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Biomedical subjects

R E Ostlund

Publications and source records attributed to R E Ostlund.

At least 19 recordsLinked to original sources
PubMed1992

Cholesteryl ester storage disease: complex molecular effects of chronic lovastatin therapy.

To better characterize the in vivo effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition on human lipid metabolism, an adolescent male with cholesteryl ester storage disease (CESD) was treated chronically with lovastatin. Therapy was associated with decreased liver-spleen size, improved but not normal serum lipids, a 26% decrease in hepatic cholesteryl ester, a 12% decrease in unesterified hepatic cholesterol, and a fourfold increase in hepatic low density lipoprotein (LDL) receptor protein. Hepatic mRNA levels for the LDL receptor and apolipoprotein (apo) B standardized to levels of hepatic gamma actin mRNA were unchanged with therapy. Kinetic studies revealed no change in the LDL fractional catabolic rate and a decrease in the LDL production rate. Size exclusion chromatography showed striking reductions in plasma very low density lipoprotein (VLDL) cholesterol and intermediate density lipoprotein (LDL) cholesterol but not LDL cholesterol with therapy. Mean LDL particle size and the LDL particle size range were increased by treatment. However, there was no difference in the ability of pretreatment or treatment LDL to bind to the LDL receptor on cultured cells consistent with previous studies in animals, indicating that lovastatin may alter LDL particles to impair interaction with the LDL receptor in vivo but not in vitro. Lovastatin therapy in CESD appears to be clinically beneficial and has complex effects on lipid metabolism that may include a dominant inhibitory effect on hepatic lipoprotein production, posttranscriptionally mediated induction of the LDL receptor, and alterations of LDL particles that interfere with their clearance by the LDL receptor in vivo.

Actins↗
PubMed1990

A protein partially expressed on the surface of HepG2 cells that binds lipoproteins specifically is nucleolin.

Nucleolin, a major nucleolar protein of rapidly growing eukaryotic cells, has been thought to be predominantly if not exclusively located in the nucleolus. Recent data however [Borer, R.A., Lehner, C.F., Eppenberger, H.M., & Nigg, N.A. (1989) Cell 56, 379-390] suggest that the protein shuttles constantly between the nucleus and cytoplasm. Ligand blotting studies of whole cell extracts of HepG2 cells identified, in addition to the LDL receptor, another LDL binding protein of Mr 109,000. The 109-kDa protein was partially purified by HPLC and, like the LDL receptor, bound apoB- and apoE-containing lipoproteins but not HDL. However, unlike the LDL receptor, the 109-kDa protein bound lipoproteins in the presence of EDTA and reducing agents, had a lower affinity for lipoproteins than the LDL receptor, and did not react with two antibodies raised against the LDL receptor. The protein sequences of three separate peptides derived from the partially purified 109-kDa species were determined and were identical except for one residue to three separate regions of the published sequence of nucleolin. On immunoblot analysis the 109-kDa protein reacted with a nucleolin-specific antibody, and purified nucleolin reacted both with anti-109-kDa antibody and with LDL. When intact HepG2 cells were treated with Pronase before harvest, there was a 46% decrease in 109-kDa protein while recovery of actin, an intracellular protein, was unaffected. When intact HepG2 cells were surface iodinated and the proteins subjected to HPLC fractionation, the 109-kDa protein was found to be iodinated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

The ratio of waist-to-hip circumference, plasma insulin level, and glucose intolerance as independent predictors of the HDL2 cholesterol level in older adults.

High plasma levels of HDL2, a subfraction of high-density lipoprotein (HDL) cholesterol, are associated with a reduced risk of coronary heart disease. To investigate the characteristics related to HDL2 cholesterol levels, we measured lipoprotein levels and several metabolic and anthropometric variables in 146 healthy subjects (77 men and 69 women) in the seventh decade of life. The level of HDL2 cholesterol was inversely correlated with the ratio of the waist-to-hip circumference (r = -0.335 for men; r = -0.370 for women; P less than 0.01) and the plasma insulin level (r = -0.400 for men; r = -0.398 for women; P less than 0.001). In a multiple regression model including both sexes, 41 percent of the variance in the HDL2 level was explained by the combined effect of the waist-to-hip ratio (P less than 0.0001), the plasma insulin level (P = 0.0003), and the degree of glucose tolerance indicated by the integrated area under the plasma glucose curve after an oral glucose-tolerance test (P = 0.05). The body-mass index, total percentage of body fat, maximal oxygen uptake, diet, and sex were not significant predictors of the HDL2 level when added to this model, whereas the original variables remained significant predictors. The HDL2 cholesterol level in subjects at the 25th percentile for waist-to-hip ratio was 153 percent of that in subjects at the 75th percentile. We conclude that HDL2 levels are inversely correlated with truncal fat, plasma insulin levels, and the presence of glucose intolerance and are not independently associated with sex or total body fat.

