Toxicological evaluation of pomegranate seed oil.

In this manuscript, the toxicology and safety of pomegranate seed oil (PSO) was evaluated by in vitro (Ames, chromosomal aberration), and in vivo toxicity tests (acute toxicity and 28-day toxicity in Wistar rats). No mutagenicity of PSO was observed in the absence and presence of metabolic activation up to precipitating concentrations of 5000 microg/plate (Ames test) or 333 microg/ml (chromosome aberration test). The acute oral toxicity study revealed no significant findings at 2000 mg PSO/kg body weight. In the 28-day dietary toxicity study PSO was dosed at concentrations of 0, 10,000, 50,000 and 150,000 ppm, which resulted in a mean intake of 0-0, 825-847, 4269-4330 and 13,710-14,214 mg PSO/kg body weight per day in males-females, respectively. At 150,000 ppm dietary exposure to PSO, a much higher dose than the level of PSO that elicits antidiabetic and anti-inflammatory efficacy, increased hepatic enzyme activities determined in plasma (aspartate, alanine aminotransferase and alkaline phosphatase) and increased liver-to-body weight ratios were observed. However, these effects might be the result of a physiological response to exposure to a very high level of a fatty acid which is not part of the normal diet, and are most likely not toxicologically relevant. The no observable adverse effect level (NOAEL) was 50,000 ppm PSO (=4.3 g PSO/kg body weight/day).

Safety evaluation of polydextrose in infant formula using a suckling piglet model.

Oligosaccharides, the third largest component in human milk, are virtually absent from cow's milk and most infant formula. Prebiotic carbohydrates like polydextrose (PDX) have been proposed as surrogates for human milk oligosaccharides. Safety assessments of novel infant formula ingredients include dose-response experiments in appropriate neonatal animal models such as the suckling pig. To further substantiate the safety of the ingredient, one-day old pigs were fed a cow's milk-based formula supplemented with PDX (1.7, 4.3, 8.5 or 17 g/L) for 18 days (n=13/dose) and compared to appropriate control (unsupplemented formula; n=13) and reference groups (day 0 pigs, and sow-reared pigs; n=13). Growth rate, formula intake, stool consistency, behavior score, blood chemistry and hematology, relative organ weights (% of body weight), tissue morphology (i.e. liver, kidney and pancreas) and pancreas biochemistry did not differ among formula-fed pigs (P>0.1). Polydextrose mimicked other prebiotic carbohydrates and had no adverse effect at the highest tested level 17.0 g PDX/L, equivalent to a dose of 8.35 g/kg of body weight per day.

Transfer of cholesterol and diacylglycerol from lipophorin to Bombyx mori ovarioles in vitro: role of the lipid transfer particle.

The objective of this study was to characterize the transfer of diacylglycerol (DAG) and cholesterol from larval Bombyx mori lipophorin to ovarioles. Transfer studies were carried out by incubating pupal ovarioles (5-day) with [(3)H]-cholesterol and [(3)H]-DAG-labeled lipophorin under different conditions. Transfer of both cholesterol and DAG exhibited hyperbolic dependency on lipophorin concentration with apparent Km values of 0.83 +/- 0.17 mg/ml and 0.74 +/- 0.16 mg/ml, respectively. Pretreatment of ovarioles with anti-lipid transfer particle (LTP) IgG significantly inhibited transfer of labeled DAG to ovarioles (75%) and not cholesterol. Injection of B. mori pupae (day 4) with anti-LTP IgG significantly affected the weight (65%), number of eggs (49%), amount of lipid (74%), and protein (65%) of the adult ovaries. Matured eggs had a very faint yellow color and deformed shape compared to controls. The inhibitory effect demonstrates the active role LTP plays in growth of ovaries, development, and oogenesis. The effect on vitellogenin shortage on egg development and maturation was determined by implanting ovaries in male recipients that lack vitellogenin. An 80% decline in egg production was observed. However, the mature eggs were normal in shape, color, and lipid content. Thus, restricting lipid or protein delivery to developing ovaries would dramatically affect choriogenesis.

