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Biomedical subjects

R E Morton

Publications and source records attributed to R E Morton.

At least 19 recordsLinked to original sources
PubMed1992

Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content.

The bicinchoninic acid (BCA) assay method for the determination of protein has been investigated for its utility in measuring the protein content of plasma lipoproteins. Although other methods, principally those based on the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275) have been extensively used for this purpose, the tolerance of the BCA method to many commonly encountered detergents and buffers offers a definite advantage over the Lowry-based methods. In this study, lipoprotein protein values obtained by the BCA method were compared to a standard modification of the Lowry et al. procedure since this assay forms the basis of much of the relevant literature. The standard BCA assay was found to overestimate the protein content of very low density lipoprotein by approximately 70% and low density lipoprotein by approximately 30%; high density lipoprotein values compared favorably. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions. This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis. In the presence of this detergent, BCA assay measurements for these three lipoprotein fractions were 97, 90, and 98%, respectively, of the reference assay values.

Humans↗
PubMed1991

Modified low density lipoprotein isolated from atherosclerotic lesions does not cause lipid accumulation in aortic smooth muscle cells.

Foam cells in atherosclerotic lesions are derived not only from blood monocytes but also from smooth muscle cells (SMC). To better understand the mechanisms by which SMC may become lipid-laden, we have studied the catabolism by cultured rabbit aortic SMC of LDL derived from atherosclerotic lesions (A-LDL) previously shown to be chemically modified. A-LDL was isolated either from homogenates of atherosclerotic plaques in human aortas by affinity chromatography and gel filtration, or from nonhomogenized extracts of plaque minces by ultracentrifugation and gel filtration. Internalization of A-LDL by SMC or fibroblasts appeared to be mediated primarily via the LDL receptor since: 1) either unlabeled LDL or A-LDL could inhibit the degradation of 125I-labeled A-LDL or of 125I-labeled LDL, 2) the uptake of both A-LDL and LDL, as estimated by their abilities to stimulate cholesterol esterification, was reduced in cells in which LDL receptor expression was down-regulated; and 3) the uptake of both [3H]cholesteryl ether-labeled A-LDL and LDL by normal fibroblasts was significant and could be inhibited by excess LDL, but was negligible in receptor-negative fibroblasts. At saturating concentrations of lipoproteins, maximum cholesterol esterification in SMC was greater for LDL than for A-LDL. Over a 48-h incubation, A-LDL, like LDL, was unable to induce cellular cholesteryl ester accumulation. Cross-competition studies suggested that either the affinity of A-LDL for the LDL receptor was less than that of LDL, or that some particles in A-LDL are not internalized by SMC. The latter alternative was supported by the observations that some A-LDL particles had undergone aggregation, especially at high concentrations, and that aggregated forms of A-LDL or plasma LDL failed to be internalized and degraded by SMC. Collectively, these results are consistent with recognition of some of the A-LDL particles by the LDL receptor, but also suggest that, at least under in vitro conditions, A-LDL is unlikely to induce lipid accumulation in SMC resulting in SMC-derived foam cells.

Adult↗
PubMed1990

Interaction of lipid transfer protein with plasma lipoproteins and cell membranes.

The hydrophobic lipid components of lipoproteins, cholesteryl ester and triglyceride, are transferred between all lipoproteins by a specific plasma glycoprotein, termed lipid transfer protein (LTP). LTP facilitates lipid transfer by an exchange process in which cholesteryl ester and triglyceride compete for transfer. Thus, LTP promotes remodeling of the lipoprotein structure, and plays an important role in the intravascular metabolism of these particles and in the lipoprotein-dependent pathways of cholesterol clearance from cells. The properties of LTP, its mechanisms of action, its roles in lipoprotein metabolism, and its modes of regulation are reviewed along with recent data that suggest a possible role for this protein in directly modifying cellular lipid composition.

Animals↗
PubMed1990

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity.

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.

Carbon Radioisotopes↗
PubMed1988

Free cholesterol is a potent regulator of lipid transfer protein function.

