Lipofundin 20% induces hepatic lipid peroxidation in New Zealand white rabbits

Objective. The aim of the present work was to evaluate the effects of Lipofundin 20% on lipid peroxidation markers in the liver of New Zealand white rabbits. Materials and methods. The animals were treated with an intravenous injection (2 ml/kg) of the lipid emulsion during 8 days through the marginal ear vein. At the end of the experiment some lipid peroxidation parameters and lipid profile were tested through spectrophotography. Results. Lipofundin was found to induce a significant (p<0.05) increase of malondialdehyde, total hydroperoxides, and peroxidation potential. Also, high levels of total cholesterol, triglycerides, LDL cholesterol and HDL-cholesterol were observed in treated animals compared with the control group (p<0.05). Conclusions. Data proved that Lipofundin induces hepatic lipid peroxidation in rabbits, mainly through a mechanism which involves an induction of hyperlipidemia


INTRODUCTION
Lipid peroxidation (LPO) was first studied in the 1930's in relation to food deterioration, but since then, there has been increasing evidence showing the involvement of free radicals in biology, leading to renewed attention on LPO with a wider scope in the fields of chemistry, biochemistry, nutrition and medicine (1,2), amongst others.Further studies revealed that, like proteins, carbohydrates, and nucleic acids, lipids are targets of reactive oxygen species (ROS) and become oxidized to render cytotoxic products (3).Several oxidized products have been studied and also used as LPO biomarkers, such as malondialdehyde (MDA) and lipoperoxides (LOOH) (4).
Artificial fat emulsions are widely used in parenteral nutrition.The soya oil-based fat emulsions represent a major part of energy and are also a necessary source of essential fatty acids in the mentioned therapy (5,6).Lipofundin 10% constitutes a frequently indicated fat emulsion as a source of calories for patients requiring parenteral nutrition, but preclinical investigations demonstrated that Lipofundin 20% induces atherosclerotic lesion formation in rabbits (7).Our group also demonstrated that this fat emulsion induces a systemic LPO in rabbits (8), but the effects on hepatic LPO have not been assessed.Therefore, the purpose of the present work is to evaluate the effects of Lipofundin 20% on lipid profile and hepatic biomarkers of LPO in New Zealand White rabbits (NZB).

MATERIALS AND METHODS
Animals.Standard NZW male rabbits, weighing 2.0-2.5 kg and 12 weeks old, were obtained from CENPALAB (Mayabeque, Cuba).Rabbits were housed under conventional conditions exposed to a 12 hr light-dark cycle with free access to water and food.Animal studies were performed with approval of the Pharmacy and Food Sciences College Institutional Animal Ethical Committee.All procedures were in accordance with the European Union Guidelines for animal experimentation.
Experimental design.Two groups of 10 rabbits were used in the study.The first group received an intravenous injection of phosphatebuffered saline solution (PBS), pH 7,4 (control group), and the second one received a slow intravenous injection of 2 ml/kg of Lipofundin MCT/LCT 20%, as an infusion during 1-2 min (7).This procedure was repeated daily during a period of 8 days.On day 9, the animals were anesthetized with ketamine hydrochloride (5 mg/kg i.m.), and euthanized with an overdose of sodium pentobarbital (90 mg/kg, i.v.).(Abbott Laboratories, México SA de CV, México).Then, the liver was perfused with NaCl 0.9% solution at 4ºC.

Liver homogenate preparation.
The hepatic right lobe of each animal was extracted and homogenized in 20mM KCl/histidine buffer, pH 7.4, 1:10 w/v using a tissue homogenizer (Edmund Bühler LBMA, Germany) at 4ºC and centrifuged for 10 min at 12000 g.Supernatants were taken for biochemical determination.

Serum sample collection.
Blood samples (1 ml) were obtained on day 0 and 9 (at the end of the study), for biochemical analyses.Blood was withdrawn from the rabbit's marginal ear vein.These samples were immediately centrifuged at 2500 g, at 4ºC for 10 min.The serum was collected and aliquots were stored at -80ºC until analysis.

