Evaluation of epirubicin-induced acute oxidative stress toxicity in rat liver cells and mitochondria, and the prevention of toxicity through quercetin administration.

Anticancer therapy with epirubicin (EPI) results in acute hepatotoxicity, likely due to the generation of free radicals. However, the oxidative status of rat liver cells and mitochondria after EPI toxicity has not been investigated. In the present study, we first investigated the pro-oxidant effect of EPI on both hepatic cells and mitochondrial function. Injection of EPI into rats at a dose of 9mg/kg (cumulative dose in human chemotherapy), induced hepatic dysfunction, as revealed by a significant increase in serum glutamate oxaloacetate transaminases (SGOT) and glutamate pyruvate transaminases (SGPT). Oxidative stress in liver cells and mitochondria was provoked by EPI because a statistically significant reduction of catalase (CAT), superoxide dismutase (SOD) and cytosolic glutathione (GSH) levels, and a significant increase in malonedialdehyde (MDA) levels - an indicator of lipid peroxidation that can perforate biological membranes - were observed. Second, the protective effect of quercetin (QE) (0.33mg/kg) against EPI-induced oxidative stress was also investigated. Indeed, the pretreatment of rats with QE protected liver cells and mitochondria from oxidative stress. This treatment prevented hepatic dysfunction by maintaining normal levels of serum transaminases following the inhibition of their hepatic leakage by preventing lipid peroxidation. Thus, QE works through the prevention of cellular membrane perforation and the antioxidant defense system of mitochondria from liver cells, which represent compartments for the permanent production of reactive oxygen species (ROS) through the respiratory chain.

Decreased lipolytic activity in tissues during infectious and inflammatory stress.

The clearance rate of endogenous and exogenous circulating lipids during the septic or inflammatory state remains a controversial subject. Thus, we have developed rat models of gram-negative and gram-positive sepsis and of sterile inflammation to study this problem. In addition to the febrile response, these stresses induced some of the following metabolic changes in the blood: decreased total protein, albumin, and ketone body levels and increased lactate, pyruvate, alanine, cholesterol, and triacylglycerol levels. The activities of heart, diaphragm, and adipose tissue lipoprotein lipase and of hepatic lipase decreased to differing extents depending on whether the enzyme substrate was a long-chain or a medium- and long-chain triglyceride-based emulsion. However, the latter emulsion was always hydrolyzed faster than the former. This observation suggests that, during infection/inflammation, the medium- and long-chain triglyceride-based emulsion would be cleared more quickly, would induce less hypertriglyceridemia, and would thus deliver lipid energy more rapidly than a traditional long-chain triglyceride-based emulsion.

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).

Gram-negative bacteria sepsis in the rat and tissue lipolytic activity on LCT and MCT/LCT-based commercial parenteral emulsions.

The aim of this study was to evaluate the effect of a gram-negative bacteria sepsis on the activity of the enzymes lipoprotein lipase (LPL) and hepatic lipase (HL), involved in the clearance of circulating triacylglycerol-rich fat particles. Fasting rats were intravenously injected with NaCl9 g.l-1, live or heat-killed Pseudomonas aeruginosa bacteria. After 18 h the animals were killed. When compared to controls, the 2 treated groups showed an increase in body temperature, cholesterolemia, triglyceridemia and a decrease in ketonemia, proteinemia, albuminemia and in the in vitro activity of diaphragm, heart and adipose tissue LPL and of HL. The decrease in the enzyme activities occurred independent of the type of emulsion used as in vitro substrate, whether it was based on long-chain triglycerides or on medium- and long-chain triglycerides, but in any case the activity was lower with the first than with the second type of fat emulsion.

Gram-positive bacterial sepsis in rat and tissue lipolytic activity on commercial parenteral fat emulsions.

To study the influence of a gram-positive sepsis on the metabolism of circulating lipids, fasted rats were injected with saline (control group) or with a suspension of heat-killed or live Staphylococcus aureus. 18 h later, body temperature was increased, while albuminemia and ketonemia were decreased in the group injected with heat-killed bacteria, as opposed to the control group. Passing from these groups to the group injected with live bacteria, more differences appeared: increase of triglyceridemia and free cholesterolemia; decrease of esterified cholesterol levels and especially of the in vitro activity of diaphragm, heart and adipose tissue lipoprotein lipase and of hepatic lipase. The decrease of lipolytic activities occurred whether they were measured on a fat emulsion containing long-chain or medium- and long-chain triglycerides. The fact that for the latter the activity was always higher than for the former suggests that the host infected with gram-positive bacteria would clear exogenous fat more easily in the case of medium-chain triglycerides.

