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Biomedical subjects

M E Bouma

Publications and source records attributed to M E Bouma.

16 recordsLinked to original sources
PubMed1992

Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia.

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.

Abetalipoproteinemia↗
PubMed1992

Alterations in lipoprotein density classes in infantile visceral leishmaniasis: presence of apolipoprotein SAA.

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.

Apolipoproteins↗
PubMed1992

Liver fibrosis in a patient with familial homozygous hypobetalipoproteinaemia: possible role of vitamin supplementation.

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.

Adult↗
PubMed1990

Description of two different patients with abetalipoproteinemia: synthesis of a normal-sized apolipoprotein B-48 in intestinal organ culture.

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.

Abetalipoproteinemia↗
PubMed1989

Plasma lipoproteins in infantile visceral leishmaniasis: deficiency of apolipoproteins A-I and A-II.

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.

Apolipoprotein A-I↗
PubMed1989

Further cellular investigation of the human hepatoblastoma-derived cell line HepG2: morphology and immunocytochemical studies of hepatic-secreted proteins.

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.

Albumins↗
PubMed1986

Isolation and characterization of three monoclonal antibodies to human serum low density lipoprotein apoprotein B.

Human serum low density lipoprotein (LDL) is a large (Mr = 2-3 X 10(6), complex particle composed of lipid, protein and carbohydrate. We obtained about 40 mouse spleen-myeloma hybrid cell lines which produce antibodies against LDL. Three of them, SC2, SC3 and SC10, have been cloned and subcloned and their antibody products characterized. They recognize three non-overlapping epitopes in native LDL. Two of them, SC3 and SC10, also are capable of recognizing very low density lipoprotein, (VLDL), whereas SC2 reacts only weakly with VLDL. All three antigenic determinants remain intact, and accessible to antibodies on the LDL protein apo B, prepared by delipidation in a 'non-denaturing' detergent, sodium deoxycholate. However, apo B prepared by organic solvent, ether-ethanol, or sodium dodecyl sulfate (SDS) delipidation, while reacting strongly with SC10, is only poorly recognized by SC2 or SC3. Proteolysis of LDL with trypsin, chymotrypsin, Staphylococcus aureus protease, papain or thermolysin gives, in each case, several non-identical protein fragments which are separable by SDS-polyacrylamide gel electrophoresis. Upon immunoblotting, some of these fragments are now recognized by either SC3 or SC10 but not SC2, some are recognized by both SC3 and SC10, and others are immunologically unreactive. The protein bands that are separated by SDS gel electrophoresis are composed of several non-identical fragments and contain the antigenic sites to differing degrees. Some of the immunologically reactive fragments do not appear to contain carbohydrate. Reduction and carboxymethylation do not destroy the immunoreactivity of LDL toward any of the antibodies; however, modification of lysine residues by citraconic anhydride markedly diminishes the reactivity of LDL toward SC3. It is likely that the two antibodies SC3 and SC10 are directed against different linear amino acid sequences or very stable domains, whereas the third, SC2, is directed against a more fragile conformational domain of apo B.

Animals↗
PubMed1986

Hypobetalipoproteinemia with accumulation of an apoprotein B-like protein in intestinal cells. Immunoenzymatic and biochemical characterization of seven cases of Anderson's disease.

We describe here seven cases (from five kindreds) of Anderson's disease, which is characterized by diarrhea, steatorrhea, hypobetalipoproteinemia with low levels of cholesterol, triglycerides, and phospholipids, and failure to secrete chylomicrons after a fat meal. Enterocytes isolated from intestinal biopsies of patients after overnight fast showed numerous fat droplets, a histological picture resembling that of abetalipoproteinemia. Immunoenzymatic staining of the enterocytes demonstrated large amounts of material that reacted with a polyclonal antiserum to apolipoprotein B. Further, the immunoreactive material was found to react with several different monoclonal antibodies capable of recognizing both the B100 and B48 forms of apoprotein B, but not with any of several monoclonal antibodies capable of recognizing only B100. This suggests that the material in the enterocytes is the B48 form of apoprotein B or a fragment thereof. Additional findings included decreased low density lipoprotein levels with an abnormal chemical composition, abnormal high density lipoprotein2 (HDL2) and HDL3 particle size distributions, and an abnormal HDL apoprotein composition. Increased amounts of proteins having electrophoretic mobilities similar to apo E and the E-AII complex were present. Finally, some cases exhibited additional protein components of apparent molecular weights between 17,000 and 28,000, which was similar to some cases of abetalipoproteinemia. These findings demonstrate that Anderson's disease is not due to the absence of synthesis of intestinal apo B and suggest that it is more complex than previously thought, affecting all the lipoprotein classes.

