In vivo protein metabolism utilizing stable isotopes and mass spectrometry: a new approach to the study of mutant proteins in humans.
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Adult↗
Biomedical subjects
R E Gregg
Publications and source records attributed to R E Gregg.
Molecular species of cholesteryl esters formed in abetalipoproteinemia: effect of apoprotein B-containing lipoproteins.
In order to study the effects of very low density (VLDL) and low density (LDL) lipoproteins on the activity and specificity of lecithin:cholesterol acyltransferase (LCAT), we determined the molecular species of cholesteryl esters (CE) synthesized in the plasma from three abetalipoproteinemic (ABL) patients, before and after supplementation with normal VLDL or LDL. The patients' plasma had significantly lower concentration of 18:2 CE and higher concentrations of 16:0 CE and 18:1 CE compared to normal plasma. Incubation of ABL plasma with [4-14C]cholesterol at 37 degrees C and the subsequent analysis of labeled CE formed by high performance liquid chromatography revealed that the major species formed was 16:0 CE (34% of total label), whereas similar incubation of the d greater than 1.063 g/ml fraction of normal plasma resulted in the formation of predominantly 18:2 CE (45% of total label). Addition of normal VLDL or LDL to ABL plasma stimulated the total LCAT activity by 30-80% and normalized the CE species synthesized. The LCAT activity of a normal d greater than 1.063 g/ml fraction also was stimulated by the normal VLDL or LDL, but there was no alteration in the species of CE formed. Most of the CE synthesized was found in the added VLDL or LDL with both ABL and normal plasma, indicating that the CE transfer (CET) activity was not affected in ABL plasma. These results suggest that while the VLDL and LDL are required for the maximal activity of LCAT, the species of CE formed are primarily determined by the molecular species composition of phosphatidylcholine in the plasma.
Abetalipoproteinemia↗
Apolipoprotein E-1Harrisburg: a new variant of apolipoprotein E dominantly associated with type III hyperlipoproteinemia.
Apolipoprotein E (apoE) is important in the modulation of the catabolism of chylomicron and very low density lipoprotein (VLDL) remnants. ApoE has three major genetically determined isoproteins in plasma, designated apoE-2, apoE-3 and apoE-4, with homozygosity for the allele coding for apoE-2 being associated with dysbetalipoproteinemia or type III hyperlipoproteinemia (HLP). We describe a new variant of apoE, apoE-1Harrisburg, which is, in contrast to apoE-2, dominantly associated with type III HLP. Five of twelve members of the affected kindred are heterozygous for the mutant form of apoE, and four of the five have type III HLP, while the fifth member has dysbetalipoproteinemia on diet therapy. Neuraminidase digestion, which removes charged sialic acid residues, did not alter the electrophoretic position of the apoE-1Harrisburg isoprotein, indicating that the altered charge of apoE-1Harrisburg was not due to sialic acid addition to the apolipoprotein. Cysteamine modification, which adds a positively charged group to cysteine, resulted in a shift of apoE-1Harrisburg from the E-1 to the E-2 isoform position, indicating that there is one cysteine in apoE-1Harrisburg as is the case for apoE-3. These results are consistent with apoE-1Harrisburg originating in the allele for apoE-3 with the mutation leading to a negative two-unit charge shift. The definitive identification of a kindred with an apoE variant, apoE-1Harrisburg, dominantly associated with dysbetalipoproteinemia and type III HLP provides a unique opportunity to gain important insights into the structure-function requirements of the E apolipoprotein as well as the mechanisms by which apoE modulates lipoprotein metabolism.
Adolescent↗
Homozygous hypobetalipoproteinemia: transcriptional regulation and 5'-flanking sequence analysis in an apolipoprotein B deficiency state.
