RESUMEN
Anderson's disease (AD) or chylomicron retention disease (CMRD) is a rare hereditary lipid malabsorption syndrome linked to SARA2 gene mutations. We report in this study a novel mutation in two sisters for which the Sar1b protein is predicted to be truncated by 32 amino acids at its carboxyl-terminus. Because the SARA2 gene is also expressed in the muscle, heart, liver and placenta, extraintestinal clinical manifestations may exist. For the first time, we describe in this study in the two sisters muscular as well as cardiac abnormalities that could be related to the reported expression of SARA2 in these tissues. We also evaluated six other patients for potential manifestations of the SARA2 mutation. The creatine phosphokinase levels were increased in all patients [1.5-9.4 x normal (N)] and transaminases were moderately elevated in five of the eight patients (1.2-2.6 x N), probably related to muscle disease rather than to liver dysfunction. A decreased ejection fraction occurred in one patient (40%, N: 60%). The muscle, liver and placental tissues that were examined had no specific abnormalities and, in particular, no lipid accumulation. These results suggest that myolysis and other extraintestinal abnormalities can occur in AD/CMRD and that the clinical evaluation of patients should reflect this.
Asunto(s)
Cardiopatías Congénitas/etiología , Síndromes de Malabsorción/complicaciones , Síndromes de Malabsorción/genética , Proteínas de Unión al GTP Monoméricas/genética , Músculos/anomalías , Mutación , Adolescente , Adulto , Femenino , Humanos , Masculino , Músculos/patología , Adulto JovenRESUMEN
We investigated, for the first time, the expression of I- and L-FABP in two very rare hereditary lipid malabsorption syndromes as compared with normal subjects. Abetalipoproteinemia (ABL) and Anderson's disease (AD) are characterized by an inability to export alimentary lipids as chylomicrons that result in fat loading of enterocytes. Duodeno-jejunal biopsies were obtained from 14 fasted normal subjects, and from four patients with ABL and from six with AD. Intestinal FABP expression was investigated by immuno-histochemistry, western blot, ELISA and Northern blot analysis. In contrast to normal subjects, the cellular immunostaining for both FABPs was clearly decreased in patients, as the enterocytes became fat-laden. In patients with ABL, the intestinal contents of I- (60.7 +/- 13.38 ng/mg protein) and L-FABP (750.3 +/- 121.3 ng/mg protein) are significantly reduced (50 and 35%, P < 0.05, respectively) as compared to normal subjects (I-135.3 +/- 11.1 ng, L-1211 +/- 110 ng/mg protein). In AD, the patients also exhibited decreased expression (50%, P < 0.05; I-59 +/- 11.88 ng, L-618.2 +/- 104.6 ng/mg protein). Decreased FABP expression was not associated with decreased mRNA levels. The results suggest that enterocytes might regulate intracellular FABP content in response to intracellular fatty acids, which we speculate may act as lipid sensors to prevent their intracellular transport.
Asunto(s)
Abetalipoproteinemia/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Mucosa Intestinal/metabolismo , Errores Innatos del Metabolismo Lipídico/metabolismo , Síndromes de Malabsorción/metabolismo , Abetalipoproteinemia/genética , Adolescente , Adulto , Niño , Proteínas de Unión a Ácidos Grasos/genética , Femenino , Humanos , Inmunohistoquímica , Errores Innatos del Metabolismo Lipídico/genética , Síndromes de Malabsorción/genética , Masculino , ARN Mensajero/metabolismoRESUMEN
BACKGROUND & AIMS: Abetalipoproteinemia and Anderson's disease are hereditary lipid malabsorption syndromes. In abetalipoproteinemia, lipoprotein assembly is defective because of mutations in the microsomal triglyceride transfer protein. Here, we evaluated the intracellular transport of apolipoprotein B48 to localize the defect in Anderson's disease. METHODS: Asparagine-linked oligosaccharide processing of apolipoprotein B48 in normal and affected individuals was determined by the endoglycosidase H and F sensitivities of the protein after metabolic labeling of intestinal explants in organ culture. Cell ultrastructure was evaluated with electron microscopy. RESULTS: In Anderson's disease as in normal individuals, there was a time-dependent transformation of high mannose endoglycosidase H-sensitive oligosaccharides, of endoplasmic reticulum origin, to complex endoglycosidase H-resistant oligosaccharides, added in the Golgi network. In contrast, despite the translocation of apolipoprotein B48 into the endoplasmic reticulum in patients with abetalipoproteinemia and in biopsies treated with Brefeldin A, which blocks anterograde transport between the endoplasmic reticulum and the Golgi network, there was no transformation of endoglycosidase H-sensitive oligosaccharides. CONCLUSIONS: In abetalipoproteinemia and Anderson's disease, apolipoprotein B48 is completely translocated into the endoplasmic reticulum, but only in Anderson's disease is the protein transported to the Golgi apparatus. This suggests that Anderson's disease is caused by a post-Golgi cargo-specific secretion defect.
