RESUMEN
Newborn screening is an accepted public health measure to ensure that appropriate health care is provided in a timely manner to infants with hereditary/metabolic disorders. Alpha-thalassemia is a common hemoglobin (Hb) disorder, and causes Hb H (beta4) disease, and usually fatal homozygous alpha(0)-thalassemia, also known as Hb Bart's (gamma4) hydrops fetalis syndrome. In 1996, the State of California began to investigate the feasibility of universal newborn screening for Hb H disease. Initial screening was done on blood samples obtained by heel pricks from newborns, and stored as dried blood spots on filter paper. Hb Bart's levels were measured as fast-moving Hb by automated high-performance liquid chromatography (HPLC) identical to that currently used in newborn screening for sickle cell disease. Subsequent confirmation of Hb H disease was done by DNA-based diagnostics for alpha-globin genotyping. A criterion of 25% or more Hb Bart's as determined by HPLC detects most, if not all cases of Hb H disease, and few cases of alpha-thalassemia trait. From January, 1998, through June, 2000, 89 newborns were found to have Hb H disease. The overall prevalence for Hb H disease among all newborns in California is approximately 1 per 15,000. Implementation of this program to existing newborn hemoglobinopathy screening in populations with significant proportions of southeast Asians is recommended. The correct diagnosis would allow affected infants to be properly cared for, and would also raise awareness for the prevention of homozygous alpha(0)-thalassemia or Hb Bart's hydrops fetalis syndrome.
Asunto(s)
Pruebas Genéticas , Hemoglobina H/análisis , Hemoglobinas Anormales/análisis , Tamizaje Neonatal , Talasemia alfa/epidemiología , Asia Sudoriental/etnología , Asiático , California , Cromatografía Líquida de Alta Presión , Femenino , Frecuencia de los Genes , Genotipo , Globinas/deficiencia , Globinas/genética , Hemoglobina H/genética , Hemoglobinas Anormales/genética , Humanos , Hidropesía Fetal/genética , Hidropesía Fetal/prevención & control , Recién Nacido , Masculino , Mutación Missense , Prevalencia , Eliminación de Secuencia , Talasemia alfa/diagnóstico , Talasemia alfa/etnología , Talasemia alfa/genéticaRESUMEN
Although hematopoietic stem cell transplantation has curative potential for selected patients with sickle cell disease (SCD), most patients who are eligible for transplantation do not have a suitable donor. Cord blood (CB) from a sibling could provide an alternative stem cell source that, while not as well established as marrow, may offer certain advantages for selected families. These potential advantages include low risk to the infant donor, the possibility that mismatched CB units from sibling donors may be acceptable for transplantation, prompt availability of a stored CB unit for transplant, and decreased risk of clinically significant graft-versus-host disease. When families with SCD (or other transplant-treatable condition) conceive a sibling, no comprehensive research resource exists to assist the family in collecting the new infant's CB. With support from the National Heart Lung and Blood Institute, we are developing a noncommercial research-based CB Banking Program specifically for medically indicated sibling donations. In preliminary experience, we have collected CB from 52 SCD families across 19 states. Of these, 2 CB units have thus far been used for transplantation and 9 others are HLA-identical. We conclude that a CB bank focusing on sibling-donations may be feasible, but further study is required to determine whether such a bank can collect CB units of sufficient quantity and quality to support controlled trials of sibling CB transplantation. Families with a specific medical need, such as those already caring for a child with SCD, should consider collecting sibling CB as part of comprehensive care if the opportunity becomes available.
Asunto(s)
Anemia de Células Falciformes/terapia , Bancos de Sangre , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal , Donantes de Tejidos , Obtención de Tejidos y Órganos/organización & administración , California , Manejo de Caso , Niño , Femenino , Control de Formularios y Registros , Histocompatibilidad , Humanos , Recién Nacido , Control de Infecciones , Masculino , National Institutes of Health (U.S.) , Selección de Paciente , Embarazo , Derivación y Consulta , Seguridad , Hermanos , Estados UnidosRESUMEN
Bone marrow transplantation has curative potential for patients with thalassemia major who have a matched sibling marrow donor, but usefulness of alternative stem cell sources is undergoing investigation. Cord blood (CB) from a sibling has different characteristics from marrow and has potential advantages and disadvantages as a stem cell source. Whereas many families caring for a child with thalassemia major (or other transplant-treatable condition) experience an additional pregnancy, most give birth at hospitals without the infrastructure needed to collect and process the new infant's CB. To address this, and with funding from the National Institutes of Health, we have developed the first noncommercial CB program, operating across the United States, designed specifically to facilitate medically indicated CB collections from sibling donors. Using a case-management model, we have collected CB for 25 thalassemia families in eight states. Three of these CB units have now been used for transplantation; two others are human leukocyte antigen-identical and contain adequate nucleated cell dose to perform transplantation in their intended recipient. We conclude that a CB bank focused on sibling donations may be a useful stem cell resource and that families with specific medical need, such as a child with thalassemia, should consider preserving CB from siblings.
