Distribution of Tocopherols and Tocotrienols in Guinea Pig Tissues Following Parenteral Lipid Emulsion Infusion.

BACKGROUND: Tocopherols and tocotrienols possess vitamin E activity and function as the major lipid-soluble antioxidants in the human body. Commercial lipid emulsions are composed of different oils and supply different amounts of vitamin E. The objective of this study was to measure all 8 vitamin E homologs within 4 different commercial lipid emulsions and evaluate their distribution in guinea pig tissues. MATERIALS AND METHODS: The distribution of vitamin E homologs within plasma and guinea pig tissues was determined using a high-performance liquid chromatography (HPLC) system. Lipid hydroperoxides in lipid emulsions were determined using a commercial kit (Cayman Chemical Company, Ann Arbor, MI), and malondialdehyde tissue levels were determined using an HPLC system. RESULTS: The lipid emulsions contained variable amounts of tocopherols, which were significantly different between emulsions. Tocotrienols were present at very low concentrations (≤0.3%). We found no correlation between the amount of vitamin E present in the lipid emulsions and lipid peroxidation. Hydroperoxides were the lowest with an olive oil-based emulsion and highest with a fish oil emulsion. The predominant vitamin E homolog in guinea pig tissues was α-tocopherol. No tissues had detectable levels of tocotrienols. Vitamin E levels (primarily α-tocopherol and γ-tocopherol) were highly variable among organ tissues. Plasma levels were a poor reflection of most tissue levels. CONCLUSION: Vitamin E levels within different lipid emulsions and plasma/tissues are highly variable, and no one tissue or plasma sample serves as a good proxy for levels in other tissues. All study emulsions were well tolerated and did not significantly increase systemic lipid peroxidation.

Comparative study of the modulation of fructose/sucrose-induced hepatic steatosis by mixed lipid formulations varying in unsaturated fatty acid content.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in developed countries. NAFLD encompasses a spectrum of diseases, ranging from hepatic steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis, and liver failure. The etiology of NAFLD remains unclear but is thought to relate to increased fatty acid flux within the liver that results in toxic fatty acid metabolite production. One source of increased fatty acid flux is fructose/sucrose-induced hepatic lipogenesis. Current treatment for NAFLD encompasses dietary modifications. However, little scientific evidence exists on which to base many dietary recommendations, especially the intake of different types of carbohydrates and fats. We hypothesized that lipid mixtures of unsaturated fatty acids would inhibit lipogenesis and subsequent hepatic steatosis induced by high carbohydrate diets. The aim of this study was to examine the effects of different complex mixtures of fatty acids upon the development of fructose/sucrose-induced hepatic steatosis. METHODS: C57BL/6 mice were randomized to normocaloric chow-based diets that varied in the type of carbohydrate (starch, sucrose, fructose). Animals in each carbohydrate group were further randomized to diets that varied in lipid type (no additional lipid, soybean oil, fish oil, olive/soybean oil, macadamia nut oil). These oils were chosen based upon their content of omega-6 polyunsaturated fatty acids, omega-3 polyunsaturated fatty acids, omega-9 monounsaturated fatty acids, or omega-7 monounsaturated fatty acids. Fatty acid flux in the liver was determine by assessing hepatic lipid content (steatosis). We also assessed fatty acid levels in the plasma and liver of the animals, hepatic lipogenesis activity, hepatic stearoyl-CoA-1 desaturase activity, and hepatic elongase activity. RESULTS: Animals consumed similar amounts of the diets and maintained normal body weights throughout the study. Both sucrose and fructose induced hepatic lipogenesis and steatosis, with fructose being more potent. All mixed lipids similarly inhibited steatosis, limiting lipid content to levels found in the control (starch) animals. Lipogenesis and stearoyl-CoA-1 desaturase activity were increased in the sucrose and fructose groups. Levels of these enzymatic processes remained at baseline in all of the lipid groups. CONCLUSION: This is the first study to compare various complex lipid mixtures, based upon dietary oils with different types of long-chain fatty acids, upon development of sucrose/fructose-induced steatosis. Both carbohydrate source and lipid content appear important for the modulation of steatosis. Moderate intake of complex lipids with high unsaturated to saturated fatty acid ratios inhibited both lipogenesis and steatosis.

