Jacqueline Férézou

Lipid composition and structure of commercial parenteral emulsions

In order to study the influence of the phospholipid/triacylglycerol (PL/TG) ratio of parenteral emulsions on the distribution and the physico-chemical properties of their fat particles, commercial 10, 20 or 30% fat formulas were fractionated by centrifugation into an upper lipid cake (resuspended in aqueous glycerol) and a subnatant or mesophase, from which a PL-rich subfraction (d = 1.010-1.030 g/l) was purified by density gradient ultracentrifugation. Chemical and 31P-NMR analyses of these fractions indicated that at least two types of fat particles coexist in parenteral emulsions: (i) TG-rich particles (mean diameter: 330, 400, 470 nm in the 10, 20, 30% emulsion) which contain practically all the TG and esterified phytosterols of native emulsions, but only a fraction of their PL, unesterified cholesterol and phytosterols, and other minor lipids; (ii) PL-bilayer particles or liposomes (mean diameter: 80-100 nm) which are constituted with the remaining PL and relatively very small amounts of TG and other lipids. The higher the oil content of the emulsion, the lower the amount of these PL-rich particles, which represent the major particle population of the mesophase. Indeed, minute amounts of TG-rich particles (probably the smallest ones) are also present in the mesophase, even in the PL-rich subfraction which contains the bulk of liposomal PL. Since the PL-rich particles of the infused emulsion generate lipoprotein X-like particles, only the large TG-rich particles can be considered as true chylomicron counterparts.

EFFECTS OF INTRAVENOUS INFUSIONS OF COMMERCIAL FAT EMULSIONS (INTRALIPID 10 OR 20 %) ON RAT PLASMA LIPOPROTEINS : PHOSPHOLIPIDS IN EXCESS ARE THE MAIN PRECURSORS OF LIPOPROTEIN-X-LIKE PARTICLES

Like most commercial parenteral emulsions, Intralipid contains the same amount of phospholipids (12 mg/ml) to stabilize 100 or 200 mg of soybean oil (10 or 20% formula, respectively). By centrifugation, 10 or 20% Intralipid was separated into a supernatant, fat particles containing the bulk of triacylglycerols stabilized by a fraction of phospholipids and an infranatant--called mesophase--consisting mainly of phospholipids used in excess as emulsifier. We observed that the initial triacylglycerol/phospholipid ratio of the emulsion (100/12 and 200/12, respectively) determines the size of the triacylglycerol-rich particles (260 and 350 nm) as well as the phospholipid content of the mesophase (6.02 and 4.67 mg/ml). To understand the mechanism of the lipoprotein-X (LPX) accumulation generally reported after intravenous fat infusions, plasma lipid levels and lipoprotein profiles were first compared in the rats after infusion (at a constant rate of 0.5 or 1 ml/h for 43 h) of Intralipid 10 or 20%. For the same intravenous triacylglycerol load (100 mg/h), rats infused with Intralipid 10% at 1 ml/h displayed higher triacylglycerol levels than rats infused with the 20% emulsion at 0.5 ml/h, suggesting that the size of exogenous fat particles modulated the catabolic rate of their triacylglycerols. The plasma levels of LPX varied according to the infusion rate of phospholipids not associated with triacylglycerol-rich particles of the emulsion. Moreover, an apo E and apo B enrichment of plasma and an elevation of the apo B48/apo B100 ratio was always observed after Intralipid infusions. In order to confirm that phospholipids of the mesophase are the main LPX precursors, lipoprotein profiles were then compared in the rats after intravenous infusion, at a constant rate of 1 ml/h, of either the mesophase or a suspension of triacylglycerol-rich particles isolated from Intralipid 20%. As expected, significant LPX amounts were only detected in rats infused with the pure mesophase of the emulsion. It was concluded that products of the lipolysis of exogenous fat particles play only a minor role in the formation of LPX. In fact these abnormal lipoproteins are generated by phospholipids of the mesophase which, like infused liposomes, actively mobilize endogenous free cholesterol. Consequently, in order to be considered as true chylomicron models for safe fat delivery in parenteral nutrition and in order to prevent some detrimental effects on cholesterol metabolism, commercial emulsions should be cleared of phospholipid excess.

