Effect of cholesteryl 14-methylhexadecanoate on the activity of some amino acid-transfer ribonucleic acid ligases from mammalian tissues

1. l-Tyrosine-, l-alanine-, l-tryptophan- and l-threonine-tRNA ligases (where tRNA is transfer RNA) were purified from mammalian tissues and the relative contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during the isolation. Purified enzymes were extracted with various organic solvents. 2. Cholesteryl 14-methylhexadecanoate contents in purified ligases were up to 210-fold that in the starting material. Different enzymes showed different contents of this cholesteryl ester. 3. Extracted enzymes lost in most cases their ability to catalyse formation of the aminoacylhydroxamate and aminoacyl-tRNA complexes. Enzymes extracted with various solvents showed a variable decreased activity. 4. The original activity could be restored to 70-100% by the addition of cholesteryl 14-methylhexadecanoate. Cholesteryl palmitate, cholesteryl margarate and cholesteryl stearate were inactive in this respect. 5. Incubation mixtures of extracted enzymes with cholesteryl 14-methylhexadecanoate added showed an initial delay in the time-course of both reactions assayed. 6. It is concluded that the effect of cholesteryl 14-methylhexadecanoate on the activity of amino acid-tRNA ligases seems to be specific and that this compound may play some role in the function of these enzymes.     

Cholesteryl esters are bound by a peptide-initiation and a peptide-elongation factor.

Significantly higher quantities of cholesteryl 14-methylhexadecanoate than of cholesteryl laurate and cholesteryl palmitate are bound by a homogeneous peptide-initiation factor and purified peptide-elongation factor 1. Cholesteryl 14-methylhexadecanoate may function as a specific allosteric modifier changing the conformation of protein synthesis factors and thus modulating the activity of their binding sites.     

The role of cholesteryl 14-methylhexadecanoate in the function of eukaryotic peptide elongation factor 1

The binding of [3H]cholesteryl 14-methylhexadecanoate by a highly purified peptide elongation factor 1 from rabbit reticulocytes is significantly enhanced by GTP and CTP, much less by guanosine 5'-[beta, gamma-methylene]-triphosphate and not at all by ATP or UTP. Removal of endogenous cholesteryl 14-methylhexadecanoate present in the molecule of the factor [Hradec, J. et al. (1971) Biochem. J. 123, 959-966] by digestion with immobilized cholesterol esterase resulted in an almost complete loss of GTPase activity and this could be restored to nearly normal values by the addition of the ester. The same holds true for the GTP-dependent autophosphorylation of the protein-synthesis factor. Cholesteryl 14-methylhexadecanoate was bound only by the beta subunit of the factor. Addition of the alpha subunit, which was inactive on its own, stimulated the binding of the ester to the beta subunit in a sigmoid dependence. The binding of the ester was significantly stimulated by aminoacyl-tRNA but this effect was fully abolished by sodium fluoride, indicating a relation of cholesteryl 14-methylhexadecanoate to the dephosphorylation of the peptide elongation factor. Treatment of the factor with cholesterol esterase decreased its activity in the poly(U)-dependent binding of phenylalanyl-tRNA to ribosome and this activity was again restored by the addition of cholesteryl 14-methylhexadecanoate. The ester thus interacts with the GTP-dependent autophosphorylation of peptide elongation factor 1 and in this way modulates the activity of the factor. A putative scheme is presented explaining the mode of action of cholesteryl 14-methylhexadecanoate.     

Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The interaction of cholesteryl 14-methylhexadecanoate

1. Transferase I from rat liver extracted with iso-octane binds significantly less aminoacyl-tRNA than the non-extracted enzyme. The original activity can be fully restored by the addition of cholesteryl 14-methylhexadecanoate. The binding capacity for GTP is not affected by the extraction. 2. In the presence of extracted transferase I the binding of aminoacyl-tRNA to ribosomes is decreased to 11-26% and the simultaneous binding of GTP to 32-43%. Cholesteryl 14-methylhexadecanoate induces a full reactivation of the extracted enzyme in both respects. 3. Extracted complexes A (aminoacyl-tRNA-GTP-transferase I) become bound to ribosomes to the same extent as the corresponding non-extracted preparations. 4. It is concluded that cholesteryl 14-methylhexadecanoate interacts with the binding site of transferase I for aminoacyl-tRNA and secondarily with that for GTP. It does not affect the binding site for ribosomes.     

