John D. Palombo

The antiproliferative effects of biologically active isomers of conjugated linoleic acid on human colorectal and prostatic cancer cells

The antiproliferative effects of two commercial preparations of conjugated linoleic acid (CLA) and their constituent isomers, cis-9, trans-11 (c9,t11)-CLA, c9,c11-CLA, and t10,c12-CLA, were determined in vitro using human colorectal (HT-29, MIP-101) and prostate (PC-3) carcinoma cells adapted to serum-free medium. The antiproliferative effects of the preparations were dependent upon the type and concentration of CLA isomer present. The t10,c12-CLA isomer exhibited the greatest potency against colorectal cancer proliferation, and the c9,t11 and t10,c12 isomers were moderately effective against prostate cancer. The t10,c12 isomer induced caspase-dependent apoptosis in MIP-101 and PC-3 cells. The results are the first to demonstrate that physiologic levels of two CLA preparations, their constituent isomers, and the c9,t11-CLA elongation product, c11,t13-conjugated eicosadienoic acid, induce dose-dependent inhibitory effects on cancer proliferation in vitro. Novel CLA preparations may prove effective as chemopreventive supplements for individuals at risk of or diagnosed with colorectal or prostate cancer.

Immunologic Effects of Acute Hyperglycemia in Nondiabetic Rats

BACKGROUND This study was designed to determine the consequences of acute hyperglycemia on the immune function of peripheral neutrophils, peritoneal macrophages, and alveolar macrophages in nondiabetic rats. METHODS The animals were randomly divided into nonsurgical (normal) and surgical groups. The postoperative rats were further divided into normoglycemic (control) and hyperglycemic (glucose) groups. The hyperglycemic condition was maintained by constant infusion of glucose to raise plasma glucose concentration to 300 mg/dL for 3 hours. The immune cells were then harvested to determine their phagocytic and oxidative capacities via flow cytometry. RESULTS The results showed that hyperglycemia significantly decreased the respiratory burst of alveolar macrophages (p < .05). In contrast, hyperglycemia enhanced phagocytosis in these cells (p < .002). There was a significant activation of the respiratory burst in peripheral neutrophils by surgery (p < .002), but no effect of hyperglycemia. CONCLUSIONS We conclude that hyperglycemia itself can influence immune function in some phagocytic cells, which may be an important factor in postsurgical infection.

A rat fatty liver transplant model

A rat model of fatty liver transplantation has been developed to study primary nonfunction in fatty liver grafts. ACI rats were fed with a diet deficient in choline and methionine for 7, 14, 28, and 42 days. Fat content in the pretransplant livers was examined by gas chromatography and histology. The main constituent of the fatty droplets was determined to be triglyceride. The triglyceride concentration reached a maximum by day 14 and remained constant for an additional 28 days. Histology revealed an absence of necrosis in 14- and 28-day fatty livers but scattered hepatocytic necrosis and inflammation in 42-day fatty livers. After being given cold (UW stored, 4 degrees C) or warm (37 degrees C) ischemia, the fatty liver was orthotopically transplanted into normal ACI rats. The one-week survival of fatty liver grafts after 6, 12, 18, and 24 hr cold preservation was 5/5, 5/6, 3/8, 0/6 for 14-day fatty liver and 5/5, 4/6, 0/8, 0/6 for 42-day fatty livers. The survival of normal liver grafts was 5/5, 6/6, 5/9, 2/8, respectively. Increased survival rate was correlated with the absence of hepatocytic necrosis. The survival after 15 and 30 min warm ischemia prior to transplant was 5/5, 2/6 for normal liver grafts and 4/7, 0/6 for 28-day fatty liver graft, respectively. Fatty livers were less resistant to damage induced by cold or warm ischemia.