Modulation of endothelial cell integrity and inflammatory activation by commercial lipid emulsions.

BACKGROUND: Thrombosis and immune dysfunction are two important complications that result from the administration of parenteral nutrition. Endothelial cells within the vasculature are crucial components necessary for maintenance of normal coagulation and immune function. METHODS: We compared the effects of three commercial lipid emulsions (LEs; Intralipid®, ClinOleic® [or Clinolipid®], and Omegaven®) differing in the levels of omega-6 polyunsaturated fatty acids, omega-3 polyunsaturated fatty acids, omega-9 monounsaturated fatty acids, and saturated fatty acids upon endothelial cell fatty acid composition using Gas chromatography, endothelial cell integrity by assessing measurement of apoptosis and necrosis using flow cytometry, endothelial cell inflammatory activation by assessing the induction of ICAM-1 by lipopolysaccharide [LPS]), and transcription factor activation (phosphorylation of NF-κB) using western blot analysis. RESULTS: Gas chromatographic analysis confirmed cellular uptake of the fatty acids within the LEs; furthermore, these fatty acid changes reflected the composition of the oils and egg phosphatides used in the manufacturing of these emulsions. However, the kinetics of fatty acid uptake and processing differed between LEs. Fish oil LE negatively impacted cell viability by doubling the percentage of apoptotic and necrotic cell populations quantified by flow cytometry using Annexin V/Fluorescein and propidium iodide. The soybean oil LE did not alter cell viability, while the olive oil-predominate emulsion improved cell viability. All LEs were capable of suppressing LPS-induced ICAM-1 expression; however, the fish oil LE was more potent than the other emulsions. Fish oil LE supplementation of cells also suppressed LPS-induced phosphorylation of NF-κB, while the soybean oil and olive predominant LE had no effect upon NF-κB phosphorylation. CONCLUSIONS: Lipid emulsions are readily incorporated and stored in the form of triacylglycerols. Soybean oil-based, olive oil-predominant and fish-oil based LEs differentially affected endothelial cell integrity. Importantly, these three LEs were capable of suppressing endothelial cell inflammatory response despite their fatty acid content.

Tocopherol and tocotrienol homologs in parenteral lipid emulsions.

Parenteral lipid emulsions, which are made of oils from plant and fish sources, contain different types of tocopherols and tocotrienols (vitamin E homologs). The amount and types of vitamin E homologs in various lipid emulsions vary considerably and are not completely known. The objective of this analysis was to develop a quantitative method to determine levels of all vitamin E homologs in various lipid emulsions. An HPLC system was used to measure vitamin E homologs using a Pinnacle DB Silica normal phase column and an isocratic, n-hexane:1,4 dioxane (98:2) mobile phase. An optimized protocol was used to report vitamin E homolog concentrations in soybean oil-based (Intralipid®, Ivelip®, Lipofundin® N, Liposyn® III, and Liposyn® II), medium- and long-chain fatty acid-based (Lipofundin®, MCT and Structolipid®), olive oil-based (ClinOleic®), and fish oil-based (Omegaven®) and mixture of these oils-based (SMOFlipid®, Lipidem®) commercial parenteral lipid emulsions. Total content of all vitamin E homologs varied greatly between different emulsions, ranging from 57.9 to 383.9 µg/mL. Tocopherols (α, ß, γ, δ) were the predominant vitamin E homologs for all emulsions, with tocotrienol content < 0.3%. In all of the soybean emulsions, except for Lipofundin® N, the predominant vitamin E homolog was γ-tocopherol, which ranged from 57-156 µg/mL. ClinOleic® predominantly contained α-tocopherol (32 µg/mL), whereas α-tocopherol content in Omegaven® was higher than most of the other lipid emulsions (230 µg/mL). PRACTICAL APPLICATIONS: The information on the types and quantity of vitamin E homologs in various lipid emulsions will be extremely useful to physicians and healthcare personnel in selecting appropriate lipid emulsions that are exclusively used in patients with inadequate gastrointestinal function, including hospitalized and critically ill patients. Some emulsions may require vitamin E supplementation in order to meet minimal human requirements.

