Levels of cholesteryl esters and other lipids in the plasma of patients with end-stage renal failure.

BACKGROUND: The importance of plasma lipid abnormalities in chronic renal failure (CRF) is well recognized, but surprisingly little attention has been given to the study of some plasma lipid fractions, including cholesteryl esters (CE) and phospholipids, which might be expected to be important factors in the pathogenesis of the disease. MATERIALS AND METHODS: Fasting blood samples were taken from 25 control subjects and 53 CRF patients (29 predialysis and 24 on hemodialysis). Samples were analyzed for urea nitrogen, creatinine, triacylglycerols, total and individual phospholipids, total and free cholesterol, as well as cholesterol bound to very low-, low- and highdensity lipoproteins (VLDL, LDL and HDL). Plasma CE was calculated and expressed as a percentage of total cholesterol. RESULTS: Over half of the patients had CE levels more than two standard deviations below the control value. In this subgroup of low CE patients, total, LDL- and HDL-cholesterol levels were also significantly lower than for controls, while levels of phosphatidylcholine and lysophosphatidylcholine were decreased and increased, respectively. In patients with high CE, no significant lipid abnormalities were observed. CONCLUSION: In this study, CE was an excellent marker for lipid disturbances--if CE was high, then the other lipid fractions were normal, but if CE was low, most other lipid fractions were abnormal. The changes noted appear to be consequences of or related to deficiency of the plasma enzyme lecithin-cholesterol acyltransferase.

Lecithin: cholesterol acyltransfer, dyslipoproteinaemia and membrane lipids in uraemia.

BACKGROUND: Dyslipoproteinaemia is the most important complication linked to the increased morbidity and mortality of uraemic patients from cardiovascular disease. Many factors contribute to the dyslipoproteinaemia, including increased production of very low density lipoproteins (VLDL), decreased lipolysis and impaired low density lipoprotein (LDL) receptor activity. In this study, the role of decreased lecithin:cholesterol acyltransferase (LCAT) activity in relation to plasma and membrane lipid changes is examined. METHODS: Fasted blood samples were taken from 65 uraemic patients, including roughly equal numbers of haemodialysis, peritoneal dialysis and undialysed subjects, and from 29 apparently healthy individuals. Plasma total and free cholesterol, cholesteryl esters (CE), total and individual phospholipids, high density lipoprotein (HDL)-, LDL- and VLDL-cholesterol were all measured, as were erythrocyte and lymphocyte free cholesterol and phospholipids. RESULTS: More than half of all patients, including those both on haemodialysis and peritoneal dialysis, as well as untreated individuals, had relative plasma concentrations of CE below the normal mean - 2SD. These patients had significantly decreased LDL- (2.62 +/- 1.04 compared to 3.61 +/- 0.97 mmol/L; p < 0.001) and HDL-cholesterol (0.71 +/- 0.30 compared to 0.94 +/- 0.27 mmol/L; p < 0.01) and increased VLDL-cholesterol (0.60 +/- 0.50 compared to 0.47 +/- 0.26 mmol/L; p < 0.05) as well as significant increases in membrane cholesterol and cholesterol/phospholipid molar ratio in erythrocytes (3.30 +/- 0.49 and 0.87 +/- 0.08 compared to 2.95 +/- 0.18 mmol/g wet weight and 0.76 +/- 0.04 mol/mol respectively, both p < 0.001) and cholesterol/phospholipid molar ratio of lymphocytes (0.58 +/- 0.14 compared to 0.45 +/- 0.04 mol/mol; p < 0.001). They were markedly deficient in LCAT activity (56.1 +/- 20.4 compared to 105.5 +/- 17.5 nmol/ml/h; p < 0.001). The LCAT activity in plasma of patients with high CE was higher than for those with low CE, but it was also significantly less than normal and this group showed smaller changes in other lipid parameters. CONCLUSIONS: LCAT deficiency is common in uraemia and is associated with changes not just in plasma lipids, but also in membrane lipids which may be relevant to the progression of the disease.

The phospholipase activities present in preheparin mouse plasma are inhibited by antiserum to hepatic lipase.