The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

1. Peptide-elongation factors were purified from rat liver and human tonsils and the contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during enzyme purification. The relative contents of this compound in purified enzyme preparations was several times higher than that in the crude starting material. Elongation factors from human tonsils contained a significantly larger quantity of the cholesteryl ester than enzyme from rat liver. 2. Transfer enzymes extracted with various organic solvents showed variable decreased activities in both binding and peptidization assay. The decrease of enzymic activity was proportional to the amount of cholesteryl 14-methylhexadecanoate extracted from a given enzymic preparation. In systems containing both extracted elongation factors the polyphenylalanine synthesis was limited by the residual activity of the less active transfer factor. 3. The original enzymic activity of extracted transferases was fully recovered by the addition of pure cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. Increase of the relative contents of this cholesteryl ester during enzyme purification, decrease of the enzymic activity after the extraction and its recovery by the addition of this compound indicates that the presence of this ester in elongation factors is essential for the normal function of these enzymes.     

Influence of cholesteryl 14-methylhexadecanoate on some ribosomal functions required for peptide elongation

1. Polyribosomes and ribosomal subunits from rat liver were adsorbed on a cellulosic ion-exchange adsorbent, freeze-dried and extracted with organic solvents. The activity of extracted particles in peptide elongation was tested in the presence of purified peptideelongation factors. 2. Chloroform-methanol mixture (2:1, v/v) extracted 1.87+/-0.15 pmol of cholesteryl 14-methylhexadecanoate/pmol of the smaller ribosomal subunit and 0.92+/-0.11 pmol/pmol of the larger subunit. 3. In the presence of transferase I, extracted polyribosomes and 40S subunits bound more phenylalanyl-tRNA than did control non-extracted particles. The same binding as in control mixtures was obtained with extracted particles supplemented with cholesteryl 14-methylhexadecanoate in quantities corresponding to those extracted. 4. The polymerization of phenylalanine was greatly decreased with extracted polyribosomes and subunits and addition of the cholesteryl ester could not fully restore the original activity. 5. Extraction significantly decreased the activity of the P site of peptidyl transferase and normal activity was recovered after the addition of the ester. The A site of peptidyl transferase in extracted polyribosomes showed an increased activity when compared with non-extracted polyribosomes. 6. Cholesteryl 14-methylhexadecanoate apparently affects the function of the ribosomal A site and peptidyl transferase site and probably also that of the guanosine triphosphatase site and P site. The presence of different amounts of the ester in polyribosomes may be one of the mechanisms modulating peptide elongation at the ribosomal level.     

Protein synthesis in the liver of rats injected with cholesteryl 14-methylhexadecanoate

1. Rats were injected intraperitoneally with cholesteryl 14-methylhexadecanoate and killed after various intervals of time up to 3 days; ribosomes and cell sap were isolated from their liver tissue. These fractions were tested for their ability to participate in protein synthesis. 2. Protein synthesis in complete systems containing ribosomes, cell sap and all necessary cofactors was significantly enhanced at 12 and 72h after the injection and significantly inhibited at 24h. At early times after injection isolated ribosomes had a slightly enhanced ability to bind nRNA. Peptide-elongation processes (i.e. binding of aminoacyl-tRNA to ribosomes, peptidyl transfer and polyphenylalanine synthesis) showed significant stimulation or inhibition depending on the time after injection of the ester. 3. A correlation was found between the ability of cell sap to stimulate polyphenylalanine synthesis and the relative cholesteryl 14-methylhexadecanoate content in the postmicrosomal supernatant at different time-intervals after administration of the ester. No significant changes were found in its content in the whole liver tissue. 4. Since the injected ester has previously been shown to accumulate in some enzymic fractions, the changes in its relative content may represent a regulatory mechanism modulating the rate of protein synthesis.     

Decreased activity of peptide-elongation factors after treatment with cholesterol esterase.

1. Peptide-elongation factors were purified from rat liver and treated with cholesterol esterase and phospholipase A2 immobilized on Sepharose 4B. 2. Binding of L-[3H]-phenylalanyl-tRNA to 40S ribosomal subunits was decreased by approx. 70% and to polyribosomes by 30% in the presence of the binding factor incubated with cholesterol esterase. Treatment of this factor with immobilized phospholipase A2 decreased the binding to smaller ribosomal subunits by only about 15%. 3. Poly(U)-dependent phenylalanine polymerization by ribosomal subunits was decreased to approx. 30% of its original value by treatment of both elongation factors with cholesterol esterase. 4. The normal activity of esterase-treated elongation factor in both the binding reaction and peptide-elongation assay was fully recovered by the addition of cholesteryl 14-methyl-hexadecanoate. 5. Different classes of lipids present in peptide-elongation factor 1 have apparently different functions. Whereas phospholipids are required to maintain the strcture of heavy aggregates of this factor, the presence of cholesteryl 14-methylhexadecanoate is obviously necessary for the normal function of peptide-elongation factors.     

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.     