Effect of short-term enteral feeding with eicosapentaenoic and γ-linolenic acids on alveolar macrophage eicosanoid synthesis and bactericidal function in rats

OBJECTIVES Because vasoactive eicosanoids derived from arachidonic acid present in immune cell phospholipids promote lung inflammation in critically ill patients, novel experimental diets containing eicosapentaenoic acid from fish oil and gamma-linolenic acid from borage oil have been designed to limit arachidonic acid metabolism. However, excess dietary eicosapentaenoic acid impairs superoxide formation and bacterial killing by immune cells. The present study determined whether short-term enteral feeding with diets enriched with either eicosapentaenoic acid alone or in combination with gamma-linolenic acid would modulate alveolar macrophage eicosanoid synthesis without compromising bactericidal function. DESIGN Prospective, randomized, controlled, blinded study. SETTING University medical center. SUBJECTS Adult male Sprague-Dawley rats. INTERVENTIONS Rats underwent surgical placement of a gastroduodenal feeding catheter and were randomly assigned to receive one of three high-fat (55.2% of total calories), low-carbohydrate diets containing isocaloric amounts of lipids for 4 days. The control diet was enriched with linoleic acid, whereas the two test diets were low in linoleic acid and enriched with either 5 mole % eicosapentaenoic acid alone or in combination with 5 mole % gamma-linolenic acid. Alveolar macrophages were then procured to assess phospholipid fatty acid composition, eicosanoid synthesis after stimulation with endotoxin, superoxide formation and phagocytosis by flow cytometry, and killing of Staphylococcus aureus MEASUREMENTS AND MAIN RESULTS Alveolar macrophage levels of arachidonic acid were significantly (p < .01) lower and levels of eicosapentaenoic and dihomo-gamma-linolenic acids were higher after feeding the eicosapentaenoic and gamma-linolenic acid diet vs. the linoleic acid diet. Ratios of thromboxane B2,/B3, leukotriene B4/B5, and prostaglandin E2/E1 were reduced in the macrophages from rats given either the eicosapentaenoic acid or eicosapentaenoic acid with gamma-linolenic acid diet compared with ratios from rats given the linoleic acid diet. Macrophages from rats given the eicosapentaenoic with gamma-linolenic acid diet released 35% or 24% more prostaglandin E1 than macrophages from rats given either the linoleic acid or the eicosapentaenoic acid diet, respectively. Macrophage superoxide generation, phagocytosis of opsonized zymosan, and killing of S. aureus were similar irrespective of dietary treatment. CONCLUSION Short-term enteral feeding with an eicosapentaenoic acid-enriched or eicosapentaenoic with gamma-linolenic acid-enriched diet rapidly modulated the fatty acid composition of alveolar macrophage phospholipids, promoted a shift toward formation of less inflammatory eicosanoids by stimulated macrophages, but did not impair alveolar macrophage bactericidal function relative to responses observed after feeding a linoleic acid diet.

Fatty acid composition of lung, macrophage and surfactant phospholipids after short-term enteral feeding with n-3 lipids.

Utilization of enteral feeding modalities may prove clinically relevant for rapid modulation of lung phospholipid polyunsaturated fatty acids (PUFA) that serve as substrates for the formation of vasoactive dienoic eicosanoids. We compared the effects of short-term enteral feeding with formulations enriched with either fish (n−3) or corn (n−6) oil PUFA on the fatty acid composition of rat lung, alveolar macrophage and surfactant phospholipids. The diets were infused continuously for 72 h through a surgically placed gastroduodenal feeding catheter by a syringe pump. The n−3 PUFA derived from the fish oil enriched diet were readily incorporated into the phospholipid membranes of the alveolar macrophages, lung tissue and pulmonary surfactant. The relative percentages of the n−3 PUFA were significantly higher and individual and total n−6 PUFA significantly lower in the macrophage, lung and surfactant phospholipids from the n−3-supplemented rats in comparison with those present in the rats infused enterally with the n−6 diet or untreated, chow-fed rats (baseline). In contrast, there was a significant increase in linoleic acid (18∶2n−6) without modification of arachidonic acid (20∶4n−6) in the alveolar macrophages, lung tissue and surfactant from rats enterally receiving the n−6 diet relative to levels measured in the rats at baseline. The results suggest that short-term continuous delivery of n−3-enriched enteral preparations can foster rapid modification of membrane phospholipid PUFA composition of lung tissue, alveolar macrophages and lung surfactant. Utilization of similar infusion modalities to deliver n−3-enriched enteral formulations may prove beneficial to critically ill or postoperative patients with persistent lung inflammation secondary to uncontrolled formation of vasoactive eicosanoids derived from arachidonic acid.