Asunto(s)
Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Talasemia beta/terapia , Recolección de Muestras de Sangre , Niño , Femenino , Humanos , Recién Nacido , Núcleo Familiar , Placenta , Embarazo , Diagnóstico Prenatal , Donantes de Tejidos , Recolección de Tejidos y ÓrganosRESUMEN
Phospholipid asymmetry is well maintained in erythrocyte (RBC) membranes with phosphatidylserine (PS) exclusively present in the inner leaflet. The appearance of PS on the surface of the cell can have major physiologic consequences, including increased cell-cell interactions. Because increased adherence of PS-exposing RBCs to endothelial cells (ECs) may be pathologically important in hemoglobinopathies such as sickle cell disease and thalassemia, we studied the role of PS exposure in calcium ionophore-treated normal RBC adherence to human umbilical vein endothelial cell (HUVEC) monolayers. When HUVEC monolayers were incubated with these PS-exposing RBCs, the ECs retracted and the RBCs adhered primarily in the gaps opened between the ECs. A linear correlation was found between the number of PS-exposing RBCs in the population and the number of adhering RBCs to the monolayer. Pretreatment of RBCs with annexin V significantly decreased adherence by shielding PS on the RBCs. Similarly, PS-containing lipid vesicles decreased RBC binding by competing for the PS binding sites in the monolayer. PS-exposing RBCs and PS-containing lipid vesicles adhered to immobilized thrombospondin (TSP) and matrix TSP, respectively, and adherence of PS-exposing RBCs to EC monolayers was reduced by antibodies to TSP and to its EC receptor, alpha(v)beta(3). Together, these results indicate a role for PS and matrix TSP in the adherence of PS-exposing RBCs to EC monolayers, and suggest an important contribution of PS-exposing RBCs in pathologies with reported vascular damage, such as sickle cell anemia. (Blood. 2000;95:1293-1300)
Asunto(s)
Anemia de Células Falciformes/sangre , Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Eritrocitos/fisiología , Fosfatidilserinas/farmacología , Trombospondinas/fisiología , Proteína 1 de Intercambio de Anión de Eritrocito/fisiología , Antígenos CD/sangre , Antígenos CD36/sangre , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Niño , Endotelio Vascular/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Matriz Extracelular/fisiología , Histamina/farmacología , Humanos , Técnicas In Vitro , Valores de Referencia , Venas UmbilicalesRESUMEN
Erythrocyte transfusion can impair detection of sickle-cell disease, galactosemia, or biotinidase deficiency with newborn screening. We report on 4 infants with SCD in whom delayed diagnosis was associated with neonatal transfusion. In 2 cases, the initial newborn screening showed no hemoglobin S. In no case was the recommended screening >/=120 days from the last transfusion obtained. Two children had significant SCD-related morbidity before diagnosis.
Asunto(s)
Anemia Neonatal/terapia , Anemia de Células Falciformes/diagnóstico , Incompatibilidad de Grupos Sanguíneos/terapia , Transfusión de Eritrocitos , Tamizaje Neonatal , Preescolar , Humanos , Lactante , Recién NacidoRESUMEN
Full-length cDNA species encoding two forms of acyl-CoA synthetase from a K-562 human erythroleukaemic cell line were cloned, sequenced and expressed. The first form, named long-chain acyl-CoA synthetase 5 (LACS5), was found to be a novel, unreported, human acyl-CoA synthetase with high similarity to rat brain ACS2 (91% identical). The second form (66% identical with LACS5) was 97% identical with human liver LACS1. The LACS5 gene encodes a highly expressed 2.9 kb mRNA transcript in human haemopoietic stem cells from cord blood, bone marrow, reticulocytes and fetal blood cells derived from fetal liver. An additional 6.3 kb transcript is also found in these erythrocyte precursors; 2.9 and 9.6 kb transcripts of LACS5 are found in human brain, but transcripts are virtually absent from human heart, kidney, liver, lung, pancreas, spleen and skeletal muscle. The 78 kDa expressed LACS5 protein used the long-chain fatty acids palmitic acid, oleic acid and arachidonic acid as substrates. Antibodies directed against LACS5 cross-reacted with erythrocyte membranes. We conclude that early erythrocyte precursors express at least two different forms of acyl-CoA synthetase and that LACS5 is present in mature erythrocyte plasma membranes.