The activity of cholesterol ester hydrolase in the enzymatic determination of cholesterol: comparison of five enzymes obtained commercially

The use of five cholesterol ester hydrolases (CEH), numbered 1 to 5, for the enzymatic determination of total cholesterol of human and rat serum are compared. All CEH gave approximately the same value (no statistical difference) for human serum. However, when rat serum cholesterol was determined, CEH-2 yielded a value significantly lower when compared to the four other CEH. The ability of each CEH to hydrolyze individual cholesterol esters was tested. During a 15-min incubation, all CEH were capable of hydrolyzing nearly 100% of cholesteryl oleate and linoleate. In contrast, the hydrolysis of cholesteryl arachidonate was only partial and varied from 20 to 80% depending on the CEH used. The highest hydrolysis was obtained by CEH-1 while the value given by CEH-2 was only 22% of that obtained by CEH-1. The rate of hydrolysis of cholesteryl arachidonate differed markedly among the CEH. The CEH-2-hydrolyzed the cholesteryl arachidonate at a rate seven times lower than the rate obtained with CEH-1. The data suggest that, Under our incubation conditions, CEH-2 did not properly hydrolyze the cholesteryl arachidonate. This phenomenon may be crucial whenever total cholesterol has to be determined enzymatically in the serum of species that contain large amount of cholesteryl arachidonate such as rat, mouse, or dog serum.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.     

Effects of dietary supplementation with creatine monohydrate during the finishing period on growth performance,carcass traits,meat quality and muscle glycolytic potential of broilers subjected to transport stress

A total of 320 male Arbor Acres broiler chickens (28 days old) were randomly allotted to one of the three experimental diets supplemented with 0 (160 birds), 600 (80 birds) or 1200 mg/kg (80 birds) creatine monohydrate (CMH) for 14 days. On the morning of 42 day, after an 8-h fast, the birds of CMH-free group were divided into two equal groups, and all birds of these four groups were transported according to the follow protocol: 0.75-h transport without CMH supplementation (as a lower stress control group), 3-h transport without CMH supplementation, 3-h transport with 600 or 1200 mg/kg CMH supplementation. Each treatment group was composed of 8 replicates with 10 birds each. The results showed that supplementation of CMH for 14 days before slaughter did not affect the overall growth performance and carcass traits of stressed broilers (P>0.05). A 3-h transport decreased plasma glucose concentration, elevated plasma corticosterone concentration, increased bird live weight loss, breakdown of muscle glycogen, as well as the accumulation of muscle lactate (P<0.05), which induced some detrimental changes to breast meat quality (lower ultimate pH and higher drip loss, P<0.05). Nevertheless, supplementation of 1200 mg/kg CMH reduced chicken weight loss, decreased the contents of lactate and glycolytic potential in pectoralis major of 3-h transported broilers (P<0.05), which is beneficial to maintain breast meat quality by reducing the drip loss (P<0.05). These findings suggest that the reduction of muscle glycolysis is probably the reason for maintainance of meat quality by supplementation of CMH in transported broilers.     

Hepatic processing and biliary secretion of the cholesteryl esters from beta very-low-density lipoproteins in the rat

beta-Migrating very-low-density lipoproteins (beta-VLDL) are cholesteryl-ester-enriched lipoproteins which accumulate in the serum of cholesterol-fed animals or patients with type III hyperlipoproteinemia. In the rat, beta-VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, beta-VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after beta-VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded. It is concluded that beta-VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from beta-VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from beta-VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low-density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.     

An alternative procedure for incorporating radiolabelled cholesteryl ester into human plasma lipoproteins in vitro.

A simple method has been developed for labelling human plasma lipoproteins to high specific radioactivity with radioactive cholesteryl esters in vitro. After isolation by preparative ultracentrifugation, the selected lipoprotein was incubated for 30 min at 4 degrees C in human serum (d greater than 1.215) that had been prelabelled with [4-14C]cholesteryl oleate or [1,2-3H]cholesteryl linoleate, and was then re-isolated by ultracentrifugation. All major lipoprotein classes were labelled by the procedure. Specific radioactivities of up to 18 d.p.m. . pmol-1 (46 d.p.m. . ng-1) were achieved. When radiolabelled high-density lipoprotein was infused intravenously, the radioactive cholesteryl ester behaved in vivo indistinguishably from endogenous cholesteryl esters produced by the lecithin (phosphatidylcholine): cholesterol acyltransferase reaction.     

The preferential uptake of very-low-density lipoprotein cholesteryl ester by rat liver in vivo.