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.

Carbon Radioisotopes↗
PubMed1988

Protein metabolism during treatment of chest infection in patients with cystic fibrosis.

Six cystic fibrosis patients with pulmonary exacerbations were studied to determine the effect of antibiotic treatment on protein nutritional status. Indirect calorimetry, nitrogen balance, protein turnover, urinary 3-methylhistidine, plasma albumin, prealbumin transferrin, and cortisol were measured before and after treatment. N loss averaged 16 and 17% on each balance. N in the sputum was up to 4.5% of absorbed N intake. At the peak of infection, protein synthesis, degradation, and urinary 3-methylhistidine were significantly higher than during recovery (31%, 28%, and 60%, respectively). On recovery a significant fall in blood sugar, albumin, morning cortisol and sputum N and a rise in prealbumin was found. Basal metabolic rate and N balance did not change. For patients in the fed state, active infection is associated with higher rates of protein synthesis and degradation. Antimicrobial treatment alters protein dynamics but does not alter measured N balance or the difference between measured protein synthesis and breakdown.

Adolescent↗
PubMed1988

The cost-effectiveness of maintenance therapy for duodenal ulceration with an H2-antagonist.

Data from several sources are used to quantify the expected direct medical costs of a recently healed duodenal ulcer patient prescribed an H2-antagonist (famotidine) for a 6-month period. These costs are compared to the expected direct medical costs associated with not using maintenance therapy. Our results indicate that the estimated direct cost of patients prescribed a 6-month regimen of an H2-antagonist (famotidine) is 30.3% lower than patients who receive no H2-antagonist therapy. Most of the savings result from a reduced risk of hospitalization and surgery. The results of the sensitivity analysis of four varying scenarios indicate that H2-antagonist maintenance therapy remains less costly even when the assumptions underlying the model are varied enormously. We conclude that the decision to withhold maintenance therapy with H2-antagonists should not be based on economics.

Cost-Benefit Analysis↗
PubMed1988

Atlantoaxial instability and abnormalities of the odontoid in Down's syndrome.

Radiography of the lateral neck was performed on all children with Down's syndrome aged 4-15 in the Southern Derbyshire health district (n = 67) and a random selection of adults (n = 94). Atlantoaxial instability, defined as a gap of over 4 mm, was present in seven (10%) children and two (2%) adults. Odontoid hypoplasia, defined as an odontoid peg two standard deviations below the mean in an age matched population, was present in 15 (22%) children and 14 (15%) adults, with accessory odontoid ossicles present in two (2%) and two (3%) respectively. No one was found to have symptoms or clinical signs of spinal cord compression. Atlantoaxial instability was therefore found to be commoner in children than adults. Different programmes of management are suggested, in terms of regular clinical examination for signs and symptoms and by radiographic screening. Particular care should be taken with those who have both atlantoaxial instability and odontoid hypoplasia or accessory ossicles as they are at particular risk of spinal cord damage.

Adolescent↗
PubMed1988

Interaction of plasma-derived lipid transfer protein with macrophages in culture.