Asunto(s)
Abetalipoproteinemia/metabolismo , Apolipoproteínas B/metabolismo , Metabolismo de los Lípidos , Abetalipoproteinemia/patología , Apolipoproteína B-48 , Apolipoproteínas B/deficiencia , Transporte Biológico , Brefeldino A/uso terapéutico , Proteínas Portadoras/fisiología , Retículo Endoplásmico/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Humanos , Mucosa Intestinal/patologíaRESUMEN
Incorporation of coconut oil (CO) rich in lauric acid into the milk diet induces a lipid infiltration of the liver (steatosis) in 1-month-old calves. Among possible steps involved in diet-induced liver steatosis, the ability of the calf liver to synthesize apolipoprotein (Apo) B and to secrete it as part of VLDL particles was investigated. Liver samples were taken from calves fed for 17 d on a conventional milk replacer containing CO (n 5) and beef tallow (BT, n 4) as reference. Samples were cut into slices 0.5 mm thick and subsequently incubated for 12 h in a medium containing a [(35)S]methionine-[(35)S]cysteine mix and 0.8 mm-sodium laurate or oleate, the major fatty acids of CO and BT diets respectively. Concentrations of total [(35)S]proteins, [(35)S]albumin and [(35)S]ApoB in liver cells were 2-fold lower 0.0004 and 0.03 respectively) in CO- than in BT-fed calves. Although the total amount of proteins secreted (including albumin) was similar in both groups of calves, the amount of VLDL-[(35)S]Apo secreted was 2-fold lower (P = 0.004) in CO- than in BT-fed calves. These results suggest that a CO-enriched milk diet induces in preruminant calves a lipid infiltration of the liver by decreasing ApoB synthesis, leading to a reduction in secretion of VLDL particles.
Asunto(s)
Apolipoproteínas B/biosíntesis , Enfermedades de los Bovinos/metabolismo , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Animales , Disponibilidad Biológica , Bovinos , Enfermedades de los Bovinos/inducido químicamente , Aceite de Coco , Técnicas de Cultivo , Grasas de la Dieta/administración & dosificación , Hígado Graso/veterinaria , Masculino , Aceites de Plantas/administración & dosificaciónRESUMEN
Anderson's disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48-containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/mu(2)) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/mu(2), 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.
Asunto(s)
Salud de la Familia , Hipobetalipoproteinemias/genética , Hipobetalipoproteinemias/metabolismo , Triglicéridos/metabolismo , Adulto , Edad de Inicio , Apolipoproteínas A/biosíntesis , Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Apolipoproteínas B/biosíntesis , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Apolipoproteínas C/biosíntesis , Apolipoproteínas C/genética , Apolipoproteínas C/metabolismo , Apolipoproteínas E/biosíntesis , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Biopsia , Cromosomas Humanos Par 4 , Quilomicrones/metabolismo , ADN Satélite/análisis , Femenino , Ligamiento Genético , Genotipo , Heterocigoto , Humanos , Hipobetalipoproteinemias/patología , Absorción Intestinal/genética , Mucosa Intestinal/metabolismo , Intestinos/patología , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/metabolismo , Síndromes de Malabsorción/patología , Masculino , Técnicas de Cultivo de Órganos , Linaje , Polimorfismo Genético , Triglicéridos/biosíntesisRESUMEN
Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [35S]methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo B but rather to a defect in VLDL assembly and/or secretion.