Asunto(s)
Coenzima A Ligasas/sangre , Eritrocitos/enzimología , Células Madre Hematopoyéticas/enzimología , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Coenzima A Ligasas/química , Coenzima A Ligasas/genética , Cartilla de ADN/genética , ADN Complementario/genética , Membrana Eritrocítica/enzimología , Sangre Fetal/citología , Sangre Fetal/enzimología , Humanos , Técnicas In Vitro , Recién Nacido , Células K562 , Datos de Secuencia Molecular , Peso Molecular , ARN Mensajero/sangre , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Distribución TisularAsunto(s)
Bancos de Sangre/organización & administración , Sangre Fetal , Recolección de Muestras de Sangre/métodos , Criopreservación , Ética Médica , Sangre Fetal/microbiología , Sangre Fetal/fisiología , Política de Salud , Prueba de Histocompatibilidad/métodos , Humanos , Control de Infecciones/métodos , Rol del Médico , Garantía de la Calidad de Atención de SaludRESUMEN
BACKGROUND: There is increasing use of highly sensitive testing with polymerase chain reaction (PCR) to study white cell microchimerism after transfusion and transplantation. This study investigated possible artifactual sources of allogeneic sample contamination before PCR testing. STUDY DESIGN AND METHODS: Quantitative Y-chromosome PCR was used to study microchimerism among transfused patients with sickle cell disease (SCD) and thalassemia by using residual specimens from the clinical laboratory. High levels of circulating male white cells among transfused patients with SCD but not thalassemia led to concern over the artifactual origin of male cells. To investigate, paired specimens were collected from 26 female SCD patients: one specimen underwent processing only for PCR, while the other underwent testing in the clinical laboratory before PCR as a process control. All laboratory instruments were also assessed for their ability to impart male allogeneic cells to aliquots of female blood. RESULTS: Thirty-three (31%) of 107 SCD samples, but 0 of 20 thalassemia samples, gave a high-level PCR signal. One of 26 paired samples that was not exposed to clinical laboratory equipment had low-level PCR positivity while 10 of the 26 became strongly positive after testing on a blood cell analyzer and a reticulocyte analyzer. Sixteen of 32 female samples became positive after reticulocyte analysis, while none became positive after blood cell analysis. Samples from thalassemia patients tested PCR-negative because reticulocyte counts had not been performed. CONCLUSION: Allogeneic cell contamination is common with clinical laboratory equipment. These samples may not be suitable for microchimerism studies. In addition to method controls, process controls should be employed where appropriate.
Asunto(s)
Pruebas Hematológicas/instrumentación , Leucocitos/química , Reacción en Cadena de la Polimerasa , Manejo de Especímenes , Anemia de Células Falciformes/genética , Transfusión Sanguínea , ADN/análisis , Contaminación de Medicamentos , Equipo Reutilizado , Reacciones Falso Positivas , Femenino , Humanos , Análisis por Apareamiento , Talasemia/genética , Quimera por Trasplante/genética , Cromosoma Y/genéticaRESUMEN
Acute chest syndrome (ACS) is associated with significant morbidity and is the leading cause of death in patients with sickle cell disease (SCD). Recent reports suggest that bone marrow fat embolism can be detected in many cases of severe ACS. Secretory phospholipase A2 (sPLA2) is an important inflammatory mediator and liberates free fatty acids, which are felt to be responsible for the acute lung injury of the fat embolism syndrome. We measured SPLA2 levels in 35 SCD patients during 20 admissions for ACS, 10 admissions for vaso-occlusive crisis, and during 12 clinic visits when patients were at the steady state. Eleven non-SCD patients with pneumonia were also evaluated. To determine if there was a relationship between sPLA2 and the severity of ACS we correlated SPLA2 levels with the clinical course of the patient. In comparison with normal controls (mean = 3.1 +/- 1.1 ng/mL), the non-SCD patients with pneumonia (mean = 68.6 +/- 82.9 ng/mL) and all three SCD patient groups had an elevation of SPLA2 (steady state mean = 10.0 +/- 8.4 ng/mL; vaso-occlusive crisis mean = 23.7 +/- 40.5 ng/mL; ACS mean = 336 +/- 209 ng/mL). In patients with ACS sPLA2 levels were 100-fold greater than normal control values, 35 times greater than values in SCD patients at baseline, and five times greater than non-SCD patients with pneumonia. The degree of SPLA2 elevation in ACS correlated with three different measures of clinical severity and, in patients followed sequentially, the rise in SPLA2 coincided with the onset of ACS. The dramatic elevation of SPLA2 in patients with ACS but not in patients with vaso-occlusive crisis or non-SCD patients with pneumonia and the correlation between levels of SPLA2 and clinical severity suggest a role for SPLA2 in the diagnosis and, perhaps, in the pathophysiology of patients with ACS.
