Isolation of very low density lipoprotein phospholipids enriched in ethanolamine phospholipids from rats injected with Triton WR 1339

Phospholipids carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases and lecithin-cholesterol acyltransferase. We have previously demonstrated [J.J. Agren, A. Ravandi, A. Kuksis, G. Steiner, Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis, Eur. J. Biochem. 269 (2002) 6223-6232] that the infusion of Triton WR 1339 (TWR), which inhibits these lipases, leads in 2 h to five-fold increase in VLDL triacylglycerol concentration along with major differences in the composition of their molecular species. The present study demonstrates that the accumulation of triacylglycerols is accompanied by major changes in the content of the VLDL phospholipids, of which the most significant is the enrichment of phosphatidylethanolamine (PtdEtn). This finding coincides with the enrichment in PtdEtn demonstrated in the VLDL of a hepatocytic Golgi fraction but it had not been demonstrated that the Golgi VLDL, along with its unusual phospholipid composition, can be directly transferred to plasma. Aside from providing an easy access to nascent plasma VLDL, the TWR infusion demonstrates that lipoprotein and hepatic lipases are also responsible for the degradation of plasma VLDL PtdEtn, as independently demonstrated for plasma phosphatidylcholine. Our results indicate also, with the exception of lysophosphatidylcholine, that preferential basal hydrolysis no dot lead to major differences in molecular species composition between circulating and newly secreted VLDL phospholipids. The comparison of the molecular species composition of VLDL and liver phospholipids suggests a selective secretion of PtdEtn and sphingomyelin molecular species during VLDL secretion.     

Formation of triton WR 1339-filled rat liver lysosomes : I. Properties and intracellular distribution of [H]Triton WR 1339

Triton WR 1339 was found to contain a high molecular weight fraction with globular polymers of ˜ 10⁵ D and a diameter of ˜80 Å (‘macrotriton’) and a low molecular weight fraction (‘microtriton’). The intracellular distribution of subfractions of [³H]Triton WR 1339 was followed by cell fractionation and by gel chromatography techniques in parallel with electron microscopy and autoradiography. Macrotriton is selectively stored in lysosomes and all evidence supports a slowly working endocytotic uptake mechanism. Microtriton permeates quickly into the cells and is rapidly and efficiently released into the bile. The data presented suggest some intracellular leaking of lysosomal contents due to the action of Triton WR 1339.     

Effects of gamma-terpinene on lipid concentrations in serum using Triton WR1339-treated rats

We studied lipid metabolism to evaluate the effects of gamma-terpinene on suppression of increases in serum lipid concentrations using Triton WR1339-treated rats. At 6 hr after Triton WR1339 injection, the total cholesterol and triglyceride concentrations in the gamma-terpinene group underwent statistically significant decreases (18.3 and 30.3%, respectively) compared with those of the Triton-treated group.     

Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339.

The proteolytic degradation of 125I-labeled low density lipoprotein by monolayers of cultured human fibroblasts was prevented by exposure of the cells to chloroquine, an agent that has been reported previously to inhibit lysosomal degradative processes. Chloroquine did not inhibit the binding of low density lipoprotein to its cell surface receptor. However, the two regulatory actions that normally follow low density lipoprotein binding to its receptor, namely suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation, were both prevented when degradation of the lipoprotein was inhibited by chloroquine. Two other agents affecting lysosomal function, Triton WR 1339 and concanavalin A, also inhibited the proteolytic degradation of low density lipoprotein in intact fibroblasts and simultaneously prevented low density lipoprotein-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation. Unlike chloroquine, however, these two agents also affect the binding of low density lipoprotein to the cells. The inhibitory action of chloropuine, concanavalin A, and Triton WR 1339 could each be reversed by removal of the agent from the culture medium. These in vivo culture data, together with the observation that cell-free extracts of fibroblasts maximally degrade 125I-labeled low density lipoprotein at pH 4 and do not form acid-soluble material above pH 6, are consistent with the hypothesis that the proteolytic degradation of low density lipoprotein by monolayers of fibroblasts occurs within lysosomes. The data also suggest that normal lysosomal function is required in order for low density lipoprotein to regulate cholesterol synthesis and cholesteryl ester formation in the fibroblast system.     