Asunto(s)
Apolipoproteínas B/metabolismo , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Albúminas/metabolismo , Animales , Bovinos , Medios de Cultivo , Cisteína/metabolismo , Dipéptidos/metabolismo , Técnicas In Vitro , Masculino , Metionina/metabolismo , Ácido Oléico/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Radioisótopos de AzufreRESUMEN
The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297 --> Gln, Asp384 --> Ala, and Arg540 --> His. Transient expression of each mutant showed that only the Arg540 --> His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540 --> His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.
Asunto(s)
Apolipoproteínas B/sangre , Proteínas Portadoras/genética , Glicoproteínas , Isomerasas/metabolismo , Errores Innatos del Metabolismo Lipídico/genética , Mutación , Apolipoproteínas B/genética , Proteínas Portadoras/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Clonación Molecular , ADN Complementario , Genotipo , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patología , Errores Innatos del Metabolismo Lipídico/sangre , Proteína Disulfuro IsomerasasRESUMEN
Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes: ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.
Asunto(s)
Intestinos/química , Lípidos/análisis , Síndromes de Malabsorción/metabolismo , Abetalipoproteinemia/genética , Abetalipoproteinemia/metabolismo , Apoproteínas/análisis , Biopsia , Crioultramicrotomía , Substitución por Congelación , Inmunohistoquímica , Intestinos/citología , Intestinos/ultraestructura , Síndromes de Malabsorción/genética , Microscopía Inmunoelectrónica , Adhesión del TejidoRESUMEN
The microsomal triglyceride transfer protein (MTP), which catalyses the transport of triglyceride, cholesteryl ester and phospholipid between phospholipid surfaces, is a heterodimer composed of the multifunctional protein, protein disulphide isomerase, and a unique large subunit with an apparent M(r) of 88K (refs 1-3). It is isolated as a soluble protein from the lumen of the microsomal fraction of liver and intestine. The large subunit of MTP was not detectable in four unrelated subjects with abetalipoproteinaemia, a rare autosomal recessive disease characterized by a defect in the assembly or secretion of plasma lipoproteins that contain apolipoprotein B (ref. 6). We report here the isolation and sequencing of complementary DNA encoding the large subunit of MTP. A comparison of this sequence to corresponding genomic sequences from two abetalipoproteinaemic subjects revealed a homozygous frameshift mutation in one subject and a homozygous nonsense mutation in the other. The results indicate that a defect in the gene for the large subunit of MTP is the proximal cause of abetalipoproteinaemia in these two subjects, and that MTP is required for the secretion of plasma lipoproteins that contain apolipoprotein B.
Asunto(s)
Abetalipoproteinemia/genética , Proteínas Portadoras/genética , Glicoproteínas , Triglicéridos , Abetalipoproteinemia/metabolismo , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Apolipoproteínas B/metabolismo , Secuencia de Bases , Bovinos , Células Cultivadas , Proteínas de Transferencia de Ésteres de Colesterol , Clonación Molecular , ADN , Análisis Mutacional de ADN , Femenino , Fibroblastos , Humanos , Intestino Delgado/metabolismo , Lipoproteínas/metabolismo , Hígado/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Alineación de SecuenciaRESUMEN
Abetalipoproteinemia is a human genetic disease that is characterized by a defect in the assembly or secretion of plasma very low density lipoproteins and chylomicrons. The microsomal triglyceride transfer protein (MTP), which is located in the lumen of microsomes isolated from the liver and intestine, has been proposed to function in lipoprotein assembly. MTP activity and the 88-kilodalton component of MTP were present in intestinal biopsy samples from eight control individuals but were absent in four abetalipoproteinemic subjects. This finding suggests that a defect in MTP is the basis for abetalipoproteinemia and that MTP is indeed required for lipoprotein assembly.