1. Preheparin plasma from mice, but not rats or man, contains high levels of phospholipase A and lysophospholipase activities which are distinct from lecithin:cholesterol acyltransferase (LCAT). 2. Neither the phospholipase A nor the lysophospholipase activities in preheparin plasma are inhibited by incubation in the presence of protamine sulphate or high salt concentrations. 3. When mouse plasma is incubated in the presence of an antiserum specific for rat hepatic triacylglycerol lipase (HTGL), the phospholipase activities are abolished. 4. These observations suggest that the phospholipase activities are attributable to the action of HTGL, which, in the mouse appears to be a freely circulating enzyme, whereas for other species this enzyme only appears in the blood following administration of heparin.

Decreased plasma lecithin:cholesterol acyltransfer and associated changes in plasma and red cell lipids in uraemia.

Plasma lipids, lecithin:cholesterol acyltransferase (LCAT) activity and erythrocyte lipid composition were compared for a group of newly diagnosed uraemic patients and a group of healthy subjects. Plasma triacylglycerol was increased and both total and high-density lipoprotein (HDL) cholesterol were decreased. A lower percentage of total cholesterol in patients' plasma was in the esterified form and plasma values of the phospholipid, lysolecithin, were also lower. The plasma LCAT activity of uraemic patients, whether expressed as nmol or percentage of cholesterol esterified per hour, was significantly lower than for normals. Both LCAT activity and lysolecithin in uraemic plasma were inversely correlated with the concentration of urea. The lipid composition of erythrocytes from patients was also abnormal, with both free cholesterol and lecithin being increased. These results are consistent with the occurrence of an acquired deficiency of LCAT in uraemia, comparable to that previously described in hepatic disease. The LCAT enzyme is secreted by the liver, and the inverse correlation noted in this study between LCAT activity and urea suggests that the increased urea in renal disease may inhibit the synthesis and secretion of the enzyme by the liver. The resulting reduction in LCAT activity may lead to the accumulation of cholesterol and lecithin in cell membranes and contribute to the overall pathophysiology of renal disease.

Transgenic mice expressing human apolipoprotein A-I have sera with modest trypanolytic activity in vitro but remain susceptible to infection by Trypanosoma brucei brucei.

Although Trypanosoma brucei brucei fatally infects livestock in much of sub-Saharan Africa, humans are innately resistant to infection, apparently because high-density lipoproteins (HDL) in human serum lyse this unicellular protozoan parasite. Recently, we demonstrated that purified human apolipoprotein (apo) A-I, the major protein (M(r) 28,016) constituent of HDL, had full trypanolytic activity in vitro whereas the apoA-I of cattle and sheep was non-lytic. In the present study, we have sought to confirm the trypanocidal capability of human apoA-I by studying four lines of transgenic mice expressing (supra)physiological serum levels of this polypeptide. Although trypanolysis in vitro by sera from transgenic mice (15.1 +/- 1.3% [mean +/- SEM], n = 30) was considerably less than by human sera (typically 60-80%), it was nevertheless significantly greater than by control sera (8.5 +/- 1.1%, n = 10; P < 0.001) and correlated with the concentration of human apoA-I (r = 0.56, P < 0.001). When trypanosomes were incubated at 37 degrees C with human serum or with human apoA-I for 30 min (i.e., within the pre-lytic period) they lost their ability to subsequently infect mice; trypanosomes incubated with transgenic mice serum remained infective. Furthermore, transgenic mice were fully susceptible to infection when inoculated with 10(3) trypanosomes; both the initial detection of trypanosomes in the blood (3-4 days) and the time to death (5-6 days) were no longer than control mice. This apparent paradox between the action of human apoA-I in human serum and in mouse serum was investigated further.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the cytolytic effects in vitro on Trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein A-I from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection.