Fat metabolism in insects.

The study of fat metabolism in insects has received considerable attention over the years. Although by no means complete, there is a growing body of information about dietary lipid requirements, and the absolute requirement for sterol is of particular note. In this review we (a) summarize the state of understanding of the dietary requirements for the major lipids and (b) describe in detail the insect lipid transport system. Insects digest and absorb lipids similarly to vertebrates, but with some important differences. The hallmark of fat metabolism in insects centers on the lipid transport system. The major lipid transported is diacylglycerol, and it is carried by a high-density lipoprotein called lipophorin. Lipophorin is a reusable shuttle that picks up lipid from the gut and delivers it to tissues for storage or utilization without using the endocytic processes common to vertebrate cells. The mechanisms by which this occurs are not completely understood and offer fruitful areas for future research.

Lipid storage and mobilization in insects: current status and future directions.

In this paper we review the current status of research on fatty acid absorption and conversion to diacylglycerol in the midgut. We further discuss how diacylglycerol may leave the midgut and associate with lipophorin in hemolymph. We review the present understanding of the role of the lipid transfer particle and lipophorin receptors in lipid delivery between lipophorin and tissues. Finally, we discuss recent studies on the mobilization of diacylglycerol from the fat body in response to adipokinetic hormone. Several suggestions for exciting areas of future research are described.

alpha-cyclodextrin extracts diacylglycerol from insect high density lipoproteins.

alpha-Cyclodextrins are water-soluble cyclic hexamers of glucose units with hydrophobic cavities capable of solubilizing lipophiles. Incubating alpha-cyclodextrin with high density lipophorin from Manduca sexta or Bombyx mori resulted in a cloudy, turbid solution. Centrifugation separated a pale yellowish precipitate. Thin-layer chromatography analysis of the lipid extract of the precipitate showed that the major lipid was diacylglycerol, while KBr density gradient analysis of the supernatant demonstrated the presence of a lipid-depleted very high density lipophorin. Transfer of diacylglycerol from lipophorin to cyclodextrin was specific to alpha-cyclodextrin and was not observed with beta- or gamma-cyclodextrins. pH had no effect on diacylglycerol transfer to alpha-cyclodextrin. However, the transfer was strongly dependent on the concentration of alpha-cyclodextrin and temperature. Increasing the concentration of alpha-cyclodextrin in the incubation mixture was associated with the formation of increasingly higher density lipophorins. Thus, at 20, 30, and 40 mm alpha-cyclodextrin, the density of B. mori lipophorin increased from 1.107 g/ml to 1.123, 1. 148, and 1.181 g/ml, respectively. At concentrations greater than 40 mm, alpha-cyclodextrin had no further effect on the density of lipophorin. alpha-Cyclodextrin removed at most 83;-87% of the diacylglycerol present in lipophorin. Temperature played an important role in altering the amount of diacylglycerols transferred to alpha-cyclodextrin. At 30 mm alpha-cyclodextrin, the amount of diacylglycerol transferred at different temperatures was 50% at 4 degrees C, 41% at 15 degrees C, 20% at 28 degrees C, and less than 3% at 37 degrees C. We propose that diacylglycerol transfers to alpha-cyclodextrin via an aqueous diffusion pathway and that the driving force for the transfer is the formation of an insoluble alpha-cyclodextrin-diacylglycerol complex.

Human peritoneal monocytic cells: lipoprotein uptake and foam cell formation.

Human peritoneal cells isolated from dialysis effluent have in vivo maturated human macrophages that could serve as a model for studying lipoprotein metabolism and foam cell formation. We previously characterized the low density lipoprotein (LDL) and acetylated LDL (acetyl-LDL) receptor activities of human total peritoneal cells. Now, we provide evidence that both LDL and acetyl-LDL stimulate acylCoA cholesterol:acyl transferase (ACAT) activity of peritoneal cells. Prolonged incubation of cells with LDL results in suppression of ACAT activity, while incubation with acetyl-LDL results in elevated and sustained enzyme activity. When human peritoneal cells were analyzed using flow cytometry, the cell population showed reactivity for CD2, CD4, CD8, CD20, CD14 and HLA-DR antigens. Purified human peritoneal mononuclear cells degraded LDL. Human peritoneal macrophages formed foam cells when exposed to LDL or acetyl-LDL in culture, and lipid deposition increased with incubation time. Macrophages incubated in the presence of butylated hydroxy toluene and LDL did not form foam cells.