Asunto(s)
Abetalipoproteinemia/etiología , Intestinos/química , Quilomicrones/metabolismo , Duodeno/química , Duodeno/metabolismo , Duodeno/ultraestructura , Humanos , Immunoblotting , Intestinos/ultraestructura , Yeyuno/química , Yeyuno/metabolismo , Yeyuno/ultraestructura , Lipoproteínas VLDL/biosíntesis , Microsomas/química , Microsomas/metabolismo , Microsomas Hepáticos/química , Triglicéridos/metabolismoRESUMEN
This study describes the alterations in the plasma lipoproteins from nine young Tunisian children with active visceral Leishmaniasis. The plasma lipid profile from affected patients was characterized by a marked hypertriglyceridaemia associated with reduced levels of total and high density lipoprotein (HDL)-cholesterol and a significant increase in the plasma ratio of unesterified to total cholesterol. Quantitative determination of plasma apolipoproteins revealed significantly decreased levels of all measured apolipoproteins, especially of apolipoproteins A-I and A-II, with the exception of apolipoprotein E, the levels of which were markedly increased. Moreover, at least two isoforms of the apolipoprotein serum amyloid A (SAA), an acute phase protein, were detected in all patients' plasma using two-dimensional electrophoresis. Immunochemical evidence was presented that apolipoproteins E and SAA, although both primarily associated with apolipoprotein A- (A-I and A-II) as well as with apolipoprotein B-containing lipoproteins, could occur as LP-E and LP-SAA subspecies, devoid of apolipoproteins A and B. However, it should be pointed out that LP-SAA particles were found in HDL2 from only two patients whereas the abnormal LP-E particles were detected in LDL and HDL2 from all investigated patients. The polydispersity and heterogeneity of patients' HDL3 were assessed by electron microscopy. It was further suggested that the profound changes in the lipoprotein metabolism of these young patients may be due to the increased hepatic synthesis of apolipoprotein SAA and/or to their altered immune function during active visceral Leishmaniasis.
Asunto(s)
Leishmaniasis Visceral/sangre , Lipoproteínas/sangre , Proteína Amiloide A Sérica/metabolismo , Apolipoproteínas/sangre , Preescolar , Femenino , Humanos , Inmunoquímica , Inmunoelectroforesis Bidimensional , Lipoproteínas/inmunología , Lipoproteínas/aislamiento & purificación , Masculino , Microscopía Electrónica , Tamaño de la Partícula , Triglicéridos/sangreRESUMEN
A case of apolipoprotein B-related disorder is reported in which liver fibrosis developed without long term administration of medium chain triglycerides, previously incriminated in the pathogenesis of this lesion. The patient was a young woman in whom the diagnosis of familial homozygous hypobetalipoproteinaemia was made at the age of 21. A first liver specimen taken at diagnosis revealed steatosis, hypertrophic Golgi apparatus and proliferating smooth endoplasmic reticulum. The patient was treated with vitamin A and E supplementation only. Two years later, a second liver biopsy, carried out because of increased serum alanine aminotransferase concentrations, showed fibrosis, mild cytolysis and marked mitochondrial alterations. Hepatic level of vitamin A was increased. This finding supports the hypothesis that liver disease observed in our patient might be an adverse effect of vitamin supplementation. Our observation underlines the importance of including liver function tests in the follow up of patients with apolipoprotein B-related disorders.
Asunto(s)
Hipobetalipoproteinemias/tratamiento farmacológico , Cirrosis Hepática/etiología , Vitamina A/uso terapéutico , Vitamina E/uso terapéutico , Adulto , Femenino , Homocigoto , Humanos , Hipobetalipoproteinemias/genética , Cirrosis Hepática/patologíaRESUMEN
A familial study of four cases with hypobetalipoproteinemia is reported. Three members are heterozygous and one is homozygous. This congenital fat malabsorption in homozygous state is commonly associated with an absence of serum apoprotein B and LDL. Neuromuscular and ophthalmological signs are absent in this case. The major role of upper digestive endoscopy in the diagnostic procedure is emphasized. Histochemical and immunoenzymatic stains of enterocytes and intestinal organ culture show defective synthesis apo B in the homozygous patient. Studies of DNA polymorphism in the homozygous patient have shown that the apo B gene doesn't certain major insertions or deletions. These results are discussed.