Apolipoprotein (apo) B is the principal apolipoprotein of chylomicrons, very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Patients with homozygous hypobetalipoproteinemia (HBL), characterized by apoB deficiency, have markedly decreased levels of hepatocyte mRNA as well as intracellular B apolipoprotein, and a virtual absence of plasma apoB. We have cloned, sequenced and analyzed the 5' regulatory region of the human apoB gene from -899 to +121 bp in normal and hypobetalipoproteinemic subjects. TATA and CAAT boxes were located at -30 and -61, respectively, and two GC-like boxes were identified at positions +56 and +108. The analysis of the HBL sequence revealed two substitutions at positions -838 and -517, when compared to the normal sequence. These substitutions were not present in any known apoB regulatory elements. The transcriptional activities of the homozygous hypobetalipoproteinemic and normal regulatory regions were compared by chloramphenicol acetyltransferase (CAT) assays in Hep G2 cells, and were found to be the same. Therefore, we conclude that the 5' regulatory region of the HBL apoB gene in this kindred is normal, and the two base substitutions do not affect promoter activity of the apoB gene. These studies suggest that a coding region abnormality in the apoB gene may lead to HBL.
Apolipoproteins B↗
Increased urinary mevalonic acid excretion in patients with abetalipoproteinemia and homozygous hypobetalipoproteinemia.
Previous reports in which cholesterol homeostasis has been examined in patients with phenotypic abetalipoproteinemia have shown an increase in whole body cholesterol synthesis when measured by sterol balance techniques but normal rates of cholesterol synthesis when measured by isotopic cholesterol turnover. Recent studies have indicated that increases in cholesterol biosynthesis are paralleled by increases in the plasma concentrations of mevalonic acid and by higher rates of excretion of mevalonic acid in the urine. In the present report we have measured the 24-h urinary excretion of mevalonic acid in 7 patients with phenotypic abetalipoproteinemia and compared this to control subjects. Urinary excretion of mevalonic acid was significantly higher in the patients with abetalipoproteinemia (57.2 +/- 10.2 nmol/kg body weight per day, mean +/- SEM) as compared to control subjects (23.1 +/- 1.5 nmol/kg per day). The magnitude of the increase in urinary mevalonic acid excretion seen in patients with abetalipoproteinemia (148%) is greater than the increase in whole body cholesterol biosynthesis assessed by sterol balance techniques (57% increase). Our results serve to further validate the usefulness of urinary mevalonate as an indicator of relative rates of cholesterol biosynthesis in humans and suggest that this measurement provides a valuable means to potentially screen for disorders associated with an oversynthesis of cholesterol.
Abetalipoproteinemia↗
Homozygous familial hypercholesterolemia. Selective removal of low-density lipoproteins by secondary membrane filtration.
Selective plasma filtration with a hollow-fiber membrane device was compared prospectively to plasma exchange in the therapy of a patient with homozygous familial hypercholesterolemia. Four liters of patient plasma was removed biweekly during each of six consecutive plasma exchanges, after which 20 consecutive biweekly 4-liter filtration procedures were conducted. The hollow-fiber membrane retained 94 percent of the low-density lipoprotein (LDL) cholesterol presented to it, and allowed passage of 83 percent of the albumin, 68 percent of the IgG, and 47 percent of the high-density lipoprotein (HDL) cholesterol. Both plasma exchange and plasma filtration decreased the patient's total and LDL cholesterol levels by 80 percent. However, filtration removed significantly less HDL than did exchange (54 versus 71% reduction in HDL levels, respectively); preserved significantly higher levels of IgG, clotting factors, and complement components; and avoided the need for expensive albumin replacement solutions. In addition, the patient tolerated the filtration procedures significantly better than the exchanges. Newer apheresis techniques that selectively deplete plasma of LDL cholesterol, such as secondary membrane filtration, are likely to replace plasma exchange as the therapy of choice in patients with homozygous hypercholesterolemia.
Blood Coagulation Factors↗
Multifocal verruciform xanthoma of the upper aerodigestive tract in a child with a systemic lipid storage disease.
We report the clinical, light-microscopic, and ultrastructural features of a case of multifocal verruciform xanthoma in the upper aerodigestive tract of a child with a systemic lipid disorder. Lipid storage cells were found in liver, bone marrow, and as a component of verruciform xanthomas. To our knowledge this represents the first case of verruciform xanthoma reported in (a) a child, (b) as a multifocal lesion in the upper aerodigestive tract, (c) associated with a systemic lipid disorder, and (d) with ultrastructural evidence of lipid accumulation within endothelial cells. Although this patient presented with lesions involving the tongue and larnyx, subsequently lesions were found in the bone marrow and liver. Two months later more lesions were discovered on the epiglottis, posteior tongue, right glottis, and in grossly normal peritonsillar mucosa. Six months later a new oral lesion developed. Based upon these observations, we speculate that the pathogenesis of verruciform xanthoma involves accumulation of excess lipid in subepithelial sites which is scavenged by macrophages. Lipid-laden macrophages release epithelial growth factors that lead to epithelial hyperplasia. Depending on the degree of epithelial hyperplasia, the gross appearance of verruciform xanthomas may be flat, sessile, papillary, or verrucous.