Abdomen↗
PubMed1990

Apolipoprotein E-rich HDL in patients with homozygous familial hypercholesterolemia.

Ordinarily, HDL1, a fraction of HDL enriched in apoE, is a minor fraction of plasma, but in human subjects and experimental animals eating diets high in fat and cholesterol and in patients with homozygous familial hypercholesterolemia (HFH) or CETP deficiency, HDL1 (or HDLc) concentrations in plasma are increased. However, little is known about the structures, compositions and metabolic sources of HDL1 in HFH patients. To obtain HDL1 for the study, we surveyed several fractions in the HDL density range for apoE by SDS-PAGE. The ratio of apoE to apoAI in the HDL (d = 1.063-1.21 g/ml) of 8 HFH patients was 0.14 +/- 0.03 compared to 0.03 +/- 0.005 in a control group of 8 normolipidemic subjects (P less than 0.001) suggesting that an apoE-rich fraction indeed was present in increased amounts. ApoE/apoAI ratios of lipoproteins of the density range 1.050-1.090 were even higher at 1.5 and 2.0 in 2 patients compared to 0.4 +/- 0.1 in controls, indicating that this density fraction may be particularly enriched with apoE-rich lipoproteins. By contrast, d = 1.020-1.050 g/ml and d greater than 1.090 fractions contained very little apoE. Therefore, we further characterized the d = 1.050-1.090 g/ml lipoproteins of HFH patients and controls. Fractionation of an d = 1.050-1.090 fraction by concanavalin-A chromatography (CONA) yielded an unbound apoE-rich fraction that contained apoE, apoAI and apoC but no apoB, and a bound LDL-like fraction that contained mostly apoB-100, as determined by SDS-PAGE and by solid phase immunoassays, containing monoclonal antibodies directed against apoB, apoE and apoAI. The apoE/apoAI ratio of the CONA unbound fraction of HFH patients was greater, and the fraction also contained more free cholesterol and phospholipids than the fraction of control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1989

Effect of exercise training on plasma cholesterol and cholesterol kinetics in adult female rats.

Adult female rats fed laboratory chow of low cholesterol content were trained by swimming for 77 days. Plasma cholesterol concentration decreased 38% compared to sedentary controls. Whole body cholesterol metabolism was studied by injection of [14C]cholesterol tracer and determination of the plasma cholesterol-specific activity during the last 49 days of the experiment. The rate constant for elimination of cholesterol from the body was 31% higher in swimming than in sedentary animals, and the size of the rapidly-exchanging cholesterol pool was 28% smaller. These results suggest that enhanced cholesterol excretion or catabolism accompanies exercise training in the rat.

Animals↗
PubMed1989

Quantitative relationship between plasma lipids and glycohemoglobin in type I patients. Longitudinal study of 212 patients.

Plasma cholesterol, triglycerides, glycohemoglobin, and other covariates were measured in 212 type I (insulin-dependent) diabetic subjects on entry into a longitudinal study of diabetes and again after an average interval of 3.7 yr. Changes in individual cholesterol and triglyceride values over time were significantly correlated with changes in glycohemoglobin. After adjustment for potentially confounding covariates, plasma cholesterol declined by 2.2% (0.1 mM) for each percentage-point reduction in glycohemoglobin and plasma triglycerides declined by 8% (0.08 mM) per percentage point glycohemoglobin. Increased insulin dose was independently associated with increased plasma triglycerides, after adjusting for glycohemoglobin level and other covariates. However, insulin dose diabetic metabolic control, measured as declining glycohemoglobin, is the variable most closely associated with reduced plasma lipids in a population of typical type I diabetic patients.

Biomarkers↗
PubMed1989

Plasma lipids in patients with type I diabetes mellitus. Influence of race, gender, and plasma glucose control: lipids do not correlate with glucose control in black women.

Plasma lipids and hemoglobin A1 were measured in 544 type I diabetic patients. Hemoglobin A1 was positively correlated with the levels of total plasma cholesterol, total triglycerides, and low-density lipoprotein cholesterol and negatively correlated with the level of high-density lipoprotein cholesterol in the entire biracial group. These relationships between plasma lipids and hemoglobin A1 were not present in black women. In the white diabetic population a reduction in hemoglobin A1 of one percentage point was statistically associated with a decrease of 0.16 to 0.17 mmol/L in total plasma cholesterol, a decrease of 0.10 to 0.13 mmol/L in low-density lipoprotein cholesterol, and a reduction of 0.12 to 0.14 mmol/L in triglycerides. These findings suggest that race and gender are important determinants of the response of plasma lipids to glucose control in type I diabetes mellitus.