Structure and metabolic fate of triacylglycerol- and phospholipid-rich particles of commercial parenteral fat emulsions

The lipid emulsions used in parenteral nutrition are constituted of particles rich in triacylglycerols (TAG) called artificial chylomicrons (200-500 nm in diameter; monolayer of phospholipids [PL] enveloping a TAG core) and PL-rich particles called liposomes (diameter inferior to 80 nm; bilayer of PL around an aqueous phase), which represent the excess emulsifier. Introduced into the circulation, the two populations of particles come into contact with circulating lipoproteins and cell membranes and experience the same overall fate: exchanges and transfers of lipids and apolipoproteins, enzymatic hydrolysis of TAG and PL, and internalization by different tissues. The relative importance of these different metabolic processes varies depending on the type of particle. The artificial chylomicrons undergo a hydrolysis of their TAG by lipoprotein lipase, with a release of fatty acids and formation of smaller particles of remnants, which are rapidly removed by the liver. In delivering fatty acids to the tissue, artificial chylomicrons fulfill an energy transport function similar to the natural chylomicrons. The liposomes hold little energy interest, and they also have deleterious effects when infused in excess. They inhibit the lipolysis of artificial chylomicrons and, by actively capturing endogenous cholesterol, they stimulate tissue cholesterogenesis and accumulate in the blood as lipoprotein-X, a long-lived abnormal lipoprotein. To limit as much as possible the metabolic perturbations due to the intravenous administration of exogenous PL, the emulsion has to be infused at a low rate, and should contain the minimal amount of excess PL.

Modulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities by steroids and physiological conditions in hamster

Our purpose was to examine the in vitro modulation of liver mitochondrial sterol 27-hydroxylase (S27OHase) and microsomal cholesterol 7alpha-hydroxylase (CH7alphaOHase) activities by certain drugs, sterols, oxysterols and bile acids, and to compare the influence of sex, age, diet and cholestyramine on these activities, in the hamster. In vitro, 7beta-hydroxycholesterol and 5alpha-cholestan-3beta-ol (cholestanol) were strong inhibitors (at 2 microM) of both enzyme activities, while 5beta-cholestan-3alpha-ol (epicoprostanol, 2 microM) and cyclosporin A (20 microM) inhibited S27OHase, but not CH7alphaOHase. These data suggest that a hydroxyl group at the 7alpha position is not required to inhibit CH7alphaOHase and that the presence of an aliphatic CH2-CH-(CH3)2 chain appears to be structurally important for S27OHase activity. Both enzyme activities remained unchanged by hyodeoxycholic acid (40 or 80 microM) while epicoprostanol inhibited only S27OHase and chenodeoxycholic acid only CH7alphaOHase. Adult (9-week old) male or female hamsters displayed similar S27OHase activity but the CH7alphaOHase activity was lower in females than in males, suggesting that the neutral bile acid pathway has a less important role in females. In male hamsters, S27OHase activity did not change with age, while CH7alphaOHase activity significantly increased (one-year vs 9-week old). A semi-purified sucrose-rich (lithogenic) diet significantly lowered both enzyme activities compared to the commercial diet. Cholestyramine induced a stimulation of both enzymes, slightly more vigorously however for the key enzyme involved in the neutral pathway. Taken together, these data indicate that the two enzymes are separately regulated and that certain drugs or steroid compounds can be useful for specifically inhibiting or stimulating the neutral or acidic bile acid pathway.

Cholesterol and bile acid biodynamics after total small bowel resection and bile diversion in humans

BACKGROUND In humans, the patterns of cholesterol and bile acid biodynamics in the absence of the small intestine are not yet known. They are described in two parenterally fed patients several months after total enterectomy and bile diversion. METHODS After an intravenous pulse of [3H]cholesterol, a long-term study involved the analysis of both the decay in the specific activity of plasma cholesterol and the biliary outputs of sterols and bile acids. RESULTS Plasma cholesterol input reached 2-3 g/day (vs. 1 g/day in healthy patients), mostly from synthesis. As assessed by sterol balance, whole body cholesterol synthesis approximated 6 g/day (vs. 0.6-0.8 g/day). Unusually, about 60% of the newly synthesized cholesterol was eliminated, without prior transit into the bloodstream, from the liver into the bile. Bile acid conversion concerned over 90% (vs. 40%-50%) of the cholesterol meant to be excreted, issued from plasma or hepatic synthesis. In addition to cholic and chenodeoxycholic acids, one patient secreted up to 1 g/day of 7-epicholic acid. CONCLUSIONS The stimulation (up to 10-fold) of the cholesterol and bile acid synthesis, stronger than that observed following ileal bypass or resection or complete bile diversion, could well be partially linked to the absence of small bowel tissue per se.