Asunto(s)
Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/enzimología , Enfermedades Pulmonares/etiología , Fosfolipasas A/sangre , Enfermedad Aguda , Adolescente , Adulto , Niño , Preescolar , Embolia Grasa/enzimología , Embolia Grasa/etiología , Ácidos Grasos no Esterificados/metabolismo , Femenino , Humanos , Lactante , Inflamación/enzimología , Isquemia/enzimología , Isquemia/etiología , Enfermedades Pulmonares/enzimología , Enfermedades Pulmonares/fisiopatología , Masculino , Fosfolipasas A2 , Neumonía/enzimología , Embolia Pulmonar/enzimología , Embolia Pulmonar/etiologíaRESUMEN
The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could be isolated from the population using magnetic beads covered with an anti-FITC antibody. Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.
Asunto(s)
Anexina A5/metabolismo , Membrana Eritrocítica/química , Lípidos de la Membrana/análisis , Fosfolípidos/análisis , Anemia de Células Falciformes/sangre , Calcimicina/farmacología , Calcio/farmacología , Separación Celular , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/ultraestructura , Eritrocitos/clasificación , Eritrocitos Anormales/química , Eritrocitos Anormales/ultraestructura , Etilmaleimida/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Separación Inmunomagnética , Microscopía Fluorescente , Fosfatidilserinas/metabolismoRESUMEN
The enzyme acyl coenzyme A:1-acyllysophosphatidylcholine acyltransferase (acyl-CoA:lysoPC acyltransferase) can be isolated in newly formed phosphatidylcholine (PC) vesicles by solubilization of rat liver microsomes with the two substrates lysoPC and acyl-CoA. In this study, we sought to optimized the conditions for the formation of PC vesicles and analyzed the lipid composition and enzyme activity of the newly formed vesicles. Analysis of PC vesicles formed by incubation of the microsomal preparation with 1-(C16:0)lysoPC and C18:1CoA, C18:2CoA, or C20:4CoA showed that the optimal protein:lysoPC ratio was 1:5 (by weight) and the optimal lysoPC:acyl-CoA ratio was 1:1 (molar amounts). PC formation increased with incubation time; after 20 h of incubation at 37 degrees C, approximately 75% of the lysoPC was converted to PC in the incubation mixture. The phospholipid molecular species composition of the vesicles reflected almost exclusively the substrates used; the vesicles contained approximately 33% of the total acyl-CoA:lysoPC acyltransferase activity from the microsomes and demonstrated a single protein band with a molecular mass of 21 kDa by gel electrophoresis. The procedure selected for the enzyme specific for lysoPC acylation, as enzyme activity toward lysophosphatidylethanolamine (lysoPE), lysophosphatidylserine (lysoPS), and lysophosphatidylinositol (lysoPI), was very low. In addition, the utilization of different acyl-CoA substrates for acylation of lysoPC was different from that in microsomes. These results show that an enzyme specific for the formation of PC from lysoPC can be isolated in PC vesicles with a designed phospholipid molecular species composition and that the lipid environment plays an important role in the regulation of the enzyme's affinity for its substrates.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Microsomas Hepáticos/enzimología , 1-Acilglicerofosfocolina O-Aciltransferasa/aislamiento & purificación , Acilcoenzima A , Animales , Técnicas In Vitro , Liposomas , Peso Molecular , Fosfatidilcolinas/química , Ratas , Solubilidad , Especificidad por SustratoRESUMEN
Evaluation of hemolytic anemia in the newborn may be complicated owing to the physiologic changes that occur during this time; however, the newborn period is a time when congenital red cell abnormalities may first present and when maternal factors need to be considered. In this article, an approach to the diagnosis of hemolytic disease in the newborn is reviewed. The unique properties of the neonatal red cell, the normal red cell changes present in the neonate, the potential congenital defects and maternal factors that may influence the associated clinical and laboratory findings consistent with the diagnosis of hemolytic anemia, and a brief review of the red cell disorders associated with hemolytic anemia in the newborn are discussed.