Child, Preschool↗
Characterization of the binding of thyroxine to high density lipoproteins and apolipoproteins A-I.
We studied binding of T4 to the lipid-complexed apolipoproteins (apo) of high density lipoproteins (HDL), the major lipoprotein carrier of thyroid hormones in human plasma, and to lipid-free apoA-I. HDL isolated from fresh normal plasma by ultracentrifugation (density, 1.063-1.210 g/mL) was photoaffinity labeled with [3,5-(125)I]T4 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands corresponding to apoA-I (28.3K) and apoC-II or apoC-III (8.6-9.2K) were seen, and their radioactivity decreased by 50-60% when labeled in the presence of 1 mumol/L T4. Photoaffinity labeling of isolated apoA-I also was demonstrated and was decreased 74% by 1 mumol/L T4, suggesting a higher affinity of the lipid-free protein for T4. T4 binding of isolated apoA-I was optimal at pH 7-8, reached a maximum after 1 h at 23 C, and decreased after incubation at 37 C. Scatchard analysis revealed a single T4-binding site with a Ka of 7.5 x 10(7) L/mol at 23 C, pH 8.2. The potency of T4 analogs as inhibitors of T4 binding to isolated apoA-I was L-T4 = D-T4 = triiodothyroacetic acid = L-rT3 much greater than L-T3 much greater than L-thyronine. The binding of T4 to apoA-I was reduced by known inhibitors of T4 binding to serum proteins (diclofenac = mefenamic acid = furosemide = 8-anilinonaphthalene sulfonic acid much greater than dilantin greater than heparin greater than barbital) and by lipids (unsaturated fatty acids greater than cholesterol = cholesterol esters = phospholipids greater than saturated fatty acids = diglycerides = triglycerides). We conclude that the binding of T4 to HDL is mediated by a specific interaction of the hormone with apoA-I and with apoC-II and/or apoC-III. Since the lipid constituents of HDL inhibit T4 binding to apoA-I, the HDL subfraction in plasma that carries most of the HDL-bound T4 should be one with a low lipid content.
Apolipoprotein A-I↗
Primary biliary cirrhosis: management of an unusual case with severe xanthomata by hepatic transplantation.
We report a patient with advanced primary biliary cirrhosis associated with Sjögren's syndrome, xanthelasma, and extensive, painful xanthomata involving cutaneous lipid deposits on her face, abdomen, hands, and buttocks and extensor surfaces over many joints. Despite conventional dietary and drug therapy, these lesions progressed rapidly over 3 years. There was symptomatic improvement of the xanthomata, but no objective amelioration of the xanthomatosis with the use of plasmapheresis over an 18-month period. Liver transplantation was undertaken for decompensated chronic liver disease and poor quality of life due to complications of xanthomatosis. Twelve months after transplantation, all xanthomata and xanthelasma and symptoms attributable to xanthomata had disappeared. Liver transplantation is a drastic but successful remedy for complications of abnormal lipid metabolism associated with primary biliary cirrhosis.
Adult↗
Molecular species of phosphatidylcholine in abetalipoproteinemia: effect of lecithin:cholesterol acyltransferase and lysolecithin acyltransferase.