Fat emulsion particle size: influence on the clearance rate and the tissue lipolytic activity.

In lipid emulsions for parenteral use the mean particle diameter of the droplets in the 20% emulsions is larger than in the 10% emulsions. In long-chain triglyceride emulsions it is greater than in medium-chain triglyceride emulsions. As the particle diameter decreases, the total interfacial area increases, as does the lipoprotein lipase (LPL) and hepatic lipase (HL) activity. For a given quantity of triglycerides and phospholipids the lipolytic activity is proportional to the total interfacial area. A doubling of the phospholipid concentration is accompanied by a small reduction in the activity of both enzymes. In going from long-chain to medium-chain triglycerides, there is an acceleration in the clearance rate of infused lipid. For a similar emulsion, the clearance rate decreases as the particle size decreases. It seems plausible that the larger the mean droplet diameter, the greater the participation of the reticuloendothelial system in the clearance.

Activities of lipoprotein lipase and hepatic lipase on long- and medium-chain triglyceride emulsions used in parenteral nutrition.

Prolonged parenteral nutrition frequently includes lipid emulsions. This report investigates how emulsions containing triacylglycerols of different molecular weight affect the rate of clearance in vivo and the activity in vitro of the two enzymes responsible for this clearance: diaphragm lipoprotein lipase (LPL) and hepatic endothelial lipase (HL). Whatever their molecular weight, the triacylglycerols of the emulsions were hydrolyzed by LPL and HL. However, the reaction was faster with medium-chain triglycerides (MCT) than with long-chain triglycerides (LCT). To be active, LPL required the presence of serum (apolipoprotein CII); for maximum activity less serum was required for MCT than for LCT. In the case of HL, serum inhibited the effect on LCT but not on MCT. However, hydrolysis of emulsified triacylglycerols by LPL and HL required the presence of albumin as a transporter of the fatty acids released. Less albumin was needed for maximum activity with MCT than with LCT. In vivo, although MCT emulsions were eliminated more rapidly than LCT emulsions, the former resulted in a greater increase in plasma concentrations of triacylglycerols and free glycerol than did the latter. This is explained by the fact that MCT provides about 1.8 times more triacylglycerol molecules than the LCT. In vitro, LPL and HL hydrolyzed structured lipids (randomly esterified triacylglycerols of medium- and long-chain fatty acids) slightly less rapidly than they did control lipids, but there was no comparable difference in the blood lipid parameters examined in vivo. Because the MCT emulsions are cleared rapidly, their fatty acids are rapidly made available to the various tissues where they are oxidized.

In vivo and in vitro release of lipoprotein lipase and hepatic lipase by low molecular weight heparins.

The purpose of this study was to compare lipoprotein lipase (LPL) and hepatic lipase (HL) releasing activities of different low molecular weight heparins (LMWH) and of a standard heparin. In vivo, the injection of most LMWH led to a LPL and HL releasing activity inferior to that obtained with a standard heparin. The releasing of LPL by muscle in vitro and of HL by perfused liver was identical with both types of heparins, but in epididymal adipose tissue, LPL activity released by LMWH was generally higher than the activity released by unfractionated heparin. We have no explanation for this apparent contradiction between the in vivo and in vitro results.

Medium-chain triglyceride-based fat emulsions: an alternative energy supply in stress and sepsis.

Medium-chain triglycerides (MCTs) and medium-chain fatty acids (MCFAs) have special physicochemical properties such as small molecular weight, small interfacial tension against water, and for the fatty acids, solubility in biological fluids. As a result the metabolic pathways followed by these fats in an organism are different and simpler, or identical but more rapid, than those followed by long-chain triglycerides (LCTs) and long-chain fatty acids (LCFAs). Consequently the MCTs have found numerous applications in oral or enteral nutrition and, more recently, in parenteral nutrition. The infusion of conventional fat emulsions in stress and sepsis is still controversial. A main question is whether an MCT supply can be beneficial for these patients. In this review, we will discuss different aspects of modified lipid and protein metabolism: exchanges between exogenous fat particles and lipoproteins; exogenous fat clearance, storage, and oxidation; reticuloendothelial system function; nitrogen balance; and hepatic function. For each of these perturbations, the MCT/LCT and structured lipid emulsions are theoretically capable to provide an appropriate solution. The efficiency of these emulsions has been demonstrated experimentally on animal models of stress and sepsis. However, the value of MCT-based fat emulsions for these pathological states has still to be ascertained by clinical studies.