Abetalipoproteinemia↗
PubMed1985

Free fatty-acid uptake by isolated rat hepatocytes.

In isolated rat hepatocytes, the rate of palmitic acid binding and uptake is directly related to the concentration of free fatty acid (FFA) in the medium. After their entry into the cell, FFA are immediately incorporated into cellular phospholipids and triglycerides and no accumulation of free fatty acids can be demonstrated inside the cell. The rate of free fatty-acid uptake remains unchanged after incubation in a 2 mM KCN containing medium, indicating that in the range of fatty-acid concentrations used in this study, this phenomenon does not require energy.

Animals↗
PubMed1979

Ultrastructural localization of apo-b and apo-c binding to very low density lipoproteins in rat liver.

Hepatic synthesis of apo-B and apo-C and their binding to nascent very low density lipoproteins (VLDL) have been studied in fat-fed rats. Apolipoproteins were located in hepatocyte organelles by light and electron microscopy after immunoenzymatic staining using peroxidase-conjugated antibodies. Our results indicate that apo-B and apo-C are synthesized by membrane-bound ribosomes. Both apoproteins seem to be adsorbed simultaneously to the lipid core of VLDL in the lumen of the endoplasmic reticulum channels, at the junction zone between rough and smooth endoplasmic reticulum. Some additional protein presumably binds nascent VLDL in the Golgi apparatus as judged by the strong positive reaction of lipoprotein particles with peroxidase-labeled antibodies. Finally our data show that significant amounts of apo-B and apo-C are bound to the sinusoidal plasma membrane in fed rat livers which probably represent remnants of lipoprotein of intestinal origin since membrane-bound apolipoproteins virtually disappeared 24 h after lymphatic duct cannulation. It is suggested that nascent VLDL (apo-C poor) could be enriched in apo-C from lipoprotein remnants at the space of Disse.

Animals↗
PubMed1978

Ribonucleic acid synthesis in rat liver during fatty acid stimulated secretion of very low density lipoproteins.

Production of very low density lipoproteins by the liver depends on the cellular availability of fatty acids. It is stimulated by the uptake of free fatty acids from the plasma and by increased lipogenesis and is inhibited by actinomycin D, suggesting that RNA synthesis is involved in the regulation of apolipoprotein synthesis. This hypothesis has been investigated in rats in vivo and in isolated perfused livers with and without stimulation by fatty acid overload: [14C] orotate incorporation in liver polyribosomal RNA is 60 per cent greater in stimulated livers as compared to controls. This increase is primarily due to a higher incorporation in bound polysomes and in those containing at least six ribosomes and does not result from the inhibition of ribonuclease. RNase digestion of polysomal RNA (4.10(-10) M enzyme, 0 degrees C, 3 h) shows that there is twice as much radioactivity in the hydrolyzed RNA of stimulated livers as compared to controls. After partial purification of poly A-rich RNA by affinity chromatography, the mass yield and radioactivity are increased by 100 per cent in stimulated livers as compared to controls. In conclusion, de novo RNA synthesis seems to be necessary for fatty acid stimulation of VLDL production.

Animals↗
PubMed1976

Effects of glycerol on VLDL secretion by the isolated rat liver.

Stimulation of VLDL production by increasing fatty acid availability is now well established. However, a possible regulatory role of glycerol, another lipid precursor, in VLDL synthesis by the liver has not yet been substaniated. The present experiments investigate this problem using the isolated perfused rat liver. [14C] Glycerol uptake and metabolism were studied at two different glycerol concentrations: 1 mumol/perfusate (control) or 1.6 mmol/perfusate. VLDL production and lipid synthesis were investigated using [14C]leucine and several labelled fatty acids as precursors in control and glycerol-overloaded livers. Neoglycogenesis and lipogenesis from glycerol carbons are negligible in our conditions. The absolute amount of glycerol, but not the precentage, taken up by the liver, increased after raising its concentration in the perfusate. A major part of exogenous (plasmatic) glycerol was esterified with endogenous (non plasmatic) fatty acids. Incorporation of radioactive fatty acids into liver or plasma lipids was lower than in the the control group. Significant differences were observed between saturated and unsaturated fatty acids used as lipid precursors. Production of VLDL as assessed by radioactive leucine and fatty acid incorporation in the VLDL of the perfusate was depressed by glycerol. Glycerol partly inhibits the normal stimulation of VLDL production by plasmatic fatty acid overload.

Animals↗
PubMedDate not supplied

[Familial hypobetalipoproteinemia. Familial study of 4 cases].

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.

Adult↗
PubMedDate not supplied

[Anderson's disease. Clinical and morphologic study of 7 cases].

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.

Adolescent↗