The separation of apolipoprotein D from cholesteryl ester transfer protein.

This study addresses the question of whether cholesteryl ester transfer protein and apolipoprotein D are identical. The data presented show that these two proteins do not co-purify during hydrophobic and cationic exchange chromatography and are readily separated by molecular sieve chromatography or electrophoresis. Furthermore, the precipitation of apolipoprotein D by specific antisera did not diminish the transfer activity of lipoprotein-deficient plasma. We conclude that apolipoprotein D and cholesteryl ester transfer protein have significantly different physicochemical properties.

The capacity of various non-esterified fatty acids to suppress lipid transfer inhibitor protein activity is related to their perturbation of the lipoprotein surface.

Lipid transfer inhibitor protein (LTIP) regulates cholesteryl ester transfer protein (CETP) activity by selectively impeding lipid transfer events involving low density lipoproteins (LDLs). We previously demonstrated that LTIP activity is suppressed in a dose-dependent manner by sodium oleate and that its activity can be blocked by physiological levels of free fatty acids [R.E. Morton, D. J. Greene, Arterioscler. Thromb. Vasc. Biol. 17 (1997)]. These data further suggested that palmitate has greater LTIP suppressive activity than oleate. In this report we define the ability of the major non-esterified fatty acids (NEFAs) in plasma to modulate LTIP activity. The greater suppression of LTIP activity by palmitate compared to oleate noted above was also seen in lipid transfer assays with various lipoprotein substrates and in the presence of albumin, showing that the relative effects of these two NEFAs are independent of assay conditions. To assess the effect of other NEFAs on LTIP activity, pure NEFAs were added to assays containing (3)H-cholesteryl ester labeled LDLs, unlabeled high density lipoproteins (HDLs) and CETP+/-LTIP. Whereas myristate, palmitate, stearate, oleate and linoleate stimulated CETP activity to varying extents, all NEFAs suppressed LTIP activity. Among these NEFAs, LTIP suppressive activity was greatest for the long-chain saturated and monounsaturated NEFAs. In contrast, linoleate and myristate were poor inhibitors of LTIP activity. The effects of increasing amounts of a given NEFA on LTIP activity correlated well with the increase in LDL negative charge induced by that NEFA, yet this relationship was unique for each NEFA, especially stearate. Notably, as measured by fluorescence anisotropy, the suppression of LTIP was highly and negatively correlated with the decreased order in the molecular packing of lipoprotein surface phospholipids caused by all NEFAs. Long-chain, saturated and monounsaturated NEFAs appear to be most effective in this regard partly because of their preferential association with LDLs where LTIP inhibition likely takes place. We hypothesize that NEFAs suppress LTIP activity by perturbing the surface properties of LDLs and counteracting the heightened molecular packing normally caused by LTIP. Diets rich in long-chain saturated and monounsaturated fatty acids may lead to a greater suppression of LTIP activity in vivo, which would allow LDLs to participate more actively in CETP-mediated lipid transfer reactions.

Exchange of retinyl and cholesteryl esters between lipoproteins of rabbit plasma.

Normal or hypercholesterolemic rabbit plasma stimulates the transfer of retinyl ester as well as cholesteryl ester from rabbit lymph chylomicrons, chylomicron remnants or from cholesteryl ester-rich plasma VLDL to the d greater than 1.019 lipoprotein fractions. The presence of p-chloromercuriphenylsulfonate does not inhibit the transfer of these esters. Partially purified lipid transfer protein from rabbit or from human plasma also accelerates the transfer of the above esters. Whereas the rabbit plasma transfer protein preferentially accelerates the transfer of retinyl ester, the human plasma transfer protein appears to have a somewhat greater stimulating effect on the transfer of cholesteryl ester from low- to high-density lipoproteins.

The acyl-CoA desaturases of microsomes from rat liver and the Morris 7777 hepatoma.

We have investigated the role of the microsomal oxidative desaturase in defining the aberrant phosphoglyceride fatty acid composition of hepatomas. The microsomal delta 9-stearoyl-CoA, delta 6-oleoyl(linolenoyl)-CoA, and delta 5-eicosatrienoyl-CA desaturase activities were studied in control and host liver and in the poorly differentiated Morris 7777 hepatoma. The delta 9-stearoyl-CoA desaturase of the hepatoma was significantly decreased (42%) relative to control liver, yet the hepatoma specific activity was twice that of host liver. Additionally, the specific activity of the delta 9-stearoyl-CoA desaturase of the tumor was found to decrease with increasing tumor weight. Also this desaturase was inactivated by freezing and thawing. The delta 6-oleoyl(linolenoyl)-CoA and delta 5-eicosatrienoyl-CoA desaturases of the hepatoma were 39% and 4% of control, respectively. The electron transport components involved in the desaturase system were reduced, although this did not appear to be rate-limiting. In addition, two competing metabolic reactions which could lower the observed desaturase activities, hydrolysis of the thioester and incorporation of substrate acyl-CoA molecules into glycerides, did not appear to be responsible for the lowered desaturase activities of the tumor. Thus, it appears that reduced levels of the desaturases themselves may be responsible for the observed activities. These results indicate that the capacity of the hepatoma to biosynthesize polyunsaturated fatty acids is greatly reduced and this is consistent with the decreased polyene content observed in many neoplasms.