Asunto(s)
Anemia Hemolítica , Anemia Neonatal , Anemia Hemolítica/diagnóstico , Anemia Hemolítica/etiología , Anemia Hemolítica/fisiopatología , Anemia Hemolítica Congénita/diagnóstico , Anemia Hemolítica Congénita/etiología , Anemia Neonatal/diagnóstico , Anemia Neonatal/etiología , Anemia Neonatal/fisiopatología , Humanos , Recién NacidoRESUMEN
Acyl-CoA-binding protein (ACBP) has been identified in a number of tissues and shown to affect the intracellular distribution and utilization of acyl-CoA. We have detected ACBP in the cytosol but not the membrane of human red blood cells and, using an e.l.i.s.a. with antibodies prepared against human liver ACBP, found that its concentration was 0.5 microM. To investigate the role of ACBP in human red blood cells, we added purified human liver ACBP and radiolabelled acyl-CoA to isolated membranes from these cells. ACBP prevented high concentrations of acyl-CoA from binding to the membrane but could not keep the acyl-CoA in the aqueous phase at low concentrations. This suggested the presence of a pool in the membrane with a binding affinity for acyl-CoA that was greater than that of ACBP for acyl-CoA. In the presence of lysophospholipid, this membrane-bound pool of acyl-CoA was rapidly used as a substrate by acyl-CoA:lysophospholipid acyltransferase (LAT) to generate phospholipid from lysophospholipid. We also found that ACBP-bound acyl-CoA was preferred over free acyl-CoA as a substrate by LAT. These results are the first documentation that human red blood cells contain ACBP and that this protein can affect the utilization of acyl-CoA in plasma membranes of these cells. The interactions between acyl-CoA, ACBP and the membrane suggest that there are several pools of acyl-CoA in the human red blood cell and that ACBP may have a role in regulating their distribution and fate.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Eritrocitos/metabolismo , Fosfolípidos/metabolismo , Acilcoenzima A/metabolismo , Proteínas Portadoras/metabolismo , Inhibidor de la Unión a Diazepam , Membrana Eritrocítica/metabolismo , HumanosRESUMEN
Hydroxyurea can increase fetal hemoglobin (HbF) and improve the clinical course of sickle cell disease (SCD) patients. However, several issues of hydroxyurea therapy remain unresolved, including differences in patients' drug clearance, predictability of drug response, reversibility of sickle cell disease-related organ damage by hydroxyurea, and the efficacy of elevated HbF. We treated two patients with hydroxyurea for periods of 1 to 4 years, monitoring clinical course and laboratory parameters at regular intervals. The first patient (patient A) had a history of chronic pain and extensive hospitalizations. The second patient (patient B) had a history of stroke and refused to continue with chronic transfusion therapy and chelation. Both patients showed a fivefold to tenfold increase in HbF (5% to 25%, 3% to 31%). However, patient A developed an acute chest syndrome, despite an HbF level of 20%. After red blood cell transfusions for hypoxia, the HbF level decreased to 5%. When hydroxyurea dosage was increased, pancytopenia developed and was not resolved until 2 months after hydroxyurea was discontinued; Patient B developed a cerebral hemorrhage on hydroxyurea; he died shortly thereafter. His HbF level was 21% before death. We noted an increase in HbF and a general improvement in the two patients. However, both experienced major SCD-related complications despite HbF levels over 20%. Our findings also suggest that the progressive vascular changes associated with SCD are unlikely to be dramatically affected by increased HbF levels. Because neither the efficacy nor the toxicity of hydroxyurea have been thoroughly investigated, physicians should be cautious in prescribing hydroxyurea for patients with SCD before completion of the National Clinical Trial.
Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Hemoglobina Fetal/metabolismo , Hidroxiurea/efectos adversos , Pancitopenia/inducido químicamente , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/complicaciones , Transfusión Sanguínea , Hemorragia Cerebral/etiología , Trastornos Cerebrovasculares/complicaciones , Femenino , Humanos , Hidroxiurea/uso terapéutico , Masculino , Dolor , Negativa del Paciente al TratamientoRESUMEN
We studied phospholipid topology and transbilayer mobility in red cells during blood storage. The distribution of phospholipids was determined by measuring the reactivity of phosphatidylethanolamine with fluorescamine and the degradation of phospholipids by phospholipase A2 and sphingomyelinase C. Phospholipid mobility was measured by determining transbilayer movements of spin-labeled phospholipids. We were unable to detect a change in the distribution of endogenous membrane phospholipids in stored red cells even after 2-mo storage. The rate of inward movement of spin-labeled phosphatidylethanolamine and phosphatidylserine was progressively reduced, whereas that for phosphatidylcholine was increased. These changes in phospholipid translocation correlated with a fall in cellular ATP. However, following restoration of ATP, neither the rate of aminophospholipid translocation nor the transbilayer movement of phosphatidylcholine were completely corrected. Taken together, our findings demonstrate that red cell storage alters the kinetics of transbilayer mobility of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, the activity of the aminophospholipid translocase, but not the asymmetric distribution of endogenous membrane phospholipids, at least at a level detectable with phospholipases. Thus, if phosphatidylserine appearance on the outer monolayer is a signal for red cell elimination, the amount that triggers macrophage recognition is below the level of detection upon using the phospholipase technique.
Asunto(s)
Membrana Eritrocítica/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/sangre , Adenosina Trifosfato/sangre , Conservación de la Sangre , Humanos , Técnicas In Vitro , Membrana Dobles de Lípidos , Marcadores de Spin , Factores de TiempoRESUMEN
Cleavage of oxidized fatty acids by phospholipase A2 has been implicated as the first step in the repair mechanism for oxidative damage to membrane phospholipids. However, the mechanism by which this enzyme preferentially hydrolyzes oxidized fatty acyl chains is poorly understood. Using a lipid monolayer technique, we found that the molecular surface areas of 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)-phosphatidylcholine (PLPC-OOH) and 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) were increased by as much as 50% relative to the parent nonoxidized 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC). These experimental data directly indicate a drastically changed molecular conformation of oxidized phospholipids in which the hydroperoxy or hydroxy group in the sn-2 fatty acid is close to the lipid-water interface. Phospholipases A2 from porcine pancreas and from bee venom were shown to break down PLPC-OOH and PLPC-OH monolayers much faster than PLPC monolayers. In all cases, the presence of serum albumin in the subphase enhanced monolayer breakdown by extracting hydrolysis products from the monolayer, but monolayer breakdown was always much faster for oxidized than for nonoxidized PLPC. This did not appear to be due to change in the extent of monolayer penetration by phospholipase A2, since enzyme-monolayer interaction studies revealed essentially identical penetration behavior of bee venom phospholipase A2 with PLPC, PLPC-OOH, and PLPC-OH monolayers. We propose that the altered molecular conformation of oxidized phospholipids facilitates access to the sn-2 ester bond, thereby ensuring their preferential hydrolysis in the presence of a phospholipase A2.
Asunto(s)
Membranas Artificiales , Fosfolipasas A/metabolismo , Fosfolípidos/metabolismo , Hidrólisis , Modelos Químicos , Conformación Molecular , Oxidación-Reducción , Fosfolipasas A2RESUMEN
We have studied the mechanism by which calcium-loading of human erythrocytes stimulates phospholipid turnover and generates diacylglycerol and phosphatidic acid. Using quantitative measurement of individual phospholipid classes, we have demonstrated that the amount of phosphatidic acid generated during calcium-loading of intact red cells exceeds the amount of diacylglycerol formed by phospholipase-C-mediated hydrolysis of the polyphosphoinositol lipids and that addition of the diacylglycerol kinase inhibitor, R59022, only partly inhibited this increase. Thus, in contrast to current explanations, the phosphatidic acid generated following calcium-loading of erythrocytes cannot be solely explained by the action of a polyphosphoinositol-lipid-specific phospholipase C with subsequent phosphorylation of diacylglycerol to phosphatidic acid. Our data demonstrate that calcium-loading of intact erythrocytes, but not of red cell ghost membranes, causes a small but significant decrease in the relative amount of phosphatidylcholine (PtdCho). In order to identify the mechanisms responsible for calcium-mediated hydrolysis of PtdCho, we encapsulated Ptd[Me-14C]Cho-containing rat liver microsomes into erythrocytes and studied the generation of [Me-14C]choline and phospho[Me-14C]choline. We found that choline was the only detectable 14C-labeled product. Furthermore, incubation of erythrocytes with calcium under hypotonic conditions and in the presence of [14C]PtdCho vesicles and ethanol resulted in the formation of [14C]phosphatidylethanol. Together, these results suggest that the loss of PtdCho during calcium-loading of human erythrocytes is caused by a previously unrecognized PtdCho-hydrolyzing phospholipase D, resulting in direct generation of phosphatidic acid. Analysis of the molecular species composition of PtdCho, phosphatidic acid, and diradylglycerol, confirm the simultaneous actions of PtdCho-hydrolyzing and polyphosphoinositol-lipid-hydrolyzing phospholipases in calcium-loaded human erythrocytes.