Asunto(s)
Hipobetalipoproteinemias/genética , Adulto , Femenino , Heterocigoto , Homocigoto , Humanos , Hipobetalipoproteinemias/patología , Intestino Delgado/patología , Intestino Delgado/ultraestructura , Microscopía ElectrónicaRESUMEN
We describe here two patients, M. P. and S. L., with recessive abetalipoproteinemia. Analysis of restriction fragments of DNA from both patients using cDNA probes spanning the entire apolipoprotein B gene revealed no major insertions or deletions. Further, as defined by restriction fragment length polymorphism, abetalipoproteinemia, in these patients, did not appear associated with particular alleles of apolipoprotein B. Northern and dot blot analysis of intestinal mRNA of one patient (M. P.) revealed a normal-sized apolipoprotein B mRNA which was present in slightly reduced amounts. At the cellular level apolipoprotein B was detected in both intestinal and hepatic biopsies, of one patient (S. L.), by immunoenzymatic techniques using polyclonal and monoclonal antibodies to apolipoprotein B-48 and/or B-100. The level of apolipoprotein B-48 appeared to increase in the intestine after a fatty meal. In the other patient (M. P.), although no apolipoprotein B was detected in the enterocytes using similar immunoenzymatic techniques, organ culture experiments using [35S]methionine demonstrated the synthesis of a normal-sized apolipoprotein B-48 which appeared to be normally glycosylated. The glycosylation and processing of two intestinal membrane enzymes, sucrase-isomaltase and aminopeptidase N, were also normal. Although lipids and apolipoprotein B-48 were present intracellularly, no lipoprotein-like particles were observed by electron microscopy in the endoplasmic reticulum, the Golgi apparatus, or in the intercellular spaces of intestinal biopsies obtained in the fasted (M. P. and S. L.) or fed state (S. L.). The defect in these cases of abetalipoproteinemia, therefore, does not appear to involve the apolipoprotein B gene nor the synthesis or the glycosylation of the apolipoprotein but instead appears to involve some aspect of lipoprotein assembly or secretion.
Asunto(s)
Abetalipoproteinemia/metabolismo , Apolipoproteínas B/biosíntesis , Mucosa Intestinal/metabolismo , Abetalipoproteinemia/genética , Abetalipoproteinemia/patología , Adulto , Apolipoproteína B-48 , Apolipoproteínas/metabolismo , Apolipoproteínas B/genética , Femenino , Genes , Humanos , Hidrolasas/metabolismo , Técnicas para Inmunoenzimas , Intestinos/patología , Hígado/metabolismo , Hígado/patología , Masculino , Microvellosidades/enzimología , Técnicas de Cultivo de Órganos , ARN Mensajero/genéticaRESUMEN
This study describes the plasma lipoprotein system of young children with visceral Leishmaniasis (Kala-azar disease). In addition to the presence of amastigote forms in the sternal aspirates of bone marrow, the patients exhibited fever, anemia, hepatosplenomegaly, various degrees of pancytopenia and a slight liver cytolysis. Patients had normal total cholesterol levels and increased triglyceride levels in the plasma. The concentrations of HDL and LDL were 30% and 50% of these reported for normolipemic subjects, respectively. In contrast, there was a three-fold increase in the concentration of VLDL. The ratio of free to total cholesterol was high; this was further substantiated by electron microscopy of HDL showing the presence of disc-like particles. Quantitative determination of apolipoproteins revealed a three- and seven-fold decrease of apolipoproteins (Apo) A-I and A-II, respectively, whereas Apo B levels were within the normal range. The presence of LP-A-II particles was demonstrated by two-dimensional immunoelectrophoresis in most of the patients' plasma during the acute phase of disease.
Asunto(s)
Apolipoproteínas A/deficiencia , Colesterol/sangre , Leishmaniasis Visceral/sangre , Lípidos/sangre , Lipoproteínas HDL/sangre , Apolipoproteína A-I , Apolipoproteína A-II , Niño , Preescolar , Humanos , Lactante , Lipoproteínas HDL/ultraestructura , Microscopía Electrónica , Triglicéridos/sangreRESUMEN
Anderson's disease is a rare autosomic recessive condition involving the transport of fat through the intestinal mucosa, which could be due to a defect in the intestinal form (B48) of apolipoprotein B. Isolated cases and one important series only have been reported. We wish here to complete the description of the disease. Seven children (age 6 months to 13 years at time of diagnosis) were followed for one month to 15 years. They presented with a malabsorption syndrome, malnutrition, fatty diarrhea (steatorrhea 4-18 g/24 h), failure to thrive (height -1 to -5.5 SD for age) and sometimes disappearance of deep tendon reflexes. Biologically they had signs of malabsorption, hypocalciuria, osteoporosis, low serum iron, decreased levels of vitamins A and E, and hypo-alpha- (50-127 mg/100 ml) and beta- (73-175 mg/100 ml) lipoproteinemia due to decreased levels of plasma cholesterol (40-70 mg/100 ml), and phospholipids (34-67 mg/100 ml); apolipoproteins A1 (26-69 mg/100 ml and B (21-44 mg/100 ml) were also low. After a fatty meal, triglycerides and apolipoproteins did not increase and chylomicrons did not appear. Jejunal biopsies showed the characteristic aspect of enterocytes loaded with lipid droplets. On electron microscopy, these fat droplets were seen in the cytoplasm but neither in the endoplasmic reticulum and the Golgi complex nor in the intercellular spaces. They did not appear to be enclosed in membranes and differed from chylomicrons by their size and density. The disease could thus be due to an abnormal apolipoprotein B48, which would prevent its binding to triglycerides and thus the formation of chylomicrons.