Adolescent↗
PubMed1988

Removal of apolipoprotein B from dog whole blood by ex vivo hemoadsorption on antibody-agarose beads.

Antibody columns for hemoperfusion were prepared by filling 300-ml polycarbonate canisters with 2% agarose gel beads having attached goat antibodies directed toward apolipoprotein B (apo B), the major apoprotein of low and very low density lipoproteins. Blood was withdrawn from the left external jugular vein of dogs, regionally treated with anticoagulant citrate dextrose solution A, pumped through the antibody column, and returned to the right external jugular vein. Immunoreactive apo B decreased by 81% during passage of blood over the column. Adverse effects were not observed during four weekly hour-long perfusions with blank columns (agarose beads without antibody attached) followed by four weekly perfusions with antibody columns. The columns were disinfected and stored in 1 M acetic acid and reused weekly in each animal. Recovery of platelets and white blood cells over the columns was 90 and 102%, respectively, with no significant differences between blank columns and antibody-containing columns. Complement was not consumed during the hemoadsorption procedure. Hemoadsorption on antibody-agarose columns is a promising potential method for removing toxic molecules and cells from whole blood.

Animals↗
PubMed1987

Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2.

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Hepatocellular↗
PubMed1987

Immunosorbent chemistry: a study of agarose-based column sorbents for the removal of low-density lipoprotein (LDL) from blood.

Anti-LDL antibody was covalently attached to agarose beads in order to prepare an immunosorbent for the removal of LDL from plasma or blood. Both the conditions of antibody coupling and the type of agarose matrix used were critical to the optimization of LDL binding capacity. Sorbents binding 6-8 mg lipoprotein cholesterol/ml column volume were obtained using cyanogen bromide or glutaraldehyde coupling procedures and crosslinked 2% agarose beads. The sorbent could be regenerated by washing with 1 M acetic acid, a reagent that was also an effective disinfectant. In vitro perfusions of whole blood over small columns of 212-300 mu beads showed excellent flow rates (2 ml/min/cm2 under 50-100 cm saline pressure); recovery of leukocytes and platelets exceeded 90%, and complement was not activated. Leakage of antibody and bead matrix was negligible. The antibody-agarose beads could not be sterilized by conventional techniques, but withstood treatment with 0.34% phosphoric acid in 80% ethanol at 37 degrees C, a novel method of chemical sterilization.

Humans↗
PubMed1986

Long-term changes in cholesterol biosynthesis and the effect of plasmapheresis therapy in a hypercholesterolemia homozygote.

Synthesis of cholesterol was measured in a familial hypercholesterolemia homozygote on four occasions from age 1.1 to 9.9 years by the sterol balance technique. Both the fecal neutral steroid and fecal bile acid components of sterol balance were elevated initially. Over the decade of study, neutral steroid excretion/kg declined 61% whereas bile acid excretion/kg was unchanged. Chronic plasmapheresis therapy every two weeks for 3.4 years reduced plasma low-density lipoprotein cholesterol 54% but had little effect on the rate of cholesterol biosynthesis.

Bile Acids and Salts↗
PubMed1986

Diversity in expression of heterozygous familial hypercholesterolemia. Characterization of a unique kindred.

Clinical and biochemical characteristics of familial hypercholesterolemia (FH) heterozygotes possessing an abnormally high molecular weight low density lipoprotein receptor (HMWR) are reported. The disorder is transmitted as an autosomal dominant trait and is not distinguishable from classic heterozygous FH on clinical grounds. The average plasma low density lipoprotein (LDL) level is 360 mg/dl and tendon xanthomata and early coronary disease are present. LDL receptor activity is higher than expected. In skin fibroblast cultures two types of functional LDL receptors are present, one with a normal apparent native molecular weight of 140,000, and the other of 176,000. When immobilized on nitrocellulose paper both receptors bind LDL. Maximum 125I-LDL binding capacity of fibroblast monolayers is reduced only 20%, compared with 50% in typical heterozygous FH. Affinity for 125I-LDL is increased and a 38% reduction in the Michaelis constant for LDL is observed. When autologous 125I-LDL was injected intravenously, the fractional catabolic rate of LDL was 205% and the LDL apoprotein B production rate was 328% of that found in a typical heterozygous FH subject. Thus, both in vitro and in vivo testing indicated only a modest deficiency of LDL receptor activity. Kindred members possessing the HMWR had an associated abnormality of cholesterol biosynthesis. Cholesterol balance studies in three individuals with the HMWR trait demonstrated elevated cholesterol biosynthesis of two to three times the mean of normal subjects. These findings suggest that increased LDL production and increased cholesterol production may assume a significant role in the pathologic manifestations of heterozygous FH. Functional abnormalities in LDL receptor activity as measured in fibroblast culture may be relatively small.