This study investigates the ability of human plasma-derived lipid transfer protein to facilitate lipid transfer to and from intact viable cells in culture. Mouse peritoneal macrophages or J774 macrophages were preincubated with acetylated low density lipoprotein and [3H]oleate/albumin to promote the intracellular synthesis and accumulation of cholesteryl [3H]oleate and 3H-labeled triglyceride. The addition of partially purified lipid transfer protein to cultures of lipid-loaded macrophages resulted in a time and concentration-dependent transfer of radiolabeled cholesteryl ester and triglyceride from macrophages to the medium. At 48 hr, lipid transfer protein facilitated the net transfer of 16 and 11% of cellular cholesteryl ester and triglyceride radioactivity, respectively, to the medium; transfer in the absence of the lipid transfer protein was less than 2%. The transfer of cholesteryl ester radioactivity was accompanied by a similar decrease in cellular cholesteryl ester mass indicating a net transfer event. Lipid transfer from cells was not dependent on the presence of a lipoprotein acceptor in the medium; however, low and high density lipoproteins present at 200 micrograms cholesterol/ml did significantly stimulate the transfer protein-facilitated efflux of these lipids. Lipid transfer protein did not appear capable of transferring radiolabeled lipid from low density or high density lipoprotein to macrophages. Radiolabeled cholesteryl ester and triglyceride transferred from cells to the medium by lipid transfer protein were associated with large molecular weight (greater than 2 x 10(6)) components in the medium with an average density greater than 1.21 g/ml; these lipids were not associated with lipid transfer protein itself. However, these radiolabeled lipids were readily incorporated into low or high density lipoproteins when these lipoproteins were added to the medium either during or after its incubation with cells. It is concluded that lipid transfer protein can facilitate the net efflux of cholesteryl esters from intact, living macrophages. These studies suggest a novel and potentially antiatherogenic role for lipid transfer protein.

Animals↗
PubMed1986

A low density lipoprotein-sized particle isolated from human atherosclerotic lesions is internalized by macrophages via a non-scavenger-receptor mechanism.

A lipoprotein particle designated A-LDL, which contains apolipoprotein B (apoB) and which is the size of plasma low density lipoproteins (LDL), was isolated from homogenates of human aortic athersclerotic plaques by a combination of affinity chromatography and gel-filtration. Compared to plasma LDL, A-LDL was more electronegative, its hydrated density was lower and more heterogeneous, and its protein-to-lipid ratio was lower. In addition, apoB in A-LDL was highly degraded, and A-LDL was recognized by mouse peritoneal macrophages (MPM) as indicated by its ability to stimulate cholesterol esterification. Cholesterol esterification was saturable with an apparent Km of 100 micrograms of A-LDL cholesterol/ml. Stimulation of cholesterol esterification was linear with time, leading to extensive accumulation of cholesteryl ester in MPM over a 48-hr time interval. The uptake or degradation of acetyl-LDL (radiolabeled either in the protein with 125I or hydrophobic core with [3H]cholesteryl ether) was markedly decreased by excess unlabeled acetyl-LDL but not by A-LDL, and excess acetyl-LDL did not inhibit the uptake or degradation of labeled A-LDL. However, a 10-fold excess of A-LDL also failed to inhibit the uptake of labeled A-LDL. This finding was consistent with the observation that, unlike the saturable stimulation of cholesterol esterification in MPM induced by A-LDL, the uptake of cholesteryl ether-labeled A-LDL was almost linear over a 0-400 micrograms cholesterol/ml range. This discrepancy between dose response curves for A-LDL, which did not occur for acetyl-LDL, could be eliminated by a 24-hr postincubation period in the absence of lipoprotein, suggesting that A-LDL is catabolized less efficiently than acetyl-LDL following internalization. In summary, we conclude that A-LDL uptake by MPM occurs via a low affinity-high capacity process. Although the uptake of A-LDL is not readily saturated, it is of sufficient affinity to lead to lipid loading of macrophages even when A-LDL is present at relatively low concentrations. If these mechanisms are operative in vivo, they could explain how foam cells in human fatty streak lesions develop.

Amino Acids↗
PubMed1986

Specificity of lipid transfer protein for molecular species of cholesteryl ester.

The capacity of the plasma-derived lipid transfer protein to facilitate the transfer of various cholesteryl ester species has been investigated. Four different molecular species of cholesteryl ester were incorporated into either reconstituted high density lipoproteins or phosphatidylcholine liposomes, and the resulting particles were used as donors in standardized lipid transfer assays. With reconstituted high density lipoproteins as substrate, the rate of transfer of cholesteryl esters was cholesteryl oleate greater than cholesteryl linoleate greater than cholesteryl arachidonate greater than cholesteryl palmitate. The transfer rate for cholesteryl oleate was 154% of that for cholesteryl palmitate. Liposome substrates gave similar results. It is concluded that lipid transfer protein transfers all major species of cholesteryl ester found in plasma; however, the relative rates of transfer were significantly affected by acyl chain composition. The transfer rates appeared to reflect substrate specificity rather than substrate availability within the donor particle.