Complexing of Fatty Acids by Triton WR1339 in Relation to Growth of Mycobacterium tuberculosis

Mycobacterium tuberculosis grew in the presence of toxic concentrations of free fatty acids when an adequate amount of triton WR1339 was added to the medium. Triton produced a solubilizing effect on turbid suspensions of fatty acids, indicating the formation of a detergent-lipid complex. Oleic acid complexed with triton served as a source of carbon for growth of M. tuberculosis.     

Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats.

Changes in whole plasma and lipoprotien apoprotein concentrations were determined after a single injection of Triton WR 1339 into rats. Concentrations of apoproteins A-I (an activator of lecithin:cholesterol acyl transferase), arginine-rich apoprotein (ARP), and B apoprotein were measured by electroimmunoassay. The content of C-II apoprotein (an activaor of lipoprotein lipase) was estimated by the ability of plasma and lipoprotein fractions to promote hydrolysis of triglyceride in the presence of cow's milk lipase and also by isoelectric focusing on polyacrylamide gels. Apoproteins C-II and A-I were rapidly removed from high density lipoprotein (HDL) after Triton treatment and were recovered in the d 1.21 g/ml infranate fraction. A-I was then totally cleared from the plasma within 10--20 hr after injection. Arginine-rich apoprotein was removed from HDL and also partially cleared from the plasma. The rise in very low density lipoprotein (vldl) apoprotein that followed the removal of apoproteins from HDL was mostly antributed to the B apoprotein, although corresponding smaller increases were observed in VLDL ARP and C apoproteins. The triglyceride:cholesterol, triglyceride:protein, and B:C apoprotein ratios of VLDL more closely resembled nascent rather than plasma VLDL 10 hr after Triton injection. These studies suggest that the detergent may achieve its hyperlipidemic effct by disrupting HDL and thus removing the A-I and C-II proteins from a normal activating environment compirsing VLDL, HDL, and the enzymes. The possible involvement of intact HDL in VLDL catabolism is discussed in relation to other recent reports which also suggest that abnormalities of the VLDL-LDL system may be due to the absence of normal HDL.     

Subcellular localization of transferrin protein and iron in the perfused rat liver. Effect of Triton WR 1339, digitonin and temperature

The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13-1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex. However, after treatment with low concentrations of digitonin the 3H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterol-rich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 degrees C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles. On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.     

Hypolipidemic effect of intravenous pluronic L-81 in fasted rats treated with Triton WR-1339: possible inhibition of hepatic lipoprotein secretion

Pluronic L-81 (L-81), a non-ionic hydrophobic surfactant, is a powerful inhibitor of the secretion of lipid-transporting chylomicrons from intestinal epithelial cells to lymph. Since the other major organ that secretes lipoproteins into the circulation is the liver, whose principal lipid secretory product is very low density lipoprotein (VLDL), we tested the hypothesis that L-81 will also inhibit hepatic lipid secretion. Rats were fasted so that they had little lipid input from the intestine. We then administered Triton WR-1339 (tyloxapol) intravenously to block peripheral utilization of VLDL, causing plasma lipids to rise rapidly. Some animals were also given L-81 intravenously to test whether the L-81 would retard the tyloxapol-induced rise in plasma lipids. Administration of tyloxapol alone (250 mg/kg) increased plasma triglyceride, phospholipid and cholesterol concentrations considerably. Simultaneous administration of a small dose of L-81 (6 mg/kg) markedly reduced the rise in plasma triglyceride, particularly in the first hour (by 45%). L-81 also diminished the rise in plasma phospholipid and cholesterol, but to a lesser extent (30%). In the fasting rat, most of the plasma triglyceride is in VLDL; therefore, L-81 probably acts by decreasing the secretion of hepatic VLDL. Thus, Pluronic L-81 may be a useful tool for examining the secretion and metabolism of hepatic lipoproteins, in particular, VLDL.     