Markedly elevated lipid transfer inhibitor protein in hypercholesterolemic subjects is mitigated by plasma triglyceride levels.

Lipid transfer inhibitor protein (LTIP, apolipoprotein F) regulates the interaction of cholesteryl ester transfer protein (CETP) with lipoproteins and is postulated to enhance the ability of CETP to stimulate reverse cholesterol transport. The factors that regulate LTIP levels and control its biosynthesis are unknown. Here, we demonstrate that plasma LTIP is dramatically increased (3-fold) in hypercholesterolemic subjects with normal to mildly elevated plasma triglyceride (TG) levels compared with control subjects. LTIP in these subjects is not correlated with the extent of hypercholesterolemia or with low density lipoprotein (LDL), high density lipoprotein, or CETP levels. However, unlike CETP, LTIP levels correlate negatively with plasma TG levels. This association does not appear to reflect decreased LTIP synthesis, inasmuch as conditions that stimulate TG synthesis and secretion (200 micromol/L oleate) do not reduce LTIP secretion by SW872 or Caco-2 cells. In contrast, native or acetyl LDL stimulates LTIP secretion 2-fold. Importantly, although plasma LTIP typically resides on LDL, up to 25% of LTIP is bound to very low density lipoprotein when this lipoprotein is enriched in cholesteryl esters, as occurs in hypercholesterolemia. In summary, LTIP levels are markedly elevated by hypercholesterolemia; however, plasma TG levels attenuate this response. We hypothesize that this arises from an increased association of LTIP with very low density lipoprotein, leading to a more rapid clearance of the inhibitor from circulation.

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.

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.

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.

Resistance of lipoproteins from continuous ambulatory peritoneal dialysis patients to in vitro oxidation.

Patients with end-stage renal failure on continuous ambulatory peritoneal dialysis (CAPD) develop abnormalities in plasma lipoproteins that may contribute to their increased risk for atherosclerosis. The oxidative modification of lipoproteins is considered to play a central role in atherogenesis. This study examines the susceptibility to oxidation in vitro of low- and high-density lipoprotein (LDL and HDL, respectively) obtained from long-term CAPD patients. CAPD LDL was less susceptible to copper-mediated protein derivatization (fluorescence) compared with control LDL CAPD LDL and HDL displayed less copper-promoted conjugated-diene production and lipid peroxide generation, suggesting a greater resistance of CAPD lipoprotein lipids to oxidation. Autooxidation during long-term storage was also much lower in CAPD LDL and HDL. However, when 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP) was used to initiate oxidation, there was no difference in conjugated-diene generation between CAPD and the control. CAPD LDL contained slightly less oxidizable, polyunsaturated fatty acid, but the vitamin E content of CAPD and control LDL was equivalent. Our findings indicate that lipoproteins from uremic patients undergoing long-term CAPD are more resistant to in vitro oxidation than control lipoproteins.

Masking by internally generated noise and protection by middle ear muscle activity.

Gross auditory nerve action potentials to high-frequency clicks were recorded bilaterally from awake cats with unilaterally tenotomized middle ear muscles (MEM) during eating and during control quiet periods. Masking by internally generated noise associated with eating increased with decreases in click intensity, but did not differ in ears with intact and tenotomized MEM. Under the conditions of this study, non-reflex MEM activity does not appear to provide protection against masking by internally generated noise.

Decreased toxicity of liposomal amphotericin B due to association of amphotericin B with high-density lipoproteins: role of lipid transfer protein.