Parenteral lipid emulsions in guinea pigs differentially influence plasma and tissue levels of fatty acids, squalene, cholesterol, and phytosterols.

Lipid emulsions are made by mixing vegetable and/or fish oils with egg yolk and contain different types and amounts of fatty acids and sterols. This study assessed the effects of oral diet, soybean oil (SO)-, fish oil (FO)-, a mixture of olive and soybean oil (OOSO)-, and a mixture of fish, olive, coconut, and soybean oil (FOCS)-based emulsions on plasma triacylglycerols and plasma and tissue fatty acid and sterol content following acute and chronic intravenous administration in the guinea pig. Upon acute administration, peak triacylglycerols were highest with SO and lowest with OOSO. Upon chronic administration, the plasma triglyceride levels did not increase in any group over that of the controls. Fatty acid levels varied greatly between organs of animals on the control diets and organs of animals following acute or chronic lipid administration. Squalene levels increased in plasma following acute administration of OOSO, but plasma squalene levels were similar to control in all emulsion groups following chronic administration. Total plasma phytosterol levels were increased in the SO, OOSO, and FOCS groups following both acute and chronic infusions, whereas phytosterols were not increased following FO infusion. Total phytosterol levels were higher in liver, lung, kidney and adipose tissue following SO and OOSO. Levels were not increased in tissues after FO and FOCS infusion. These results indicate that fatty acid and sterol contents vary greatly among organs and that no one tissue reflects the fatty acid or sterol composition of other tissues, suggesting that different organs regulate these compounds differently.

Steroidal compounds in commercial parenteral lipid emulsions.

Parenteral nutrition lipid emulsions made from various plant oils contain steroidal compounds, called phytosterols. During parenteral administration of lipid emulsions, phytosterols can reach levels in the blood that are many fold higher than during enteral administration. The elevated phytosterol levels have been associated with the development of liver dysfunction and the rare development of liver failure. There is limited information available in the literature related to phytosterol concentrations in lipid emulsions. The objective of the current study was to validate an assay for steroidal compounds found in lipid emulsions and to compare their concentrations in the most commonly used parenteral nutrition lipid emulsions: Liposyn(®) II, Liposyn(®) III, Lipofundin(®) MCT, Lipofundin(®) N, Structolipid(®), Intralipid(®), Ivelip(®) and ClinOleic(®). Our data demonstrates that concentrations of the various steroidal compounds varied greatly between the eight lipid emulsions, with the olive oil-based lipid emulsion containing the lowest levels of phytosterols and cholesterol, and the highest concentration of squalene. The clinical impression of greater incidences of liver dysfunction with soybean versus MCT/LCT and olive/soy lipid emulsions may be reflective of the levels of phytosterols in these emulsions. This information may help guide future studies and clinical care of patients with lipid emulsion-associated liver dysfunction.

Oleic acid inhibits stearic acid-induced inhibition of cell growth and pro-inflammatory responses in human aortic endothelial cells.