Asunto(s)
Mucosa Intestinal/metabolismo , Errores Innatos del Metabolismo Lipídico/metabolismo , Adolescente , Niño , Preescolar , Familia , Femenino , Trastornos del Crecimiento/etiología , Humanos , Lactante , Absorción Intestinal , Masculino , Microscopía Electrónica , Trastornos Nutricionales/etiologíaRESUMEN
The hepatoblastoma cell line HepG2 has been a matter of many investigations; most of them include biochemical studies of lipoprotein and other hepatic protein metabolism. However, the accurate cellular features of these cells have not been emphasized. We studied the cellular histologic, histochemical, and ultrastructural characteristics of this cell line. In addition, we investigated by immunoenzymatic methods the cellular biosynthesis of several proteins: apolipoproteins-AI, -B, -D, and -E, albumin, alpha-fetoprotein, transferrin, alpha-1-antitrypsin, C-reactive protein, fibronectin, and collagens I, III and IV. The rates of accumulation, in the medium of HepG2 cells, of albumin, alpha-1-antitrypsin, transferrin, and alpha-fetoprotein were 13.2 +/- 1.9; 4.9 +/- 1.5; 3.2 +/- 0.4; and 10.7 +/- 1.7 micrograms/10(6) cells/24 h, respectively. Our results show that HepG2 cells exhibited most cellular features of normal human hepatocytes. Bile canaliculi as well as Golgi apparatus complexes were particularly developed. Except for the C-reactive protein, HepG2 cells have all retained the ability to synthesize hepatic proteins but with some variable intensity from cell to cell. This hepatoblastoma cell line seems to represent a useful tool in the understanding of hepatic protein biosynthesis, particularly for the investigation on the secretory pathway of plasma proteins.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Biosíntesis de Proteínas , Albúminas/biosíntesis , Apolipoproteínas/biosíntesis , Proteína C-Reactiva/biosíntesis , Carcinoma Hepatocelular/metabolismo , Colágeno/biosíntesis , Citoplasma/metabolismo , Citoplasma/patología , Fibronectinas/biosíntesis , Histocitoquímica , Humanos , Técnicas para Inmunoenzimas , Cinética , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Microscopía Electrónica , Coloración y Etiquetado , Transferrina/biosíntesis , Células Tumorales Cultivadas , alfa 1-Antitripsina/biosíntesis , alfa-Fetoproteínas/biosíntesisRESUMEN
In this study we prepared a pure apolipoprotein D and obtained a specific antiserum to it. The purified apolipoprotein D migrated as a single band of Mr = 29,000 but appeared as five isoforms on isoelectrofocusing. The antiserum did not cross-react with other apolipoproteins. Immunoenzymatic staining revealed the presence of apolipoprotein D in the perinuclear area of the cytoplasm of isolated normal hepatocytes and HepG2 cells. Apolipoprotein D was also localized in intestinal epithelium and in liver cells. The intracellular distribution of apolipoprotein D was similar to that of apolipoprotein B. Our results indicated that apolipoprotein D, like many other circulating apolipoproteins, is synthesized in enterocytes and hepatocytes.
Asunto(s)
Apolipoproteínas/metabolismo , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Adulto , Apolipoproteínas/inmunología , Apolipoproteínas/aislamiento & purificación , Apolipoproteínas D , Humanos , Sueros Inmunes , Técnicas para Inmunoenzimas , Mucosa Intestinal/citología , Hígado/citologíaRESUMEN
Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver fragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precursor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [3H]glycerol, more than 92% of the [3H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.