Carrier Proteins↗
PubMed1985

Binding of plasma-derived lipid transfer protein to lipoprotein substrates. The role of binding in the lipid transfer process.

Plasma-derived lipid transfer protein (LTP) facilitates the transfer of cholesteryl ester and triglyceride between all lipoproteins. Kinetic models of the transfer event have suggested that transfer is facilitated through the physical interaction (binding) of LTP with its lipoprotein substrate. Such binding has been observed previously between LTP and high density lipoprotein (HDL), but not between LTP and low (LDL) or very low (VLDL) density lipoproteins. In the present study, the interaction of LTP with plasma lipoproteins has been re-evaluated. These experiments have employed Sepharose-bound lipoproteins in order to facilitate the rapid separation of unbound and lipoprotein-associated LTP. The validity of this approach in assessing LTP-lipoprotein interactions was evidenced by the fact that free (unbound) lipoproteins could competitively inhibit or disrupt the binding of LTP to the Sepharose-bound lipoproteins. LTP was observed to bind to VLDL, LDL, and HDL. Whereas VLDL- and LDL-LTP complexes were labile and almost completely dissociated in 90 min, HDL-LTP complexes remained intact during this time. Under equilibrium conditions, LTP binding to all lipoproteins was characterized by high affinity, saturable kinetics; the apparent affinities (Kd) of VLDL, LDL, and HDL for LTP were nearly the same (congruent to 25 nM). The results of two studies correlated lipid transfer activity with LTP binding to lipoproteins: 1) LTP binding and transfer activity increased in parallel as the amount of LTP in the assay was increased and 2) the inhibition of transfer activity caused by differing amounts of an inhibitory protein correlated with similar decrements in LTP binding. The latter data also suggest that the inhibitor protein suppresses lipid transfer activity by disrupting LTP-lipoprotein interactions. It is concluded that LTP avidly binds to VLDL, LDL, and HDL via a reversible, saturable mechanism and that the binding of LTP to the lipoprotein surface is an integral component of the lipid transfer reaction.

Carrier Proteins↗
PubMed1985

Lipoproteins containing apo B extracted from human aortas. Structure and function.

We have isolated, by anti-LDL affinity chromatography, apo B-containing lipoproteins from homogenates of atherosclerotic plaques excised from the human aorta. This fraction, called A-LP, has similarities with plasma LDL, such as having similar size and relative lipid composition, along with containing apo B. However, the fraction also contains some particles larger than LDL, it is more electronegative than LDL, the relative protein content is less than in LDL, and its apo B is highly degraded. A-LP is recognized by a high affinity binding site on mouse peritoneal macrophages (MPM), as suggested by dose-response curves of stimulation of cholesterol esterification. The interaction is inhibited by negatively-charged carbohydrates such as fucoidin, but excess A-LP did not inhibit the degradation of labeled acetyl-LDL by MPM, suggesting that the binding site recognizing A-LP may not be the scavenger receptor. Finally, stimulation of cholesterol esterification by A-LP in MPM is unregulated over a 48 hr time interval, leading to massive accumulations of cholesteryl esters and a transition of these MPM to a morphology characteristic of foam cells. It is possible that when monocytes enter the arterial intima at specific sites and become tissue macrophages, they internalize A-LP in an unregulated fashion. This, in turn, would make the monocyte-macrophage lipid-laden, and could explain the etiology of foam cells in fatty streak lesions. The modification in A-LP relative to P-LDL responsible for the enhanced recognition still needs to be elucidated.

Animals↗
PubMed1984

Facilitated transfer of cholesteryl ester between rough and smooth microsomal membranes by plasma lipid transfer protein.