In order to study the role of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in determining the molecular species composition of phosphatidylcholine (PC) and the specificity of lecithin:cholesterol acyltransferase (LCAT) in human plasma, we studied the PC species composition in plasma from abetalipoproteinemic (ABL) and control subjects before and after incubation at 37 degrees C. The ABL plasma contained significantly higher percentages of sn-2-18:1 species (16:0-18:1, 18:0-18:1, and 18:1-18:1) and lower percentages of sn-2-18:2 species (16:0-18:2, 18:0-18:2, and 18:1-18:2) as well as sn-2-20:4 species (16:0-20:4, 18:0-20:4, and 18:1-20:4). Similar abnormalities were found in the PC of ABL erythrocytes, while the PE of the erythrocytes was less affected. The relative contribution of various PC species towards LCAT reaction in ABL plasma was significantly different from that found in normal plasma. Thus, while 16:0-18:2 and 16:0-18:1 contributed, respectively, 43.8% and 15.9% of the total acyl groups used for cholesterol esterification in normal plasma, they contributed, respectively, 21.5% and 37.9% in ABL plasma. The relative contribution of 16:0-20:4 was also significantly lower in ABL plasma (4.7% vs. 9.0% in normal), while that of 16:0-16:0 was higher (6.4% vs. 0.5%). However, the selectivity factors of various species (percent contribution/percent concentration) were not significantly different between ABL and normal plasma, indicating that the substrate specificity of LCAT is not altered in the absence of VLDL and LDL. Incubation of ABL plasma in the presence of normal VLDL or LDL resulted in normalization of its molecular species composition and in the stimulation of its LCAT activity. Addition of LDL, but not VLDL, also resulted in the activation of lysolecithin acyltransferase (LAT) activity. The incorporation of [1-14C]palmitoyl lysoPC into various PC species in the presence of LDL was similar to that observed in normal plasma, with the 16:0-16:0 species having the highest specific activity. These results indicate that the absence of apoB-containing lipoproteins significantly affects the molecular species composition of plasma PC as well as its metabolism by LCAT and LAT reactions.
1-Acylglycerophosphocholine O-Acyltransferase↗
Competitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of apolipoprotein A-I using a monoclonal antibody.
A semi-automated competitive enzyme-linked immunosorbent assay for human plasma apolipoprotein (Apo) A-I has been developed which utilizes nondelipidated samples, microtiter plates, commercially available monoclonal antibodies and alkaline phosphatase conjugated second antibody. The working range of the assay is 5-100 ng of Apo A-I. The range of plasma concentrations for plasma Apo A-I was 1.21 +/- 0.34 g/l for a random sample of 40 healthy adults. Intra- and inter-assay coefficients of variation (CV) were 4 and 7%, respectively. There was a good correlation between this assay and a radial immunodiffusion assay (r = 0.96). The assay is suitable for measurement of apolipoprotein A-I in either normal or pathological plasma, lipoprotein density classes, and for cell biological and molecular biological investigations.
Antibodies, Monoclonal↗
A deletion mutation in the ApoC-II gene (ApoC-II Nijmegen) of a patient with a deficiency of apolipoprotein C-II.
The apolipoprotein C-II gene from a patient with a deficiency of apoC-II was cloned and sequenced. A single base deletion of a guanosine at position 2943 in exon three of the gene of the proband was identified by sequence analysis. This point mutation results in a shift of the reading frame and introduces a premature termination codon (TGA) at a position in the gene immediately following amino acid 17 of the mature C-II apolipoprotein. This single base deletion results in the loss of a normally occurring HphI restriction enzyme site in the apoC-II gene. Amplification of the mutant DNA sequence by the polymerase chain reaction and restriction enzyme digestion with HphI established that the patient is a homozygote for the base deletion. No apoC-II was detectable in the patient's plasma by two-dimensional gel electrophoresis and immunoblotting. We propose that the guanosine deletion is the primary genetic defect in this kindred leading to premature termination and formation of a nonfunctional truncated 17-amino acid C-II apolipoprotein which ultimately results in apoC-II deficiency.
Apolipoprotein C-II↗
Apolipoprotein C-II deficiency: identification of a structural variant ApoC-II Padova.
Apolipoprotein(apo) C-II DNA, RNA and protein from a patient with a familial deficiency of apoC-II were evaluated and compared to normal individuals. No major defect of the apoC-II gene could be detected by Southern blot hybridization. Northern and slot blot analyses of total liver RNA documented normal levels of a normal sized apoC-II mRNA. Immunohistochemical studies of the liver of the apoC-II deficient patient revealed a normal to slightly elevated intracellular content of the C-II apolipoprotein. Plasma apoC-II was 3 to 5% of normal apoC-II levels and exhibited abnormal electrophoretic mobility on two dimensional gel electrophoresis and immunoblotting. We postulate that at the molecular level, the deficiency of apoC-II in the plasma of this patient results from a structural defect in the coding portion of the apoC-II gene leading to either defective secretion of cellular apoC-II or increased catabolism of a structurally defective apoC-II in plasma.
