Lipofundin 20 % induces hyperlipidemia and oxidative stress in male

Lipofundin is a lipid emulsion used in parenteral nutrition. One of adverse effects reported for this kind of pharmaceutical products is the capacity to induce oxidative stress, which is an important contributor of many diseases, such as cardiovascular diseases. The aim of the present work was to evaluate the effects of Lipofundin administration on lipid profile and serum redox biomarkers, in order to determine if both events are responsible for the undesirable effects of this lipid emulsion. Male Sprague Dawley rats were intravenously administered with 2 mL/kg of Lipofundin 20% daily, for 8 days. Then, serum lipid profile and redox biomarkers were spectrophotometrically determined. A significant increase (p<0,05) of serum lipids and biomolecules damages was observed at the end of the experiment, while a reduction of antioxidant capacity was also detected in treated rats compared with controls. Our data demonstrated that Lipofundin 20% induces hyperlipidemia, which promotes an oxidative stress state in Sprague Dawley rats.


Introduction
antioxidants generates a state of stress in the cell termed OS.Clearly, chronic OS encompasses a Artificial fat emulsions are widely used in wide variety of patho-physiological processes parenteral nutrition.The soya oil-based fat that directly or indirectly affect the cellular redox emulsions represent a major part of energy and state (4).are also a necessary source of essential fatty acids The impact of Lipofundin 20% administration in mentioned therapy (1).Lipofundin constitutes on lipid levels and redox environment in male a usually indicated fat emulsion as a source of Sprague Dawley (SD) rats had not been studied calories for patients requiring parenteral previously.In the present study we demonstrated nutrition, but researches by Jellinek et al. (2) that Lipofundin 20% induces hyperlipidemia and showed that Lipofundin induces atherosclerotic lesions in rabbits and recently, our group a systemic oxidative stress after 8 days of treatment.demonstrated that Lipofundin promotes a high

susceptibility to lipid peroxidation (3) and also
Animals : Adult male SD rats, weighing 250-300 dyslipidemia and oxidative stress (OS) in New g, were obtained from CENPALAB (Bejucal, Zealand white rabbits after 8 days of treatment Mayabeque, Cuba).Rats were housed in a (non published data).
temperature conditioned room (25 ± 1 ºC) with a An imbalance between oxidants and relative humidity of 50-52% and exposed to lightantioxidants resulting from increased production dark cycle of 12 h with free access to water and of oxidants and/or reduction in the amounts of food.Animal studies were performed with approval RANSOD kit (catalogue No. SD 125, Randox of Pharmacy and Food Sciences College Labs, Crumlin, UK), where xanthine and xanthine Institutional Animal Ethical Committee.All oxidase were used to generate superoxide anion Germany) is a lipid emulsion containing soya oil 1 min (5).100 g, medium-chain triglycerides 100 g, After precipitation of thiol proteins, the glycerol 25 g, egg lecithin 12 g, α-tocopherol 170 reduced glutathione (GSH) levels were measured ± 40 mg, and sodium oleate/water for injection in according to the method of Sedlak and Lindsay sufficient quantity to 1000 mL.
(6) with Ellman`s reagent (5,5`dithiobis-2-Experimental design: Two groups of 5 rats were nitrobenzoic acid) (Sigma St Louis, MO, USA) used in the study.The first group received an and the absorbance was measured at 412 nm.intravenous injection of phosphate-buffered Purified GSH (Sigma St Louis, MO, USA) was saline (PBS), pH 7,4 (control group), and the used to generate standard curves.second one received a slow intravenous injection The advanced oxidation protein products of 2 mL/kg of Lipofundin MCT/LCT 20 %, as an (AOPP) were measured as described previously infusion during 1-2 min (2).This procedure was (7).Briefly, samples in PBS (1 mL) were treated repeated daily during a period of 8 days.On day with 50 µL of potassium iodide 1,16 M followed 9, the animals were anesthetized with ketamine by the addition of 100 µL of acetic acid.The hydrochloride (5 mg/kg i.m.), and euthanized absorbance was immediately read at 340 nm. with an overdose of sodium pentobarbital (90 AOPP concentration was expressed as µM of mg/kg, i.v.) (Abbott Lab., Mexico SA de CV, chloramines-T.Mexico).
Concentration of malondialdehyde (MDA) was determined using the LPO-586 kit obtained Serum sample collection : Blood samples (3 mL) from Calbiochem (La Jolla, CA, USA).In the were withdrawn from the ocular plexus on day 0 assay, the production of a stable chromophore (before Lipofundin administration) and from the after 40 min of incubation at 45 ºC was measured abdominal aorta on day 9 (at the end of the study), at 586 nm.For standards, freshly prepared solutions for biochemical analyses.These samples were of malondialdehyde bis [dimethyl acetal] (Sigma immediately centrifuged at 2500g, at 4 ºC for 10 St Louis, MO, USA) were employed and assayed min.The serum was collected and aliquots were under identical conditions (8).stored at -80 ºC until analysis.
In order to determine susceptibility to lipid Serum lipid assay: Serum total cholesterol (TC), peroxidation and total reactive antioxidant power triglycerides (TG), low-density lipoproteins (TRAP), the samples were incubated with a (LDLc) and high density lipoprotein (HDLc) solution of copper sulphate (final concentration 2 were determined using commercial enzymatic mM) at 37 ºC for 24 h.The peroxidation potential kits (Randox, Crumlin, UK).
(PP) was calculated by subtracting the MDA levels before the induction of lipid peroxidation Redox biomarkers determinations: All biochemical from the one obtained at 24h (9).parameters were determined by spectropho-Finally, the total antioxidant status (TAS) tometric methods using a Pharmacia 1000 was determined using a Randox TAS kit Cat No. Spectrophotometer (Pharmacia LKB, Uppsala, 2332. In the assay ABTS (2,2 -Azino-di-[3-Sweden). SOD activity was determined by using ethylbenzthiazoline sulphonate]) is incubated CAT were significantly higher (p<0.05) in with a peroxidase (metmyoglobin) and hydrogen Lipofundin group at the end of the experiment peroxide (H O ) to produce the radical cation compared to control rats.The GSH levels decreased 2 2 •+ significantly after 8 days of Lipofundin treatment ABTS .This has a relatively stable blue-green in comparison to those of untreated animals color, which is measured at 600 nm.Antioxidants (p<0.05).In addition, in treated rats, TAS was in the added sample cause suppression of this significantly lower (p<0.05)than in controls.color production to a degree which is proportional Finally, the susceptibility to lipid peroxidation to their concentrations.
was higher in those rats who received Lipofundin.Statistical analysis: Statistical analysis was After 8 days, in these animals was observed a performed using the SPSS program for Windows significant increase of PP (p<0.05),compared to (version 11.5, SPSS Inc).Bartlett's Box-test was the one calculated in controls.used to test the homogeneity of variance.Differences