The accessibility of intracellular membrane cholesteryl esters to removal was tested with plasma lipid transfer protein as a tool. Incubation of a mixture of non-radioactive smooth microsomes + rough microsomes prelabeled with cholesteryl ester resulted in slight movement (2-4%) of radioactive cholesteryl ester into smooth microsomes. With the addition of increasing amounts of plasma lipid transfer protein to the mixture, the % transfer of cholesteryl ester into smooth microsomes progressively increased until a plateau was reached at 14%. Movement of cholesteryl ester in the reverse direction was examined with non-radioactive rough microsomes as an acceptor and smooth microsomes prelabeled with cholesteryl ester as a donor. The pattern of the % cholesteryl ester transferred in the reverse and forward direction was almost identical in the presence of plasma lipid transfer protein, showing bidirectional movement of cholesteryl ester between membranes.

Animals↗
PubMed1983

Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma.

In a previous study we demonstrated that highly purified lipid-transfer protein facilitated the transfer of triglyceride, cholesteryl ester, and phosphatidylcholine between plasma lipoproteins. It remained unclear, however, whether these lipids were transferred by independent sites on the lipid-transfer protein. To address this point, we have studied the protein-mediated transfer of triglyceride, cholesteryl ester, and phosphatidylcholine as a function of the concentration and lipid composition of donor and acceptor lipoproteins. Lipoproteins labeled in vitro, reconstituted lipoproteins of defined lipid composition, and phosphatidylcholine liposomes with or without triglyceride and/or cholesteryl ester have been used to investigate the inter-relationships of lipids transferred by the lipid-transfer protein. In studies of initial (less than or equal to 10-13%) transfer, we found that, although absolute transfer rates were affected, the ratio of cholesteryl ester to triglyceride transferred was independent of donor and acceptor lipoprotein concentrations and acceptor lipoprotein lipid composition. With reconstituted lipoproteins as donor, we demonstrated that this ratio was linearly related to the ratio of cholesteryl ester to triglyceride in the donor particle; the sum of triglyceride and cholesteryl ester transferred remained constant and independent of the lipid composition of the donor. Experiments with intact lipoproteins labeled in vitro and with small unilamellar vesicles in the presence and absence of p-chloromercuriphenylsulfonate, confirmed the interdependence of triglyceride and cholesteryl ester transfer. In contrast, under all assay conditions, no correlation was found between the amount of phosphatidylcholine transferred and the transfer of triglyceride and/or cholesteryl ester. We conclude that triglyceride and cholesteryl ester compete for transfer and that the extent of transfer for each lipid is determined by its relative concentration in the donor particle, whereas phosphatidylcholine transfer is independent of triglyceride and cholesteryl ester transfer. The data also strongly support the conclusion that lipid transfer protein promotes both the exchange and net transfer of triglyceride and cholesteryl ester and that the net transfer process proceeds by a reciprocal exchange of triglyceride and cholesteryl ester without net transfer of core lipid between lipoproteins.

Carrier Proteins↗
PubMed1982

The diagnosis of urinary tract infection: comparison of urine culture from suprapubic aspiration and midstream collection in a children's out-patient department in Nigeria.

Suprapubic aspiration (SPA) was compared with midstream urine collection (MSU) in the diagnosis of urinary tract infection (UTI) in a busy paediatric out-patient department in Northern Nigeria, where usually only one specimen can be taken before starting antimicrobial therapy. SPA in 287 children was compared with 175 MSU collections. Urine was obtained at the first attempt in 260 (91%) SPA's which were easy to perform and safe. MSU collections were more difficult, particularly in the younger child. In 51 cases MSUs were compared with SPAs taken immediately before, and gave an accurate indication of urine infection with no false negative or positive cultures. It is concluded that when only one urine specimen can be collected before starting antimicrobial therapy, a single MSU specimen gives an adequate result if correctly collected and plated out. When accuracy of diagnosis is essential or if a MSU cannot be obtained, SPA is indicated. A white cell count on unspun urine proved to be the most successful sideroom test for urinary tract. Counts in excess of ten cells per mm3 were found in 62% of patients with positive cultures.

Child↗