The African trypanosome, Trypanosoma brucei brucei causes a fatal wasting disease in livestock but does not ordinarily infect humans, apparently because this unicellular parasite is lysed by high density lipoproteins (HDL) in human serum. To assess whether there is a specific active constituent in trypanolytic HDL, we have systematically compared the cytotoxic action on T.b.brucei in vitro of native and delipidated HDL, and of individual apolipoproteins, from nonpermissive hosts (human and baboon) with their counterparts from susceptible hosts (cattle and sheep). When suspensions of trypanosomes were incubated for 2 h at 37 degrees C with human or baboon plasma most cells were lysed, but not with bovine or sheep plasma. Similarly, HDL isolated from human and baboon plasma were trypanolytic (typically about 95% and 60% lysis, respectively, at 1 mg protein/ml), whereas bovine and sheep HDL were benign (less than 8% lysis). Subfractionation of human HDL by serial isopycnic ultracentrifugation and by heparin-Sepharose affinity chromatography established that the denser and smaller particles had greater trypanolytic activity both in vitro and in vivo. When human HDL was delipidated, the trypanocidal activity was associated with the water-soluble protein (apolipoprotein) fraction and not with the lipid constituents. Bovine apolipoproteins were also weakly trypanolytic in free solution (20-40% lysis), but not when complexed with cholesterol-phospholipid liposomes (less than 10% lysis). The major apolipoprotein of human HDL, apolipoprotein (apo) A-I had full trypanolytic activity (89-95% lysis at 1 mg protein/ml) when purified, whether in solution or incorporated into liposomes, but other apolipoproteins isolated from human HDL, including apoA-II, apoC, and apoE, were nontrypanolytic. Purified baboon apoA-I was also trypanolytic, though less potent than human apoA-I, but apoA-I from permissive hosts (cattle and sheep) was inactive when presented in liposomes. Incubation of bovine or sheep HDL with purified human apoA-I, and subsequent separation of the HDL by ultracentrifugation, produced chimeric HDL containing significant amounts of the human apolipoprotein; these particles showed appreciable trypanolytic activity. By contrast, human HDL particles in which about 70% of the apoA-I had been displaced with apoA-II had markedly reduced lytic properties compared to the native HDL (30% versus 80% lysis at 0.6 mg total protein/ml). We tentatively conclude that the trypanolytic activity of native human or baboon plasma resides in the apoA-I content of the HDL particles and that, conversely, bovine and sheep plasma are inactive because the apoA-I polypeptide present in their HDL lacks trypanocidal activity.

Characteristics of the binding of human and bovine high-density lipoproteins by bloodstream forms of the African trypanosome, Trypanosoma brucei brucei.

Bloodstream forms of Trypanosoma brucei brucei are unable to synthesize cholesterol but appear to bind and take up plasma low-density lipoproteins (LDL) from their host. Whether cholesterol homeostasis of this unicellular parasite also requires interactions with host high-density lipoprotein (HDL) particles is unknown. Equilibrium binding of radioiodinated apolipoprotein E-depleted human HDL3 (d = 1.125-1.21 g/ml) and bovine HDL (d = 1.063-1.21 g/ml) by T.b.brucei was rapid (less than 30 min) at 4 degrees C and was characterized by a saturable, specific component. There were five times the number of high-affinity binding sites for human HDL3 as for bovine HDL (64,000 vs. 11,500 per trypanosome) and their binding affinity was greater with an equilibrium dissociation constant (Kd) of 157 nM compared to 315 nM for bovine HDL). Binding of rat and rabbit HDL3 was similar to bovine HDL. By contrast, equilibrium binding of human LDL was slower (approximately 6 h) and the number of high-affinity binding sites (Kd = 23 nM) was much lower for this ligand (660 per trypanosome). Total binding of HDL3 was independent of divalent cations and was only slightly inhibited by heparin, but when the trypanosomes were preincubated with trypsin or pronase the binding was markedly reduced. After 30 min at 37 degrees C, binding of bovine HDL and human HDL3 was 10-20% higher than at 4 degrees C; after 45 min trypanolysis occurred with human HDL3 but not with bovine HDL. Chemical modification of HDL3 by treatment with cyclohexanedione, by acetylation or by reductive alkylation had little effect on its ability to compete with [125I]labelled HDL3 for binding by the parasite. Nitrosylation of HDL3 with tetranitromethane increased its binding ability, suggesting that trypanosomes might possess scavenger receptors, and native HDL3 was less effective than nitrosylated HDL3 in displacing bound [125I]labelled nitrosylated HDL3. These findings suggest that, in addition to a receptor for LDL, T.b.brucei has other lipoprotein binding sites which separately recognize HDL from permissive host species such as bovine, trypanolytic HDL such as human HDL3, and more negatively charged HDL particles such as nitrosylated HDL3.