Cyclic vs Continuous Enteral Feeding With ω-3 and γ-Linolenic Fatty Acids: Effects on Modulation of Phospholipid Fatty Acids in Rat Lung and Liver Immune Cells

UNLABELLED Arachidonic acid (AA) present in lung and liver immune cell phospholipids is the precursor of eicosanoids that promote neutrophil margination, leading to tissue injury and inflammation. Administration of novel enteral formulations low in linoleic acid (LA) and containing eicosapentaenoic acid (EPA) from fish oil and gamma-linolenic acid (GLA) from borage oil displaces AA and promotes cell formation of eicosanoids with reduced inflammatory potential. The present study was undertaken to determine whether or not short-term provision of enteral diets containing GLA, EPA, or both in a cyclic fashion modulated the fatty acid composition of rat alveolar macrophage (AM) and liver Kupffer and endothelial (K&E) cell phospholipids in vivo to the extent achieved during continuous feeding. METHODS Rats were isocalorically fed through a gastrostomy catheter for 3 or 6 days with high-fat, low-carbohydrate diets that were enriched with either LA (diet A), EPA (diet B), or EPA + GLA (diet C). The rats were randomized by infusion modality, ie, continuous vs cyclic (14 hours feeding with 10 hours fasting daily) feeding. AM and K&E were isolated and phospholipid fatty acid profiles were determined by gas chromatography. RESULTS The dietary effects on AM and K&E cell phospholipid fatty acids for a given feeding period were not significantly influenced by the infusion modality. AM and K&E cells from rats receiving either diet B or diet C for 3 days had significantly lower AA and LA and higher EPA and dihomo-GLA (DHGLA), respectively, than rats given diet A regardless of the infusion modality. The mole % of EPA and DHGLA in K&E cells were higher after 6 vs 3 days of cyclic feeding with diet C. Using the eicosanoid precursor ratio (EPA + DHGLA/AA), the potential for generation of AA-derived eicosanoids was lower in rats given die B or C vs diet A regardless of infusion modality. DISCUSSION Given the rapid changes in lung and liver immune cell phospholipid fatty acids, short-term provision of EPA and GLA-enriched diets cyclically or continuously may prove clinically relevant for modulating the fatty acid composition and potential eicosanoid formation by these cells.

Metabolism of dietary α-linolenic acid vs. eicosapentaenoic acid in rat immune cell phospholipids during endotoxemia

Short-term (i.e., 3 d) continuous enteral feeding of diets containing eicosapentaenoic (EPA) and γ-linolenic (GLA) polyunsaturated fatty acids (PUFA) to endotoxemic rats reduces the levels of arachidonic acid (AA) and linoleic acid (LA) in alveolar macrophage (AM) and liver Kupffer and endothelial (K&E) cell phospholipids with attendant decreases in prostaglandin formation by these cells in vitro. Diets that contain α-linolenic acid (LNA) as a substrate for endogenous formation of EPA may not be as effective in facilitating these immune cell modifications given the limited activity of Δ6 desaturase. In the present study we compared the effectiveness of an LNA-enriched diet vs. an (EPA+GLA)-enriched diet to displace phospholipid AA from AM and liver K&E cells in vivo in endotoxemic rats fed enterally for 3 or 6 d. We determined the fatty acid composition of AM and K&E cell phospholipids by gas chromatography. We found that AM and K&E cells from rats that had received the EPA+GLA diet for 3 d had significantly (P<0.001) higher mole percentage of EPA and the GLA metabolite, dihomoGLA, than corresponding cells from rats given the LNA diet or a control diet enriched with LA. Rats given the LNA diet had relatively low levels of stearidonic acid, EPA and other n−3 PUFA, while rats given the LA diet had low levels of GLA and dihomoGLA. We conclude that diets enriched with LNA or LA may not be as effective as those enriched with FPA+GLA for purposes of fostering incorporation of EPA or dihomoGLA into and displacement of AA from macrophage phospholipids under pathophysiologic conditions commonly found in acutely septic patients.

Enhanced restoration of adenine nucleotides in rat liver following extended preservation in UW solution by provision of adenosine during reperfusion.