Previously, we have shown that liposomal amphotericin B (L-AmpB) composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) was less nephrotoxic but equally as effective as Fungizone, which consists of amphotericin (AmpB) and deoxycholate. We have also observed that AmpB predominantly associates with high-density lipoproteins (HDL) in human serum and that the amount of AmpB associated with HDL increases when AmpB is incorporated into negatively charged liposomes. Furthermore, we observe that AmpB was less toxic in vitro to pig kidney cells when associated with HDL, but still toxic when associated with LDL. To further understand why HDL-associated AmpB causes reduced renal toxicity, we first examined LLC PK1 cells for the presence of LDL and HDL receptors and then the cytotoxic effects of HDL- and LDL-associated AmpB following trypsin treatment of LLC PK1 renal cells, which removed only the high-affinity LDL receptors. We found that LLC PK1 renal cells expressed high- and low-affinity LDL receptors but only low-affinity HDL receptors. Furthermore, when LLC PK1 cells were treated with trypsin, HDL- and LDL-associated AmpB were less toxic to the cells than was AmpB. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters (CE) whose transfer among lipoproteins is regulated by lipid transfer protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next investigated. We observed that LTP facilitated the transfer of AmpB, but not L-AmpB, from HDL to LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of lycra garments in the lower limb using 3-D gait analysis and functional assessment (PEDI).

Whole body lycra garments were assessed in eight children using gait analysis, the paediatric evaluation of disability index (PEDI), and a questionnaire of parental acceptance. Seven of the children had cerebral palsy and one Duchennes muscular dystrophy. After initial assessment and fitting of the garment, there was a 2-week introduction period followed by 6 weeks of wearing the garment for at least 6 h everyday, following which they were re-assessed. The root mean square error (RMSE) was used as a measure of variability over three separate passes through the gait laboratory and was a reference figure for gait stability. Proximal stability around the pelvis improved for five children and distal stability improved for three. Five children improved in at least one aspect of the PEDI scale. Although the parents and children detected these improvements, they did not outweigh the disadvantages of wearing the suit and as a consequence only one out of eight families considered continuing with the lycra garment.

Uptake and metabolism of free fatty acids by the Morris 7777 hepatoma and host rat liver.

The relative capacity of Morris 7777 hepatomas and livers of tumor-bearing rats to take up and subsequently metabolize intravenously injected radiolabeled free fatty acids was investigated. The objective was to determine differences in lipid metabolism which may affect the lipid composition previously observed in this tumor. Both tissues demonstrated comparable selectivity in the uptake of palmitate, linoleate and arachidonate from blood, although the hepatoma took up one-tenth as much free fatty acid per g wet wt as liver. A much greater percentage of fatty acid taken up by the hepatoma was converted to aqueous soluble radioactivity, perhaps the result of oxidation. In the hepatoma, palmitate was incorporated into phospholipid molecular species in a pattern similar to that observed for diglyceride, which suggested that phospholipid synthesis occurred predominantly de novo. On the other hand, in liver, a large percentage of palmitate was incorporated into polyunsaturated phospholipid molecular species that were not present in the diglyceride pool, which suggested significant incorporation by the acylation of monoacyl phosphoglycerides. These studies indicate that the specificity for the uptake of fatty acids was not different in the two tissues; however, the subsequent metabolic processes are markedly different.

Spondyloepimetaphyseal dysplasia and abnormal dentition in siblings: a new autosomal recessive syndrome.

The clinical and radiological features are described in male and female siblings with a unique form of spondyloepimetaphyseal dysplasia. In addition to generalized platyspondyly with epiphyseal and metaphyseal involvement, these children also have thin tapering fingers with accentuated palmar creases and abnormal dentition with oligodontia and pointed incisors. Parental consanguinity suggests that this is an autosomal recessive disorder.

Dislocation of the hips in children with bilateral spastic cerebral palsy, 1985-2000.

The aim of this study was to assess the rate of hip dislocation at different ages in children with bilateral spastic cerebral palsy attending special schools in southern Derbyshire, UK, between 1985 and 2000. The medical notes of 110 individuals (68 males, 42 females) were obtained. They were divided into four groups according to the Gross Motor Function Classification System (GMFCS). We determined whether or not their hips were dislocated at the ages of 5, 10, and 15 years, and the kind of surgery performed in each case. The percentage of individuals with one or both hips dislocated increased with age and with severity of disease. Of those in GMFCS Level II (n=18), none had dislocations; Level III (n=16), none had dislocations at ages 5 and 10, but 11% had by the age of 15; Level IV (n=35), 8% had dislocations by age 5, 19% by age 10, and 30% by age 15; Level V (n=41), 22% had dislocations by age 5, 48% by age 10, and 50% by age 15. Forty-two per cent of individuals with hip dislocation had not had previous preventive surgery. Twenty-one per cent of hips operated on still proceeded to dislocation. We conclude that there was a high rate of hip dislocation, especially in GMFCS groups Levels IV and V, and that this often occurred very early. Preventive surgery avoided dislocation in many children. However, orthopaedic referral was often not made before dislocation was discovered, or the referral was made too late for surgery on soft tissue to be successful. These results may be compared with those from current programmes of hip management, involving radiological surveillance and early use of conservative and surgical interventions.

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.

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.