Purification and partial characterization of a lutein-binding protein from the midgut of the silkworm Bombyx mori.

A lutein-binding protein was purified from fifth instar larval midgut of Bombyx mori by a combination of ammonium sulfate fractionation and three chromatographic procedures, gel filtration, chromatofocusing, and anion exchange chromatography. The protein has a pI of 5.4 and an apparent molecular mass of 35,000 Da, as determined by a linear gradient SDS-polyacrylamide gel electrophoresis. The lutein-protein complex is water-soluble and more stable than the carotenoid or protein alone. The carotenoid moiety was identified by thin layer chromatography, light absorption spectroscopy, and high performance liquid chromatography as all-trans-lutein. Lutein is specifically and stoichiometrically bound to the protein, with a ratio of 3 mol of lutein per mol of protein. Binding of lutein (absorption maximum in hexane at 454 nm) to the apoprotein results in a marked red spectral shift of about 38 nm, giving rise to absorption maxima at 432, 462, and 492 nm in 20 mM Tris-HCl, pH 7.0. The lutein-protein complex is characterized by fine spectral structure indicating that lutein is in a relatively rigid environment. This protein is distributed in equal amounts throughout the midgut and in all developmental stages of the larval B. mori.

Lipoprotein metabolism in human peritoneal cells.

The feasibility of using human cells isolated from peritoneal dialysis effluent as a model for studying lipoprotein and cholesterol metabolism was investigated. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (acetyl-LDL) by saturable, high affinity receptor-mediated processes. Positive correlations of the percentage of macrophage cells with degradation rates of LDL (r = 0.742; p < 0.05) and acetyl-LDL (r = 0.931; p < 0.01) indicated that macrophage cells significantly contributed to lipoprotein degradation. LDL receptor-mediated degradation was calcium dependent, and sensitive to pronase and chloroquine treatments. The receptor exhibited specificity for lipoproteins containing apolipoprotein B (apoB) or apolipoprotein E (apoE). Exposure of cells to LDL for 24 hrs significantly down-regulated LDL receptor-mediated degradation. Acetyl-LDL receptor-mediated degradation was calcium independent, inhibited by chloroquine, and was sensitive to pronase and fucoidin treatments. The scavenger receptor exhibited specificity for only acetyl-LDL. These results demonstrate that human peritoneal cells can provide a source of human tissue macrophages suitable for studies of cholesterol and lipoprotein metabolism and offer the opportunity for comparison of metabolic characteristics of in vivo maturated macrophages with available macrophage-like cell lines.

1,25-Dihydroxyvitamin D3-induced HL-60 macrophages: regulation of cholesterol and LDL metabolism.

Differentiation of human promyelocytic leukemic HL-60 cells with 1,25-dihydroxyvitamin D3 (D3) results in macrophages which exhibit specific and saturable receptor-mediated processing of both native and modified low density lipoprotein (LDL). Analysis of binding kinetics demonstrated that macrophages bind LDL and acetyl-LDL with similar affinities, yet possess significantly different numbers of receptors (55 +/- 6 x 10(3) LDL receptors/cell vs 79 +/- 7 x 10(3) acetyl-LDL receptors/cell). D3-induced HL-60 macrophages challenged with LDL or acetyl-LDL exhibited suppression of HMG-CoA reductase activity as well as a significant induction in the incorporation of [14C]oleate into cholesteryl ester compared with macrophages incubated with lipoprotein depleted serum. Maximum increases in ACAT activity were obtained in macrophages incubated with 25-hydroxycholesterol plus LDL or acetyl-LDL. The increase in ACAT activity in macrophages challenged with acetyl-LDL paralleled the increase in cellular cholesterol content and the increase of oil red O lipid stainable material, imparting the macrophages with a foamy appearance. The data indicate that D3-induced HL-60 macrophages are a useful model for the study of lipoprotein--macrophage interactions as related to foam cell development and atherogenesis.