Influence of hypoxanthine and other purines on the lecithin:cholesterol acyltransferase reaction in the plasma of rat and other species.

1. Esterification of radiolabelled cholesterol in the plasma of rat, mouse, pig, ox and, to a lesser extent, guinea pig was partially inhibited by hypoxanthine, xanthine and guanine; esterification in human plasma and in plasma from 12 other vertebrate species was unaffected by purines. 2. Esterification of endogenous cholesterol and the formation of lysolecithin in rat plasma were decreased in the presence of purines indicating that it was the lecithin:cholesterol acyltransferase (LCAT) reaction that was inhibited rather than the isotopic equilibration of labelled cholesterol with the endogenous substrate lipoproteins. 3. Maximum inhibition of the LCAT reaction in rat plasma occurred at 1.4 mM hypoxanthine or xanthine; inhibition was not dependent upon the concentration of LCAT or plasma lipoproteins but increased with the amount of lipoprotein depleted rat plasma (LDRP) present in the incubation mixture. 4. Partial inhibition of the LCAT reaction in rat or mouse plasma by purines had no significant effect on the fatty acyl composition of the cholesteryl esters (CE) formed by LCAT. 5. In the presence of heated rat plasma, LDRP or, to a lesser extent, rat high density lipoproteins (HDL) prepared from heated plasma, the LCAT reaction in human plasma was inhibited by hypoxanthine. 6. Rat HDL and LDRP prepared from plasma pre-incubated at 37 degrees C for 4 hr before heating increased and decreased, respectively, the inhibitory effect of hypoxanthine on human plasma LCAT compared with HDL and LDRP prepared from unincubated rat plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the levels and lipid composition of plasma lipoproteins (VLDL, LDL and HDL) in Brazilian patients with hepatosplenic schistosomiasis mansoni.

1. The plasma concentrations of low- and high-density lipoproteins (LDL and HDL) were significantly reduced in Brazilian patients with compensated hepatosplenic schistosomiasis mansoni (SM) when compared with healthy individuals, but very low-density lipoprotein (VLDL) levels were unchanged. 2. All three classes of lipoproteins isolated from SM plasma had an increased content of triacylglycerol and unesterified cholesterol and decreased cholesteryl ester and phospholipid. 3. The individual phospholipid composition of patient VLDL, LDL, HDL was also altered; the amount of phosphatidylcholine was increased and that of lysophosphatidylcholine decreased. 4. The saturated and monounsaturated fatty acyl content of cholesteryl esters in patient lipoproteins was also significantly increased, and diunsaturated and polyunsaturated fatty acyl content was decreased. 5. When isolated lipoproteins were examined as negatively stained preparations by electron microscopy, the morphology of SM patient LDL was normal but the HDL fraction was abnormal and showed marked heterogeneity of size with the presence of occasional discoidal particles which resembled "nascent" HDL.

Alterations in the levels and lipid composition of plasma lipoproteins (VLDL, LDL and HDL) in Brazilian patients with hepatosplenic schistosomiasis mansoni

1. The plasma concentrations of low- and high-density lipoproteins (LDL and HDL) were significantly reduced in Brazilian patients with compensated hepatosplenic schistosomiasis mansoni (SM) when compared with healthy individuals, but very low-density lipoprotein (VLDL) levels were unchanged. 2. All three classes of lipoproteins isolated from SM plasma had an increased content of triacylglycerol and unesterified cholesterol and decreased cholesteryl ester and phospholipid. 3. The individual phospholipid composition of patient VLDL, LDL, HDL was also altered; the amount of phosphatidylcholine was increased and that of lysophosphatidylcholine decreased. 4. The saturated and monounsaturated fatty acyl content of cholesteryl esters in patient lipoproteins was also significantly increased, and diunsaturated and polyunsaturated fatty acyl content was decreased. 5. When isolated lipoproteins were examined as negatively stained preparations by electron microscopy, the morphology of SM patient LDL was normal but the HDL fraction was abnormal and showed marked heterogeneity of size with the presence of occasional discoidal particles which resembled nascent HDL

Trypanosoma brucei brucei: differences in the trypanocidal activity of human plasma and its relationship to the level of high density lipoproteins.