Apolipoprotein C-II↗
Homozygous hypobetalipoproteinemia: a disease distinct from abetalipoproproteinemia at the molecular level.
apoB DNA, RNA, and protein from two patients with homozygous hypobetalipoproteinemia (HBL) were evaluated and compared with normal individuals. Southern blot analysis with 10 different cDNA probes revealed a normal gene without major insertions, deletions, or rearrangements. Northern and slot blot analyses of total liver mRNA from HBL patients documented a normal size apoB mRNA that was present in greatly reduced quantities. ApoB protein was detected within HBL hepatocytes utilizing immunohistochemical techniques; however, it was markedly reduced in quantity when compared with control samples. No apoB was detectable in the plasma of HBL individuals with an ELISA assay. These data are most consistent with a mutation in the coding portion of the apoB gene in HBL patients, leading to an abnormal apoB protein and apoB mRNA instability. These results are distinct from those previously noted in abetalipoproteinemia, which was characterized by an elevated level of hepatic apoB mRNA and accumulation of intracellular hepatic apoB protein.
Apolipoproteins B↗
Donor splice site mutation in the apolipoprotein (Apo) C-II gene (Apo C-IIHamburg) of a patient with Apo C-II deficiency.
The DNA, RNA, and protein of apo C-II have been analyzed in a patient with apo C-II deficiency (apo C-IIHamburg). Markedly reduced levels of plasma and intrahepatic C-II apolipoprotein were demonstrated by immunoblotting and immunohistochemical analysis. Northern, slot blot, and in situ hybridization studies revealed low levels of a normal-sized apo C-II mRNA. No major rearrangement of the apo C-II gene was detected by Southern blotting. Sequence analysis of apo C-II genomic clones revealed a G-to-C substitution within the donor splice site of intron II. This base substitution resulted in the formation of a new Dde I and loss of a Hph I restriction enzyme cleavage site. Amplification of the mutant sequence by the polymerase chain reaction and digestion with Dde I and Hph I restriction enzymes established that the patient was homozygous for the G-to-C mutation. This is the initial report of the DNA sequence of an abnormal apo C-II gene from a patient with deficiency of apo C-II. We propose that this donor splice site mutation is the primary genetic defect that leads to defective splicing and ultimately to an apo C-II deficiency in this kindred.
Adult↗
Binding of thyroid hormones to human plasma lipoproteins.
The binding of T4, T3, and rT3 to plasma lipoproteins was investigated in normal subjects and patients with abnormal lipoprotein metabolism. Gel filtration on Sepharose CL-6B demonstrated iodothyronine binding to all lipoprotein classes. In the total lipoprotein fraction (density less than 1.210 g/mL), high density lipoproteins (HDL) were the major binders, accounting for 92% of lipoprotein-bound T4, 99% of lipoprotein-bound T3, and 55% of lipoprotein-bound rT3. The estimated iodothyronine binding in normal plasma to HDL, low density lipoproteins (LDL), and very low density lipoproteins (VLDL) was 3%, 0.2%, and 0.03% for T4, 6%, 0.05%, and 0.02% for T3, and 0.1%, 0.1%, and 0.01% for rT3, respectively. These estimates may be low owing to possible dissociation during chromatography and the short incubation period used to avoid changes in lipoprotein structure. In VLDL and LDL deficiency (abetalipoproteinemia), HDL deficiency (Tangier disease), LDL excess (type IIa hyperlipoproteinemia), and VLDL excess (type III, IV, and V hyperlipoproteinemia), the distribution of iodothyronines reflected the lipoprotein abnormality. Variations resulting from altered distribution within HDL subclasses were also found. Binding was saturable, with approximate dissociation constants for VLDL, LDL, and HDL of 10(-5)-10(-6) mol/L. We conclude that thyroid hormones bind specifically to apolipoproteins, although additional binding by solubilization in the lipid components of the lipoproteins may also occur.
Abetalipoproteinemia↗
The role of apolipoprotein E and lipoprotein receptors in modulating the in vivo metabolism of apolipoprotein B-containing lipoproteins in humans.
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Alleles↗
Apolipoproteins and lipoproteins in human plasma: an overview.
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Apolipoproteins↗