Reduced regulatory capacity of low density lipoproteins from patients with coronary artery disease.

To test the hypothesis whether low density lipoprotein (LDL) poor in cholesteryl ester from patients with coronary artery disease (CAD) express reduced capacity to regulate cellular sterol and lipoprotein metabolism, we compared the abilities of CAD-LDL and control-LDL to suppress receptor-mediated LDL degradation; activate acyl-CoA: cholesterol acyltransferase (ACAT); and regulate sterol synthesis rates in HL-60 promyelocytic leukemic cells. The ratio of apolipoprotein B to cholesteryl ester was 23% higher for CAD-LDL than control-LDL (P less than 0.01), whereby CAD-LDL contained less cholesterol per particle than control-LDL and would be predicted to exert a reduced regulatory effect on sterol and lipoprotein metabolism than control-LDL at the same level of apo B protein. The results indicate that receptor-mediated 125I-LDL degradation rates were 43% higher for cells pre-incubated with CAD-LDL than with control-LDL (P less than 0.04), consistent with CAD-LDL having a lower ability to down-regulate LDL (apo B/E) receptor expression. When LDL degradation rates were expressed as a percentage of the rate of HL-60 cells incubated in lipoprotein-free medium, the mean LDL degradation rate for cells pre-incubated with CAD-LDL was 56% of untreated cells, while for cells incubated with control-LDL the average value was 41%. The data indicate that the suppression of receptor-mediated LDL degradation was proportional to the LDL cholesterol concentration in the medium. ACAT activity was 42% lower in cells pre-incubated with CAD-LDL as compared to control-LDL (P = 0.002), suggesting that the entry of cholesterol into the ACAT substrate pool was lower in cells pre-incubated with CAD-LDL. There was no significant difference in the rate of sterol synthesis from [14C]acetate between cells pre-incubated with CAD-LDL versus control-LDL. The data support the hypothesis that LDL from CAD patients exhibit a decreased ability to down-regulate apo B/E receptor activity which could in part account for the previously observed increase in LDL degradation by mononuclear leukocytes from CAD patients (Shi et al., Atherosclerosis, 85 (1990) 127).

Lipoprotein receptors of HL-60 macrophages. Effect of differentiation with tetramyristic phorbol acetate and 1,25-dihydroxyvitamin D3.

The human promyelocytic leukemic cell line HL-60 is a unique model for studies of the effects of macrophage differentiation on the expression and regulation of lipoprotein receptors. Undifferentiated HL-60 cells express a regulated low density lipoprotein (LDL) receptor and lack the acetylated LDL (acetyl-LDL) scavenger receptor. HL-60 macrophages differentiated with tetramyristic phorbol acetate failed to degrade LDL and acetyl-LDL via receptor-mediated processes. Differentiation with 1,25-dihydroxyvitamin D3 (D3) induced macrophages exhibiting specific saturable receptors for LDL and acetyl-LDL. The LDL receptor of D3-induced macrophages was found to exhibit specificity for apolipoprotein B- and E-containing lipoproteins, to be calcium dependent, and to be inhibited by pronase and chloroquine. Maximal degradation of acetyl-LDL was achieved within 2 days of D3 treatment and was specific for acetyl-LDL, was calcium independent, was inhibited by chloroquine, and was sensitive to pronase and fucoidin treatment. Incubation of D3-induced macrophages with LDL or acetyl-LDL resulted in reductions in sterol synthesis and receptor-mediated degradation of LDL; the scavenger receptor pathway was unaltered. These results demonstrate that D3-induced HL-60 macrophages exhibit patterns of sterol and lipoprotein metabolism and regulation that make them a useful model system for in vitro studies of lipoprotein-macrophage interactions related to foam cell development and atherogenesis.