Although high density lipoprotein (HDL) particles purified from human serum by ultracentrifugation are known to lyse Trypanosoma brucei brucei, it is unclear whether individual differences in the trypanocidal activity of human serum reflect changes in the concentration of HDL per se. In the present study, trypanolytic activity, whether assessed in vitro or in vivo, was greater with plasma from normal healthy individuals than with plasma from patients with various hepatic diseases and associated low levels of HDL. For all subjects taken as a single group there were highly significant positive correlations between the plasma concentration of apolipoprotein (apo) A-I, the major protein constituent of HDL and trypanolysis in vivo (r = 0.93, n = 10, P less than 0.001) or in vitro (r = 0.77, n = 36, P less than 0.001). Removal of plasma apoB-containing (i.e. non-HDL) lipoproteins by precipitation revealed that the trypanocidal activity was also significantly correlated with HDL-cholesterol and HDL-apoA-II, as well as with HDL-apoA-I, but not with HDL-apoE. Depletion of all or part of plasma apoA-I by non-ultracentrifugal methods abolished or decreased the trypanolytic effect of the plasma. The findings from these experiments, which were designed to avoid alteration in the composition of HDL by ultracentrifugal forces, provide additional support for the proposal that the trypanocidal action of human plasma resides with native HDL particles.

Characterisation of the binding of low-density lipoproteins to cultured rat mesangial cells.

Mesangial cell lipid accumulation is a recognised feature of glomerular disease and has been implicated as a factor in the pathogenesis of renal injury. To investigate possible mechanisms of such accumulation, binding of 125I-labelled human low-density lipoprotein (LDL) to rat mesangial cells was studied in vitro. Experiments were performed at 4 degrees C to prevent ligand internalisation. LDL remained associated with the cells after repeated washing. Binding was time-dependent, was inhibited by addition of an excess of unlabelled LDL, but to a much lesser extent by apoprotein-A-rich high-density lipoprotein particles devoid of apoprotein E (HDL-A). Specific binding reached saturation at an LDL concentration of 21 micrograms/ml, required the presence of calcium, and was inhibited by heparin and dextran sulphate. Scatchard analysis suggested a single class of binding site (Kd 22.7 micrograms protein/ml). Higher binding affinities were obtained when rat LDL was substituted for human LDL (Kd 1.3 micrograms/ml) and when human fibroblasts were exposed to human LDL under identical experimental conditions (Kd 3.0 micrograms/ml). Further experiments at 37 degrees C demonstrated degradation of LDL by cells. These results suggest that mesangial cells possess apoprotein B, E receptors. Mesangial cell lipid accumulation may therefore result from receptor-mediated endocytosis of LDL particles.

Alterations in the fatty acyl composition of plasma cholesteryl esters in Brazilian patients with hepatosplenic schistosomiasis mansoni.

1. The dyslipoproteinemia commonly occurring in the hepatosplenic forms of schistosomiasis mansoni in Brazilian patients is characterized by low plasma levels of cholesteryl esters and of the cholesterol-esterifying enzyme, lecithin:cholesterol acyltransferase (LCATase, EC.2.3.1.43). 2. In the present study, normal healthy individuals and patients suffering from hepatosplenic schistosomiasis mansoni were compared for the fatty acyl compositions of circulating plasma cholesteryl esters and of those formed in vitro by the action of LCATase on a) the endogenous plasma lipoproteins and b) an excess of lipoprotein substrate composed of heat-inactivated plasma. 3. In patient plasma the proportions of saturated and monounsaturated cholesteryl esters were higher and those of diunsaturated and polyunsaturated esters were lower than in the control group. 4. Similar differences were observed between patients and controls in the proportions of the cholesteryl ester subclasses formed in vitro by the action of LCATase on endogenous plasma lipoproteins. 5. Incubation of fresh normal or patient plasma with excess heat-inactivated plasma as substrate for LCATase produced proportions of cholesteryl ester subclasses similar to those formed during incubation of nonheated aliquots of the appropriate substrate plasma. 6. We conclude that the alterations in fatty acyl composition of plasma cholesteryl esters in patients with hepatosplenic schistosomiasis mansoni do not appear to be a direct consequence of the low levels of LCATase activity in patient plasma.