Redox biomarkers determination.
All biochemical parameters were determined through spectrophotometric methods using a Pharmacia 1000 Spectrophotometer (Pharmacia LKB, Uppsala, Sweden).Total protein levels were determined using the method described by Bradford (9) with bovine albumin serum as standard.
MDA Concentration was determined using the LPO-586 kit obtained from Calbiochem (La Jolla, CA, USA).In the evaluation, the production of a stable chromophore after 40 min of incubation at 45ºC was measured at 586 nm.For control, freshly prepared solutions of malondialdehyde bis [dimethyl acetal] (Sigma St Louis, MO, USA) were employed and evaluated under identical conditions (10).
In order to determine susceptibility to lipid peroxidation and total reactive antioxidant power (TRAP), the samples were incubated with a solution of copper sulphate (final concentration 2 mM) at 37ºC for 24 h.The peroxidation potential (PP) was calculated by subtracting the MDA levels before the induction of LPO from the one obtained at 24h (11).
Statistical analysis.Statistical analysis was performed using the SPSS program for Windows (version 11.5, SPSS Inc).Bartlett's Box-test was used to test the homogeneity of variance.Differences between groups were determined by independent student's t-test (two-tailed).Data was expressed as the mean ± standard deviation (SD).A P-value <0.05 was considered as statistically significant.

RESULTS
Serum total cholesterol, triglycerides, LDLcholesterol and HDL-cholesterol levels showed a significant increase (p<0.05) in those animals who were treated during 8 days with the lipidrich emulsion Lipofundin, compared with the control (Table 1).These parameters were determined on day 0 and statistical differences were not shown (data not shown).
animals compared with controls.Total ROOH levels were also significantly higher (p<0.05) in the animals that were administered with lipofundin than in non-treated specimens.Besides, lipofundin treatment also caused an increase in PP compared with the control group (p<0.05).

DISCUSSION
After lipofundin administration, high levels of triglycerides, total cholesterol, LDL-cholesterol and HDL-cholesterol serums were observed.Indeed, there is a causal relationship between elevated plasma lipids and the occurrence of LPO (12).Lipofundin 20%-induced hyperlipidemia could be associated with the high content of triglycerides in this emulsion.High levels of exogenous triglycerides promote ApoB100 and cholesterol synthesis, and eventually the assembly of very low-density lipoproteins (VLDL) (13).In fact, these results are in accordance with our previous report (8), while it is known that lipofundin 10% caused a 60% increase in total serum cholesterol after parenteral administration in a human study (5).
In addition, there is a mutual lipid and apolipoprotein exchange between serum lipoproteins and infused triglyceride/ phospholipid particles (14).The increase of HDL-cholesterol may be determined by a physiological response against the elevated LDL-cholesterol levels.
This study demonstrated that lipofundininduced hyperlipidemia induces liver oxidative stress.Strong evidence of the involvement of increased free radical production in the onset of hyperlipidemia has been reported previously (15).Also, lipofundin was recently demonstrated to induce an increase of serum lipids in rabbits (7) and in rats (16).The serum lipid levels were similar between groups at the beginning of the experiment, also it was observed no differences of lipid levels between day 0 and 9 in controls (data not shown).Data are the means ± standard deviation.Asterisks represent statistical differences (p<0.05).The samples were tested for triplicate in all performed assays.TC: total cholesterol, TG: triglycerides, HDLc: cholesterol of high-density lipoprotein, LDLc: cholesterol of lowdensity lipoprotein.An increasing MDA level in those animals treated with lipofundin 20% was also detected in this study.Our data is in accordance with the criteria that this end-product of LPO and is strongly associated with the development of hyperlipidemia (20).
Lipofundin-induced high serum lipid levels, especially atherogenic ones such as cholesterol and LDL allow to explain, in part, the fact that these were higher in animals administered with LPO products than in controls.MDA is considered as a major epitope of oxidized LDL (21), suggesting that lipofundin 20% induces an increase in LPO closely associated with an elevation of LDL particles and related ApoB100containing lipoproteins.Also, PP increase in livers from treated rabbits reinforces the criteria that LPO is determinant in the loss of redox hepatic status in former animals which were under Lipofundin 20% treatment.
In conclusion, the present study demonstrated that lipofundin 20% induces hyperlipidemia, thereby promoting hepatic LPO.Our data shows novel evidences of lipofundin-induced oxidative damages on hepatic lipids.These results reinforce the attractive characteristics of lipofundin to be used as an experimental inductor of LPO in rabbits.