Table-2. Effects of Lipofundin on redox biomarkers.
between groups were determined by student's ttest (two-tailed).Data were expressed as the mean ± standard deviation (SD).The level of statistical significance employed was at least p< 0.05.

Serum lipids: Serum TC, TG, LDLc and HDLc
Values are the mean ± standard deviation.Asterisks represent statistical differences (p<0.05)levels showed a significant increase (p<0.05) in those animals who were treated during 8 days Discussion with the lipid-rich emulsion Lipofundin, while no At the end of the experiment we observed significant changes in serum lipids were high serum levels of triglycerides, total cholesterol, observed in the control rats throughout the study LDLc and HDLc in the animals treated with (Table 1).
Lipofundin 20% in comparison to control rats.

Table-1. Effects of Lipofundin on serum lipid
Lipofundin-induced hyperlipidemia could be profile.
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) (10).In fact, Lipofundin 10% caused a 60% increase in total Values are the mean ± standard deviation.Asterisks serum cholesterol after parenteral administration represent statistical differences (p<0.05) in a human study (11).On the other hand, the Redox biomarkers: Table 2 shows the behavior of increase of HDLc may be determined by a serum redox parameters in both groups at day 9. physiological response against the elevated The markers of damages to biomolecules were LDLc levels.significantly (p<0.05)modified after 8 days of In this study we demonstrated that Lipofundin-Lipofundin administration compared to non induced hyperlipidemia was associated with a treated group.At the end of the experimental systemic OS.Strong evidences for the period the MDA levels, one of the end-products involvement of free radicals production in the of lipid peroxidation, were higher in Lipofundin onset of hyperlipidemia have been reported treated animals compared with controls.Besides, previously vascular diseases (12).lipid hydroperoxides.The high content of these In hyperlipidemia, cellular damages take metabolites may in turn induce the gene place through mechanisms involving lipid expression of the enzyme and also its activity is peroxidation and oxidative modifications of incremented in response to oxidant stimulus proteins (13).On the other hand, a disruption of mediated by ox-LDL and other ROS (22).
During atherogenesis, the reactive molecules antioxidant enzymes activity and a drastic that are produced have the potential to deplete the reduction of non-enzymatic defenses are also surrounding cells of their GSH levels, affecting observed (14).High levels of MDA in the sera of their antioxidant defenses and detoxification treated rats were observed, which suggest the role pathways (23).Our results showed a significant of LPO in the loss of redox cellular status in the depletion of serum GSH levels in the animals former animals which were under Lipofundin treated with the lipid emulsion compared to the treatment.MDA levels have been considered not control rabbits.This fact could be associated with only an indicator of OS, but also as a biochemical the Lipofundin-mediated ROS generation and marker of atherogenesis and other diseases with the high concentration of biomolecules associated with the high levels of serum lipids damages detected in Lipofundin-treated animals.(3,(15)(16)(17).
Finally, TAS was affected by Lipofundin, which Oxidative modifications of proteins have is in accordance with other reports of the been also implicated in hyperlipidemia and literature (17).atherosclerosis (18).Through AOPP determination we measured the chlorinated proteins levels, Conclusions caused by myeloperoxidase-derived hypochlorous In summary, the present study demonstrated acid (HOCl).It has been shown that HOClthat Lipofundin 20% induces hyperlipidemia, modified proteins are present in hyperlipidemic thereby promoting a systemic oxidative stress.patients and predict the progression of This work shows novel evidences of Lipofundincardiovascular diseases such as atherosclerosis induced oxidative damages on lipids and proteins (19).The high levels of AOPP in those animals and the deleterious effects on antioxidant status.that received the lipid emulsion suggest an active These results reinforce the attractive characteristics role of macrophages activation and inflammation of Lipofundin to be used as an inductor of in the OS generation.
experimental hyperlipidemia and oxidative stress.Antioxidant defenses, as expression of the