The extensive reduction of adenine nucleotides during preservation coupled with the loss of salvageable precursors during initial reflow may exacerbate recovery of adenine nucleotides in allograft liver following transplantation. The objective of this study was to assess whether provision of adenosine during reperfusion of rat liver stored for 20 hr in University of Wisconsin solution could enhance adenine nucleotide restoration. ATP and total adenine nucleotide content of livers perfused with an oxygenated Krebs/fluorocarbon solution containing 1 mM adenosine were restored to levels in vivo within 30 min of perfusion. Adenine nucleotide recovery in livers perfused without adenosine was only 65% of normal. Acute nutritional deprivation of the donor rats had no effect on adenine nucleotide restoration. These results indicate that a conditional deficiency of intracellular nucleotide precursors exists during initial reperfusion of liver subjected to extended storage in UW solution. Provision of supplemental adenosine to the allograft liver during initial reflow appears warranted to promote full and rapid restoration of adenine nucleotide content following extended preservation ex vivo.

Comparison of growth and fatty acid metabolism in rats fed diets containing equal levels of γ-linolenic acid from high γ-linolenic acid canola oil or borage oil.

We have utilized transgenic technology to develop a new source of γ-linolenic acid (GLA) using the canola plant as a host. The aim of the present study was to compare the growth and fatty acid metabolism in rats fed equal amounts of GLA obtained from the transgenic canola plant relative to GLA from the borage plant. Young male Sprague-Dawley rats (n=10/group) were randomized and fed a purified AIN93G diet (10% lipid by weight) containing either a mixture of high GLA canola oil (HGCO) and corn oil or a control diet containing borage oil (BO) for 6 wk. GLA accounted for 23% of the triglyceride fatty acids in both diets. Growth and diet consumption were monitored every 2–3 d throughout the study. At study termination, the fatty acid composition of the liver and plasma phospholipids was analyzed by gas chromatography. The growth and diet consumption of the HGCO group were similar to the BO group. There were no adverse effects of either diet on the general health or appearance of the rats, or on the morphology of the major organs. There was no significant difference between the diet groups for total percentage of n−6 polyunsaturated fatty acids present in either the total or individual phospholipid fractions of liver or plasma. The relative percentage of GLA and its main metabolite, arachidonic acid, in each phospholipid fraction of liver or plasma were also similar between groups. The percentage of 18∶2n−6 in liver phosphatidylethanolamine and phosphatidylinositol/serine was higher (P<0.05) and 22∶5n−6 was lower in the HGCO group than the BO group. This finding could be attributed to the higher 18∶3n−3 content in the HGCO diet than the BO diet. Results from this long-term feeding study of rats show for the first time that a diet containing transgenically modified canola oil was well-tolerated, and had similar biological effects, i.e., growth characteristics and hepatic metabolism of n−6 fatty acids, as a diet containing borage oil.

Essential fatty acid deficiencies in patients with chronic liver disease are not reversed by short-term intravenous lipid supplementation.

The purpose of this study was to determine theplasma triglyceride and phospholipid fatty acid (FA)composition of severely malnourished patients withchronic liver disease and to examine the effects of parenteral nutrition with a total nutrientadmixture (TNA) on these profiles. Nine consecutivepatients with end-stage chronic liver disease werecompared with 35 patients admitted for elective surgery of upper gastrointestinal malignancy. Baselinelaboratory values and the FA profiles of the plasmatriglyceride and phospholipids were analyzed. FAprofiles were also performed after infusion of a TNAincluding 33 ± 7 g of lipid/24 hr for 7.9 ±4 days in the patients with chronic liver disease.Compared with control patients, the plasma phospholipidfatty acid analysis results (relative mole percentage)of patients with chronic liver disease weresignificantly lower in the two essential FA, linoleicacid (15.4 ± 3.4% vs 20.8 ± 2.9%, P <0.001) and α-linolenic acid (0.02 ± 0.05%vs 0.08 ± 0.10%, P < 0.001). Similar changeswere demonstrated in the FA composition of thetriglyceride fraction. Short-term infusion ofintravenous lipid resulted in a significant increase inlinoleic acid in the triglyceride fraction (9.9 ± 2.8% beforesupplementation vs 20.7 ± 9.4% aftersupplementation, P < 0.01) and a decrease in oleicacid (38.7 ± 5.2% before supplementation vs 29.3± 7.5 after supplementation, P < 0.01). In conclusion, acute and chronicdeficiencies of essential FA occurs in patients withchronic liver disease. The clinical significance ofthese deficiencies is unknown, but they potentially may impact on eicosanoid metabolism. Short-termsupplementation with modest amounts of intravenous lipidhas only a minimal effect on normalization oflonger-chain fatty acids.