PHOSPHOLIPID-RICH PARTICLES IN COMMERCIAL PARENTERAL FAT EMULSIONS. AN OVERVIEW

In parenteral nutrition, the infusion of a fat EMU supplies both concentrated energy and covers the essential fatty acid requirements, the basic objective being to mimic as well as possible the input of chylomicrons into the blood. This objective is well met by the TAGRP of the EMU, which behave as true chylomicrons. However, commercial EMU also contain an excess of emulsifier in the form of PLRP. The number of these PLRP depends directly on the PL/TAG ratio of the EMU. They differ from the TAGRP by their composition (PL vs TAG and PL), their structure (PL in bilayer versus monolayer), and their granulometry (mean diameter 70-100 nm for PL vs 200-500 nm). The metabolic fate of the PLRP is similar in several ways to that of the TAGRP: exchanges of PL with the PL of the different cellular membranes and of the lipoproteins; captation of free CH from these same structures; and enrichment in apolipoproteins. However, because the TAGRP are the preferred substrates of the lipolytic enzymes, their clearance is much more rapid (half-life < 1 h) than that of the PLRP. As the infusion is continued, the PLRP end up accumulating and being transformed into LP-X (free CH/PL = 1; half-life of several days). As soon as the EMU is infused, the PLRP enter into competition with the TAGRP, in the lipolysis process as well as for sites of binding and for catabolism. The sites for catabolism of the two types of PAR are not the same: adipose tissues and muscles utilize the fatty acids and monoacylglycerols released by the lipolysis of the TAGRP; hepatocytes take up their remnants; the RES and the hepatocytes participate in the catabolism of the PLRP and the LP-X. Thus, prolonged infusion of EMU rich in PLRP leads to a hypercholesterolemia, or at least a dyslipoproteinemia, due to elevated LP-X, associated with a depletion of cells in CH, stimulating thus tissue cholesterogenesis. However, parenteral nutrition has evolved towards the utilization of EMU with a low PL/TAG ratio (availability of 30% formula) and less rapid delivery. For these reasons, the hypercholesterolemias that used to be observed with the 10% EMU have become much less spectacular or have even disappeared. It is interesting to note that patients on prolonged TPN, in particular those with a short small intestine, have weak cholesterolemia, reflecting a lowering of HDL and LDL not masked by elevated LP-X. At present, it seems difficult to produce sufficiently stable parenteral EMU devoid of PLRP. Notwithstanding, all the observations made since the introduction of the EMU in TPN are in favour of the use of PLRP-poor EMU. It is clear that the 10% formulas, and generally those with a PL/TAG ratio of 12/100, are ill-advised, especially in patients with a retarded clearance of circulating lipids.

Effects of hyodeoxycholic acid and α-hyocholic acid, two 6α-hydroxylated bile acids, on cholesterol and bile acid metabolism in the hamster

