Effect of medium/ω-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

Abstract

Lipid emulsion (LE) containing medium/ω-6 long chain triglyceride-based emulsion (MCT/ω-6 LCT LE) has been recommended in the place of ω-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/ω-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/ω-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/ω-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/ω-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription.

Fig. 1

Plasma free fatty acids (a), triacylglycerol (b), cholesterol (c) and thiobarbituric acid reacting substances (TBARS) (d) concentrations. The values are presented as mean ± standard error of the mean of seven samples. *P < 0·05 for comparison between before and immediately post-infusion of medium/ω-6 long chain triglyceride-based lipid emulsion; #P < 0·05 for comparison between immediately after infusion and 18 h post-infusion of medium/ω-6 long chain triglyceride-based lipid emulsion.

Fig. 2

Plasma fatty acid composition. Fatty acids were extracted and saponified, and the composition was determined by gas chromatography (HPLC). The values are presented as mean ± standard error of the mean of seven samples; *P < 0·05 for comparison between pre- and post-infusion of medium/ω-6 long chain triglyceride-based lipid emulsion.

Fig. 3

Percentage of viable, necrotic and apoptotic lymphocytes (a) and neutrophils (b). Cells, freshly obtained and after being cultured for 24 and 48 h, were isolated from the blood of the volunteers before infusion of medium/ω-6 long chain triglyceride-based lipid emulsion, immediately post-infusion and 18 h later. Cells were stained with annexin V-fluorescein isothiocyanate (FITC) and incubated for 15 min at room temperature in the dark. Afterwards, cells were stained with propidium iodide (PI). Fluorescence of annexin V-FITC was measured in FL1 (530/30 nm) and PI in FL2 (585/42 nm). We analysed the percentage of viable (annexin V-FITC^-/PI^-), apoptotic (annexin V-FITC⁺/PI^-) and necrotic cells (annexin V-FITC⁺/PI⁺).Ten thousand events were evaluated per experiment. The values are presented as mean ± standard error of the mean of seven samples. *P < 0·05 for comparison between before and after infusion or 18 post-infusion; #P < 0·05 for comparison between after infusion and 18 h post-infusion.

Fig. 4

Proportion of lymphocytes and neutrophils with fragmented DNA. The percentage of lymphocytes (a) and neutrophils (b) with fragmented DNA is indicated. Cells, freshly obtained and after being cultured for 24 and 48 h, were isolated from the blood of the volunteers before infusion of medium/ω-6 long chain triglyceride-based lipid emulsion, immediately post-infusion and 18 h later. Cells were stained with a buffer containing citrate, Triton X-100 and propidium iodide and analysed by flow cytometry. Fluorescence was measured in the FL2 channel (585/42 nm). Ten thousand events were evaluated per experiment. The values are presented as mean ± standard error of the mean of seven experiments. *P < 0·05 for comparison between before and after infusion.

Fig. 5

Effects of medium/ω-6 long chain triglyceride-based lipid emulsion on neutral lipid accumulation in lymphocytes. Fluorescence mean of lymphocytes with neutral lipid accumulation is indicated. Cells, freshly obtained and after being cultured for 24 and 48 h, were isolated from the blood of the volunteers before infusion of medium/ω-6 long chain triglyceride-based lipid emulsion, immediately post-infusion and 18 h later. Cells were stained with Nile red and fluorescence was measured in the FL1 channel (530/30 nm). The values are presented as mean ± standard error of the mean of seven experiments. *P < 0·05 for comparison between before and after infusion.