The removal from the blood and the uptake by the liver of injected very-low-density lipoprotein (VLDL) preparations that had been radiolabelled in their apoprotein and cholesteryl ester moieties was studied in lactating rats. Radiolabelled cholesteryl ester was removed from the blood and taken up by the liver more rapidly than sucrose-radiolabelled apoprotein. Near-maximum cholesteryl ester uptake by the liver occurred within 5 min of the injection of the VLDL. At this time, apoprotein B uptake by the liver was only about 25% of the maximum. Maximum uptake of the injected VLDL apoprotein B label was not achieved until at least 15 min after injection, by which time the total uptakes of cholesteryl ester and apoprotein B label were very similar. The results suggest that preferential uptake of the lipoprotein cholesteryl ester by the liver occurred before endocytosis of the entire lipoprotein complex. The fate of the injected VLDL cholesteryl ester after its uptake by the liver was also monitored. Radiolabel associated with the hepatic cholesteryl ester fraction fell steadily from its early maximum level, the rate of fall being faster and more extensive when the fatty acid, rather than the cholesterol, moiety of the ester was labelled. By 30 min after the injection of VLDL containing [3H]cholesteryl ester, over one-third of the injected label was already present as [3H]cholesterol in the liver. When VLDL containing cholesteryl [14C]oleate was injected, a substantial proportion (about 25%) of the injected radiolabelled fatty acid appeared in the hepatic triacylglycerol fraction within 60 min: very little was present in the plasma triacylglycerol fraction at this time.     

Determination of the absolute configurations of the anteiso acid moieties of glycoglycerolipid S365A isolated from Corynebacterium aquaticum

The absolute configurations of the two acid moieties, 12-methyltetradecanoate and 14-methylhexadecanoate, of glycoglycerolipid S365A isolated from Corynebacterium aquaticum were determined by an HPLC analysis after their conversion with the chiral fluorescent labeling reagents, (1S,2S)- and (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanol. Both anteiso acids had the S configuration.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

Hydrogen bonding in DNA base complexes.

Experimental intermolecular frequencies in the DNA base complexes 1-methylthymine (1-MT) and cytosine monohydrate (CMH) are analyzed in terms of simple analytic interatomic potentials. Calculations with two different values for the constants of the nonbonded interactions are considered, and the hydrogen bond potentials are determined for each of these models. The observed frequencies in 1-MT are reasonably well described, although corresponding potentials are very different in the two models. The observed frequencies in CMH are less well described, although corresponding hydrogen bond potentials are similar in the two models. Hydration interactions are found to be important in CMH and the role of the water molecule is discussed. Possible reasons for the shortcomings of this simple analysis are considered.     

Accumulation of HDL-like lipoproteins in the plasma low-density fractions of tumor-bearing mice

Outgrowth of the transplanted GRSL lymphoma in GR mice yielded several-fold increased blood plasma levels of low- and very-low-density lipoproteins, while high-density lipoproteins were strongly reduced. Changes in cholesteryl ester fatty acid profiles indicated an accumulation of HDL-like particles rather than LDL in the low-density fractions. By intravenous injection of [14C]cholesteryl ester-labeled HDL into tumor-bearing mice, conversion of HDL into lipoproteins of low density was demonstrated.     

Processing of different liposome markers after in vitro uptake of immunoglobulin-coated liposomes by rat liver macrophages

We compared the metabolic fate of [³H]cholesteryl[¹⁴C]oleate, [³H]cholesteryl hexadecylether, ¹²⁵I-labeled bovine serum albumin and [³H]inulin as constituents of large immunoglobulin-coupled unilamellar lipid vesicles following their internalization by rat liver macrophages (Kupffer cells) in monolayer culture. Under serum-free conditions, the cholesteryl oleate that is taken up is hydrolyzed, for the greater part, within 2 h. This occurs in the lysosomal compartment as judged by the inhibitory effect of the lysosomotropic agents monensin and chloroquin. After hydrolysis, the cholesterol moiety is accommodated in the cellular pool of free cholesterol and the oleate is reutilized for the synthesis mainly of phospholipids and, to a lesser extent of triacylglycerols. During incubation in plasma, however, substantial proportions of both the cholesterol and the oleate are shed from the cells, predominantly in the unesterified form. When the liposomes are labeled with the cholesteryl ester analog [³H]cholesteryl hexadecylether only a very small fraction of the label is released from the cells, even in the presence of plasma. Similar to the label remaining associated with the cells, the released label is identified in that case as unchanged cholesteryl ether. The liposomal aqueous phase marker ¹²⁵I-labeled bovine serum albumin is also readily degraded intralysosomally and the radioactive label is rapidly released from the cells in a trichloroacetic acid-soluble form. Also, as much as 20% of the aqueous phase marker [³H]inulin that becomes cell-associated during a 2-h incubation with inulin-containing liposomes, is released from the cells during a subsequent 4-h incubation period in medium or rat plasma. The usefulness of the various liposomal labels as parameters of liposome uptake and intracellular processing is discussed.