populaçäo adulta com valores normais de ferro, ferritina e folatos séricos, na cidade de Säo Paulo / Erythrocyte values obtained from adults presenting normal iron, ferritin and folate serum levels in Säo Paulo city (800m of altitude)

Na cidade de Säo Paulo, situada a 800 metros acima do mar, foi realizado um inquérito populacional para determinar valores hematimétricos eritrocitários normais em indivíduos adultos...

Purificaçö parcial da desidrogenase lática eritrocitária / Partial purification of erythrocyte lactate dehydrogenase

A purificaçäo parcial da desidrogenase lática (LDH) de glóbulos vermelhos humanos vencidos foi efetuada utilizando-se, basicamente, a DEAE-celulose equilbrada em tampäo fosfato 10mM, pH 7,6, em que a hemoglobina näo se liga, mas a desidrogenase lática se adere à resina. A eluiçäo foi feita em gradiente fosfato 10-100mM e as fraçöes onde as atividades de LDH estavam presentes foram reunidas, dialisadas e novamente submetidas a cromatografia e eluiçäo. A preparaçäo final mostrou ausência de piruvato quinase e das enzimas da glicólise, com exceçäo de negligível contaminaçäo com monofosfogliceromutase e glutationa peroxidase, também presentes na preparaçäo Sigma L2881 obtida de coraçäo de suíno

Hemoglobina fetal no recém-nascido e tempo de gestaçäo / Fetal hemoglobin in the newborn infant and gestational age

Em 113 recém-nascidos de 31 a 44 semanas de gestaçäo foram determinados os teores de hemoglobina F do feto, hemoglobina total do feto e hemoglobina da gestante. Näo foi encontrada correlaçäo entre a hemoglobina F e as hemoglobinas totais do feto e da mäe, mas se encontrou discreta correlaçäo negativa entre o tempo de gestaçäo e os teores de hemoglobina F do feto

Erythrocyte adenosine deaminase deficiency in Brazil

A atividade da adenosina deaminase eritrocitária (E.C.3.5.4.4) foi feita em 300 doadores de sangue através de determinaçäo quantitativa em espectrofotômetro. Valores normais de 1,7 + ou - 0,7 u.i./g Hb/min/37-C foram estabelecidos, e foi encontrado um indivíduo heterozigoto que exibiu atividade de 0,5 u.i./g Hb/min/37-C, o que permitiu a determinaçäo da freqüência do gene em 0,0033 e a probabilidade da ocorrência de um homozigoto deficiente em 100.000 nascimentos