Asunto(s)
Calcio/metabolismo , Eritrocitos/metabolismo , Ácidos Fosfatidicos/biosíntesis , Fosfatidilcolinas/metabolismo , Fosfolipasa D/metabolismo , Adulto , Animales , Células Cultivadas , Diacilglicerol Quinasa , Membrana Eritrocítica/metabolismo , Humanos , Hidrólisis , Técnicas In Vitro , Lípidos de la Membrana/metabolismo , Microsomas/metabolismo , Fosfotransferasas/antagonistas & inhibidores , Ratas , Fosfolipasas de Tipo C/metabolismoRESUMEN
We investigated the role of free radicals in hemoglobin (Hb) oxidation and denaturation. To generate free radicals, we used two azocompounds, the hydrophilic 2,2'-azobis(2-amidinopropane hydrochloride and the hydrophobic 2,2'-azobis(2,4-dimethylvaleronitrile) and a drug of the quinone family, phenazine methosulfate. The radical species involved were analyzed by direct EPR and spin trapping with 5,5-dimethyl-1-pyrroline N-oxide, and N-t-butyl-alpha-phenyl-nitrone. The free radicals generated by the azocompounds were carbon radicals and, in the presence of molecular oxygen, peroxyl/alkoxyl radicals. The reaction of phenazine with Hb produced a nitrogen-centered semiquinoid radical detectable by EPR only under N2 and reactive oxygen species (O2-. and H2O2) in the presence of molecular oxygen. Azocompounds oxidized Hb to methemoglobin, hemichromes, and choleglobin while phenazine produced methemoglobin and ferrylhemoglobin. For all three drugs, low oxygen tensions (pO2 = 62 mm Hg) increased the formation of Hb oxidation products, whereas high oxygen tensions (pO2 = 540 mm Hg) reduced Hb oxidation. The formation of irreversible Hb oxidation products (irreversible hemichromes and Hb cross-linking) was observed only with the azocompounds and was reduced at high pO2. Spin traps and thiourea protected Hb from the oxidative damage induced by the azocompounds, whereas enzymes scavenging reactive oxygen species, such as superoxide dismutase and catalase, affected Hb oxidation induced by phenazine and that induced by the hydrophobic azocompound. These results indicate distinct patterns of oxidation and denaturation with each agent. Damage induced by phenazine was dependent on the formation of reactive oxygen species, whereas the damage induced by the azocompounds was due mainly to carbon-centered radicals with some involvement by reactive oxygen species only for the hydrophobic azocompound. The preferential interaction of Hb with drug radicals scavenged by molecular oxygen indicates that this protein may be more reactive under hypoxic conditions and led to the view that a good supply of oxygen can provide an important defense against drug-induced Hb oxidation.
Asunto(s)
Carbono/metabolismo , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Amidinas/farmacología , Compuestos Azo/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Depuradores de Radicales Libres , Radicales Libres , Hemoproteínas/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Cinética , Metahemoglobina/metabolismo , Metosulfato de Metilfenazonio/farmacología , Nitrilos/farmacología , Oxidación-Reducción , Oxígeno/farmacología , Desnaturalización Proteica , Espectrofotometría , Marcadores de Spin , Superóxidos/farmacologíaRESUMEN
While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-hemoglobin chains, purified alpha-hemoglobin chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-hemoglobin chain autoxidation and appearance of membrane-bound globin. In addition, alpha-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-hemoglobin chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-hemoglobin chains. In summary, entrapment of purified alpha-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-hemoglobin chains can be examined.