Saturated fatty acids (SFAs), significant components of both enteral/parenteral nutritional formulations (including diet), are linked to cardiovascular disease complications, such as atherosclerosis. We investigated whether oleic acid (C18:1n-9) reduces the growth inhibitory and pro-inflammatory effects of the stearic acid (C18:0) in human aortic endothelial cells (HAEC). Stearic acid induced growth inhibition at concentrations less than 50 µM, whereas higher concentrations invoked cytotoxicity. Stearic acid-induced growth inhibition and cytotoxic effects were eradicated upon cosupplementation with oleic acid (25 µM). Oleic acid (as low as 5 µM) also inhibited the stearic acid-induced increase in intercellular adhesion molecule-1 (ICAM-1) expression. Stearic acid-induced phosphorylation of nuclear factor-kappa B (NF-κB), a transcriptional regulator of ICAM-1, was also reduced by oleic acid. HAECs supplemented with either stearic or oleic acid resulted in cellular incorporation of C18:0 and C18:1n-9, respectively. Stearic acid primarily incorporated into phospholipids without increasing the total fatty acid content in HAECs. In contrast, oleic acid, with or without stearic acid, incorporated into both phospholipids and triglycerides, with a significant increase in total fatty acid amounts in triglycerides. Our data suggest that oleic acid has the ability to reduce the inflammatory effects of long-chain SFAs in HAECs through reducing cellular stearic acid incorporation and NF-κB activation.

An improved method for determining medium- and long-chain FAMEs using gas chromatography.

The existing protocols for analyzing fatty acid methyl esters (FAMEs) using a one-step acetyl chloride (AC) catalyzed transesterification and extraction procedure cannot accurately determine the medium- and long-chain fatty acids simultaneously in clinical (enteral, parenteral) formulations. For example: (1) addition of AC at room temperature generates an exothermic reaction that often results in loss of sample and possible injury to the analyst; (2) certain polyunsaturated fatty acids (PUFAs) are less stable at elevated temperatures during the transesterification and contribute to the over-estimation of the C16:0 and C18:1 fatty acids; and (3) the flame-ionization detector (FID) response varies depending on the carbon chain length of the fatty acids, that consequently impacts the underestimation of medium-chain fatty acid (C6-C10) recoveries. To overcome these deficiencies and accurately determine FAMEs, we have developed an improved one-step transesterification method that employs the addition of AC in tubes kept on a dry ice bath, the transesterification at room temperature, and the data analysis using relative response factors. Using this modified protocol, we determined the fatty acid composition of lipid emulsions (Omegaven and Lipidem) on a Shimadzu GC2010 gas chromatography (GC) system using a capillary GC column (Zebron ZB-WAX plus, 30 m, 0.25 mm ID, 0.25 microm). Our data suggest that the improved method can be easily used to accurately determine fatty acids (C6-C24) in functional foods and lipid emulsions.

Long-chain saturated fatty acids induce pro-inflammatory responses and impact endothelial cell growth.

BACKGROUND & AIMS: Saturated fatty acids (SFAs), significant components of enteral and parenteral formulations, have been linked to cardiovascular complications. However, the effect of SFAs upon vascular inflammation is less clear. Endothelial cells (EC) play an important role in the acute inflammatory responses. We, therefore, evaluated the acute effects of different chain-length SFAs upon EC functions. METHODS: Endothelial cells were cultured with various SFAs. Growth and cytotoxicity were determined by WST-1 assay. Apoptosis and pro-inflammatory adhesion molecule (ICAM-1) expression was assayed using flow cytometry. Activation of NF-kappaB was analyzed using western blot analysis. RESULTS: Long-chain SFAs (C14:0-C20:0) inhibited EC growth in a chain-length dependent manner. Medium-chain SFAs (C6:0-C12:0) did not significantly affect EC growth. In contrast, the short-chain SFA (C4:0) stimulated cellular growth. Stearic acid induced significantly more EC apoptosis and necrosis than palmitic acid or myristic acids. Stearic acid (>10muM) treatment also significantly increased ICAM-1 expression. Stearic acid's pro-inflammatory response was confirmed by phosphorylation of IkappaB-alpha and NF-kappaB in a dose dependent manner. CONCLUSIONS: Long-chain SFAs can induce pro-inflammatory responses and significantly impact growth and viability of EC. Our data suggest that the presence of long-chain SFAs in parenteral formulations may have harmful effects on the vascular system.