Alterations in the fatty acyl composition of plasma cholesteryl esters in brazilian patients with hepatosplenic schistosomiasis mansoni

1. The dyslipoproteinemia commonly occurring in the hepatosplenic forms of schistosomiais mansoni in Brazilian patients is characterized by low plasma levels of choleteryl esters and of the cholesterol-esterifying enzyme, lecithin:cholesterol acyltransferase (LCATase, EC.2.3.1.43). 2. In the present study, normal helathy individual and patients sufferin from hepatosplenic schistosomiasis mansoni were comapred for the fatty acyl compositons of circulating plasma cholesteryl esters and of those formed in vitro by the action of LCATase on a) the endogenous plasma lipoprotins and b) an excess of lipoprotein substrate composed of heat-inactivated plasma. 3. In patient palsma the proportions of saturated and monounsaturated cholesteryl esters were higher and those of diunsaturated and polyunsaturated esters were lower than in the control group. 4. Similar differences were observed between patients and controls in the proportions of the cholesteryl ester subclasses formed in vitro by the action of LCATase on endogenous plasma lipoprotins. 5. Incubation of fresh normal or patient plasma with escess heat-inactivated plasma as substrate for LCATase produced proportions of cholesteryl ester subclasses similar to those formed dduring incubation of nonheated aliquots of the appropriate plasma. 6. We conclude that the alterations in fatty acyl composition of palsma cholesteryl estes in patients with hepatosplenic schistosomiasis mansoni do not appear to be direct consequence of the low levels of LCATase acivity in patient plasma

Changes in the lipid composition of erythrocytes during prolonged fasting in lizard (Tropidurus torquatos) and rat (Rattus norvegicus).

During prolonged fasting in lizard and rat, plasma levels of unesterified cholesterol (UC) and phospholipids (TPL) decreased and there were reductions and increases, respectively, in the molar ratios of lecithin (PC) to sphingomyelin (SPH) and UC to TPL. Plasma lecithin: cholesterol acyltransferase (LCATase) activity in lizard and rat plasma was reduced during prolonged fasting. Erythrocyte lipid composition for fasted animals was also characterized by a reduction in the molar ratio PC/SPH and an increase in UC/TPL, and in both species there were positive correlations between these molar ratios in red cells and those in plasma. In both species these were changes in the morphology of the erythrocytes, and those from fasted rats showed alterations in osmotic fragility and permeability which correlated with alterations in lipid composition. These results suggest that changes in plasma lipoprotein lipid composition, linked to reduced LCATase activity, may cause similar alterations in the lipid composition of red cell membranes leading to altered membrane properties.

Unusual lipid composition of erythrocytes from the insectivorous bat Molossus molossus.

The lipid composition of washed erythrocytes from Molossus molossus and Molossus ater were studied. In comparison with other mammalian species, bat erythrocytes were characterized by very high cholesterol/phospholipid molar ratios, as well as by low sphingomyelin content. The lipid composition of bat erythrocytes was similar to that of patients suffering from lecithin: cholesterol acyltransferase deficiency, but the physiological significance of this in bats is unknown.

Inhibitory effect of high-density lipoproteins from plasma of schistosomiasis mansoni patients on the binding of normal low-density lipoproteins to cultured skin fibroblasts.

Total high-density lipoproteins (HDL) isolated from the plasma of patients suffering from hepatosplenic schistosomiasis mansoni have an abnormal apoprotein composition. The apoprotein E content is increased and one or two abnormal apoproteins with pI 6.0 and 6.5 are present in patient HDL. Whilst normal HDL have a negligible effect on the binding of 125I-labelled normal low density lipoproteins (LDL) by cultured human skin fibroblasts, patient HDL is inhibitory. It is concluded that the abnormal apoprotein composition of schistosomiasis HDL allows it to bind to the LDL receptor on the fibroblast membrane.