The effects of hyodeoxycholic (HDCA) and alpha-hyocholic acids (alpha-HCA), on cholesterol, bile acid and lipoprotein metabolism, were studied in hamsters. The animals were fed a low cholesterol control diet supplemented with 0.1% HDCA or alpha-HCA for 3 weeks. In both treated groups, the LDL-cholesterol concentration was significantly lowered and was associated with a global hypocholesterolemic effect. Moreover, hepatic cholesterol ester storage was reduced and HMGCoA reductase activity was respectively enhanced 13.5-times and 7.7-times in HDCA and alpha-HCA groups compared to controls. In contrast, cholesterol 7 alpha-hydroxylase activity and LDL-receptor activity and mass were not modified. In bile, the cholesterol saturation index was increased 5-fold (HDCA group) and 2-fold (alpha-HCA group) as a consequence of an enlarged proportion of biliary cholesterol. The two 6-hydroxylated bile acids induced an enhanced fecal excretion of neutral sterols (HDCA group: 11.6-times, alpha-HCA group: 3.2-times versus controls) which was consistent with a 59% decrease in intestinal cholesterol absorption in the HDCA group. The major effects due to bile acid treatments were a decrease in LDL-cholesterol concentration, a strong stimulation of hepatic cholesterol biosynthesis and an excessive loss of cholesterol in feces. These perturbations might be the result of the enrichment of bile with hydrophilic bile acids, leading to a limited return of endogenous cholesterol from the intestine to the liver.

The mesophase of parenteral fat emulsion is both substrate and inhibitor of lipoprotein lipase and hepatic lipase

Six 10% and 20% parenteral fat emulsions were separated by centrifugation into two fractions: (1) a supernatant containing the bulk of triacylglycerols (Tg) as fat particles stabilized by phospholipids (PL); and (2) an infranatant, called mesophase, consisting essentially of PL (one third of the original PL in the 10% formula, one sixth in the 20% formula, in the case of emulsions containing 12 g PL.L-1) and small amounts of Tg and free sterols, probably in the form of liposomes. The lipolytic enzymes, lipoprotein lipase (LPL) and hepatic lipase (HL), involved in the Tg-rich lipoprotein clearance, hydrolyze both types of particles, although Tg-fat particles are their preferred substrate. Inactivated serum (providing apo C-II) is needed to ensure the maximum LPL hydrolysis rate of both types of particles. It partially inhibits the HL activity on the mesophase. Substrate of the lipolytic enzymes, the mesophase, is also an inhibitor of their activity, the inhibition being directly proportional to the amount of PL contained in the mesophase. This inhibition is of uncompetitive type. For LPL, it seems that the mesophase acts on a site distinct from that of the apo C-II binding site. These results partly explain the low PL clearance after a fat emulsion infusion. But in particular, they help to explain the lower clearance of a 10% emulsion (larger PL excess) compared with a 20% emulsion (with the same amount of Tg, but less PL excess).

Intralipid 10%: Physicochemical Characterization

OBJECTIVES Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase. The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g), and a liposome-enriched fraction (ring of density 1.010-1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be < or = 3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Lipolytic Activities in Rats Fed a Sucrose-Rich Diet Supplemented with either Cystine or Cholesterol: Relationships with Lipoprotein Profiles

To study the relationships between lipolytic activities and plasma lipoprotein levels in rats, three diets were given for 8 weeks: a semipurified diet (based on sucrose, casein and lard) and this diet enriched with 5% cystine or with 1% cholesterol. Both supplemented diets induced hypercholesterolemia. Lipoprotein analysis by density gradient ultracentrifugation of plasma indicated that hypercholesterolemia of cystine-fed rats (+52%) was characterized by an increased cholesterol level in high-density lipoprotein (HDL; +131%) and low-density lipoprotein 2 (LDL2; +147%), the lipoprotein fraction containing essentially apolipoprotein-E-rich high-density lipoproteins (HDL1), and was associated with a decreased cholesterol level in triglyceride-rich lipoproteins (TRL: -69%). That obtained by cholesterol feeding (+28%) was due to a large increase in the TRL cholesterol level (+315%) whereas cholesterol was reduced in HDL (-40%) and in LDL2 (-60%). Under these dietary conditions, the activity of hepatic lipase (HL) was measured in liver homogenates and those of both HL and lipoprotein lipase were measured in plasma after heparin injection. The activity of HL (1,783 +/- 132 mU/g liver in control rats) was increased by 48% in cystine-fed rats and decreased by 40% in cholesterol-fed rats. Similar changes were observed in the activity of both lipases measured in postheparin plasma. Highly significant positive correlations linked each lipolytic activity with the level of cholesterol, phospholipids and proteins in LDL2 (HDL1-rich fraction) and in HDL. In contrast, significant negative correlations were found between all of the TRL components and the activity of the lipases.(ABSTRACT TRUNCATED AT 250 WORDS)