Comparison of plasma clearance of low density lipoprotein with beta-very low density lipoprotein or acetoacetylated low density lipoprotein in cholesterol-fed rabbits

The plasma clearance and tissue distribution of radioiodinated low-density lipoprotein (LDL), beta-very low density lipoprotein (beta-VLDL), and acetoacetylated LDL were studied in cholesterol-fed rabbits. Radioiodinated LDL ([125I]LDL) was cleared more slowly than either [125I]beta-VLDL or acetoacetylated-[125I]LDL and its fractional catabolic rate was one-half that of [125I]beta-VLDL and one-ninth that of acetoacetylated-[125I]LDL. Forty-eight hours after the injection of the labeled lipoproteins, the hepatic uptake was the greatest among the organs evaluated with the uptake of [125I]LDL being one-third that of either [125I]beta-VLDL or acetoacetylated-[125I]LDL. The reduction in the hepatic uptake of LDL due to a down-regulation of the receptors would account for this retarded plasma clearance.     

Effect of Triton WR-1339 on lecithin-cholesterol acyltransferase in the rat

The effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase was measured in rat serum following addition of Triton to the serum in vitro or after intravenous injection of the detergent. The inhibitory effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase when the detergent was added in vitro was dose dependent and appeared to result from a direct action on the enzyme rather than from a physical modification of the substrate by the detergent. The serum half-life ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of Triton WR-1339 injected intravenously in the rat was 23.1 ± 1.0 h. The inhibitory effect of Triton on serum LCAT activity when the detergent was given intravenously was also dose dependent and was reversed when the serum concentration of Triton decreased; under specific conditions, LCAT activity reached values higher than control. This behavior after treatment of the animal may be explained by increased concentration of the enzyme in the plasma, by stimulation of LCAT activity by the very low density lipoprotein or metabolites accumulating in the plasma of rats treated with Triton WR-1339, or by a combination of these factors.     

Plasma lipoprotein changes attending the intravenous administration of Triton WR-1339 in normolipidemic dogs: preferential effect on high density lipoproteins

The nonionic detergent Triton WR-1339 was injected intravenously into normolipidemic dogs in a single dose of 150 mg/kg body weight followed by three other injections (75 mg/kg) on days 2, 6, and 12. The Triton produced a significant elevation of the plasma cholesterol of these animals, but not of their triglyceride levels, and profound changes of their plasma lipoproteins, particularly of the high density lipoprotein class. These changes were dependent on the concentration of Triton attained in plasma; when the levels were above 1.5 mg/ml, density gradient ultracentrifugation, electrophoretic, and chemical analyses indicated that an interaction between Triton and HDL had occurred. This interaction was attended by a gradual loss of the surface components of HDL, namely apoA-I, phospholipids, and unesterified cholesterol, and by the appearance of two cholesteryl ester-rich lipoproteins of d 1.019-1.024 g/ml and d 1.038-1.058 g/ml containing apoA-I and proteins with electrophoretic mobilities of apoB, apoE, and apoA-IV. At the time that these changes had occurred, the activities of the enzymes lecithin: cholesterol acyltransferase and post-heparin lipase were unaffected. When 125I-labeled apoA-I was injected intravenously into animals receiving Triton, the residence time of the radiolabeled protein in plasma increased from a control value of 3.1 days to 7.2 days. However, the apparent half-times of the radiolabeled apoA-I varied among the lipoprotein fractions it was associated with: d 1.119-1.159 g/ml, 5.28 days; d 1.019-1.024 g/ml, 7.55 days, and d 1.038-1.058 g/ml, 5.39 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial damage and inhibition of respiration in animal cell cultures treated with Triton WR-1339