Apolipoproteins B↗
PubMed1986

The low density lipoprotein receptor on human peripheral blood monocytes and lymphocytes: visualization by ligand blotting and immunoblotting techniques.

Western blotting and immunoprecipitation techniques were used to study low density lipoprotein (LDL) receptors from cultured human monocytes, lymphocytes, and fibroblasts. After incubation in lipoprotein-deficient media to allow induction, receptors were solubilized, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and detected by incubation with apolipoprotein B-containing lipoproteins followed by radiolabeled antiapolipoprotein B antibody. LDL receptors of identical apparent mol wt were demonstrated in monocyte and lymphocyte cell extracts; the receptors bound LDL as well as human post-prandial lipoprotein (density, less than 1.0063) on Western blots, but did not bind acetyl-LDL. LDL receptors in mononuclear cells could also be detected by immunoblotting using immunoglobulin G-C7, a monoclonal antibody raised against the bovine LDL receptor. When cell extracts were blotted, the mononuclear cell receptors had a lower mol wt than the fibroblast receptor in unreduced sodium dodecyl sulfate-polyacrylamide gels, with an apparent mol wt difference of 5,000 [134,000 +/- 1,400 (+/- SE) for mononuclear cells vs. 139,000 +/- 1600 for fibroblasts]. Immunoprecipitation and electrophoresis of L-[35S]methionine-labeled cell extracts revealed more rapid conversion of the receptor precursor to the mature receptor in monocytes than in fibroblasts. Western blotting of mononuclear cells from a patient with an abnormally high mol wt receptor characterized in fibroblasts demonstrated that the same mutation was expressed in monocytes and lymphocytes. This report represents the first visualization of human mononuclear cell LDL receptors by Western blotting and immunoprecipitation. These techniques may find application in population screening for LDL receptor variability.

Antibodies, Monoclonal↗
PubMed1985

The effects of amphotericin B on lipid metabolism in cultured human skin fibroblasts.

Amphotericin B inhibits hydroxymethylglutarylcoenzyme A (HMGCoA) reductase activity and incorporation of [3H]acetate into sterols and fatty acids of human skin fibroblasts. Delivery of cholesterol to cells via the low density lipoprotein receptor pathway is not altered. The effects of amphotericin B on cell lipid metabolism are partially reversed by both potassium and insulin.

Amphotericin B↗
PubMed1985

Effect of cholesterol and growth factors on the proliferation of cultured human skin fibroblasts.

A cholesterol-deficient growth medium for human skin fibroblasts was prepared by adding to Eagle's Minimum Essential Medium a bovine serum treated with ultracentrifugation to remove bulk lipoproteins followed by silicic acid adsorption to remove residual lipoproteins and cholesterol. Cell growth was slow, but the daily cell doublings could be increased by 76% by including 7.5 micrograms purified cholesterol/ml in the medium. Cell growth in cholesterol-deficient culture medium could be increased to that seen with medium containing 15% untreated fetal bovine serum by the inclusion of the following growth factors: epidermal growth factor (EGF), cortisol, non-essential amino acids, insulin, transferrin and selenium. Cholesterol increased the proliferation of these rapidly-growing cultures by 19%. No effect of cholesterol was observed in transformed L-cell mouse fibroblasts.

Amino Acids↗
PubMed1985

Demonstration of functional low-density lipoprotein receptors by protein blotting in fibroblasts from a subject with homozygous receptor-negative familial hypercholesterolemia.

We report the detection of low-density lipoprotein (LDL) receptors by the technique of receptor blotting in fibroblasts from a patient with homozygous familial hypercholesterolemia (FHC) previously classified as "receptor negative." Solubilized receptors were electrophoresed, transferred to nitrocellulose paper, treated with LDL followed by radiolabeled antibody to LDL, and visualized by autoradiography. GM 2000 FHC fibroblasts revealed LDL receptors with an apparent molecular weight of approximately 140,000, the same as in normal cells. LDL receptor activity by blotting in GM 2000 cells was greatly diminished in comparison with normal cells, but was calcium dependent. Receptor activity was also detectable by conventional monolayer binding and degradation assays. Thus, GM 2000 cells have profoundly diminished LDL receptor activity, but retain the genetic capacity to make LDL receptor material of normal molecular weight that is capable of binding LDL. Previous studies have demonstrated the presence of trace amounts of immunoreactive LDL receptor protein in fibroblasts from some receptor-negative FHC homozygotes. Our work extends these studies by demonstrating the ability of this material to bind LDL.

Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts.

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).

Binding, Competitive↗