Inhibition of cellular respiration by treatment with the nonionic detergent Triton WR-1339 was found to be related to the cytotoxic response of cell to the surfactant. Respiration of sensitive cell lines (AV-3 and HeLa) markedly inhibited by Triton concentrations as low as 125 μgm/ml. Conditionally sensitive lines (BHK-21 and L-929) were affected by 500 μgm/ml while the respiration of insensitive cultures (primary rat and chick embryo cells) was unaffected by this concentration. Macrocyclon, a cyclic analogue of Triton, failed to alter the respiration rate of any of the above cell cultures. The levels of isocitric and succinic dehydrogenases in sensitive and conditionally sensitive cells were depressed within 2 hours after treatment with 500 μgm/ml of Triton was initiated and by 6 hours the activity was only 25% of the untreated controls. Similar results were obtained with mitochondrial preparations from these cells. Enzyme levels in insensitive cells were unaffected by Triton treatment. Mitochondrial damage was the most striking characteristic noted in treated cells examined by electron microscopy. The mitochondria were quite distorted and had lost most of their cristae formation. This mitochondrial damage was seen in all cell types examined although the rate at which it occurred varied. With sensitive cultures, damage was pronounced within 6 hours after the addition of Triton while mitochondria from conditionally sensitive cells were not grossly affected until 48 hours and they appeared to repair the damage following the removal of Triton.     

Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339

Triglyceride (TG), a water-insoluble energy-rich lipid, is secreted by the liver as part of very low density lipoproteins (VLDLs) to supply energy to extrahepatic tissues. Overproduction of VLDL is associated with increased risk of cardiovascular heart disease; this has renewed an interest in factors that affect hepatic TG production. The TG production rate is determined by measuring temporal increases in plasma TG under conditions in which TG hydrolysis by lipoprotein lipase (LPL) is inhibited. The nonionic detergent, Triton WR-1339 (Triton), has commonly been used to inhibit LPL for this purpose. Triton, in addition to inhibition of TG hydrolysis, has properties that have the potential to adversely influence lipoprotein metabolism. Another nonionic detergent, poloxamer 407 (P-407), also inhibits LPL. In these studies, we demonstrate that P-407 is comparable to Triton in the determination of TG production but without the unwanted side effects of Triton.     

Evidence that use of Triton WR1339 underestimates the triacylglycerol entry rate into the plasma of lactating rats owing to continued accumulation of lipid in the mammary gland.

An analysis is presented of the catalytic factors responsible for the rate-enhancement that may be observed when a protein modification reaction is compared with a reaction of the same modifying agent with a model micromolecular compound exhibiting the same reactive group as the protein under study. It is seen that affinity-mediated rate-enhancement of protein modification is realized by the loss of activation entropy. On the assumption that attainment of maximal affinity-mediated rate-enhancement presents with an activation entropy of the protein modification reaction equal to zero, whereas the activation enthalpy of the reaction remains unchanged, it is shown that the value for maximal affinity-mediated rate-enhancement is equal to e-delta s++/R. Accordingly, protein modification reactions may be differentiated into (i) reactions the rate-enhancement of which (relative to the reaction of the same modifying agent with a model compound) is primarily entropy-controlled and (ii) reactions the rate-enhancement of which is primarily enthalpy-controlled. It is seen that modifying agents of low reactivity towards model compounds, but with a high, i.e. highly negative, activation entropy are better suited as prospective affinity-based protein-modifying agents, since the potential affinity-mediated rate-enhancement, and hence the selectivity, of these compounds is necessarily high. Kinetic and thermodynamic constants of the reaction of modifying agents with proteins, and with model compounds, and values of maximal affinity-mediated rate-enhancement, based on published data of the reaction of several modifying agents with model compounds, are presented and discussed.