Suppression of cholesterogenesis and reduction of LDL cholesterol by dietary ginseng and its fractions in chicken liver.

The effects of ginseng root powder and of serially extracted solvent fractions of ginseng on avian hepatic cholesterol metabolism and lipogenesis and on avian serum lipoprotein cholesterol levels were examined. In one study, White Leghorn females were fed for 4 weeks a corn-based diet (control) or an experimental diet in which was incorporated 0.25% Wisconsin ginseng or an equivalent quantity of a serial solvent fraction [petroleum ether (PESF), methyl alcohol (MESF), water (WASF)] or of the residue. beta-hydroxy-beta-methylglutaryl-CoA (HMG-CoA) reductase activity was significantly lower (P less than 0.01) in each of the treatment groups (31-37% of control activity) except that fed the extracted residue (90% of control, N.S.). Cholesterol 7 alpha-hydroxylase activity was lowered in parallel (45-64% of control, P less than 0.01) by all treatments except the residue (100% of control). Also with the exception of the residue treatment, each ginseng treatment effected a lowering of the serum total cholesterol level (67-83% of control, P less than 0.01) and of serum low density lipoprotein cholesterol level (53-81% of control, P less than 0.01). Lipogenic activities and serum triglycerides levels were lowered (P less than 0.01) by two of the ginseng treatments. The PESF treatment was the most effective suppressor of each parameter, 74% and 68% respectively, of the control values. The WASF also had significant impact. Not one of the experimental diets influenced the serum high density lipoprotein level. The PESF, the potent source of suppressors, effected a change in the ratio of low to high density lipoprotein cholesterol from 1.46 (control) to 0.88. The levels of cholesterol and triglycerides in liver under these conditions showed a similar pattern as that of serum. In companion studies, broiler females were fed 0.28% Chinese red ginseng root powder or its various fractions. The results confirmed those recorded above. The factor(s) responsible for lowering the serum total and low density lipoprotein cholesterol levels were generally more concentrated in the PESF and WASF of ginseng and each was significantly more effective than was ginseng root powder. Ginsenosides (saponins) are considered to be the active agents for the suppression of cholesterogenesis and lipogenesis.

Suppression of cholesterol biosynthesis by constituents of barley kernel.

Hepatic beta-hydroxy-beta-methylglutaryl CoA (HMG-CoA) reductase, cholesterol 7 alpha-hydroxylase (7 alpha-hyd), and fatty acid synthetase (FAS) activities and cholesterol levels were determined in chicks fed isonitrogenous corn- and high-protein barley flour (HPBF) based diets. HMG-CoA reductase (-27%), 7 alpha-hyd (-30%), and serum cholesterol (-13%) were reduced, whereas FAS increased (28%) in comparison to a corn-based (control) diet. fractions obtained by serial extractions of HPBF with solvents of increasing polarity were fed at levels equivalent to 20% HPBF in a corn-based diet to female White Leghorn (WHL) chickens for 3 weeks. A petroleum ether-soluble fraction of HPBF produced 3 effects: an increase in body weight (18%), a strong suppression of HMG-CoA reductase (-36%) and FAS (-40%) accompanied by decreases in serum triglyceride (-9%) and cholesterol levels (-23%). The methanol-soluble fraction produced a significant suppression of HMG-CoA reductase (-49%) and serum cholesterol level (-29%), and an increase in FAS activity (95%). These effects were duplicated in 7-week-old broiler chickens which also showed a significant decrease in chol-LDL (low density lipoprotein) levels by these fractions. The factor(s) lowering serum cholesterol concentration was about equally divided between the polar and nonpolar fractions, and each was significantly more effective than the 20% HPBF in the corn-based diet. The observed effects on lipogenesis and cholesterogenesis might be attributed to a number of chemical constituents of HPBF, but cannot be attributed to the water-insoluble plant fibers.

Effect of AMO 1618 on cholesterol and fatty acid metabolism in chickens and rats.

AMO 1618 (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride) was added to corn-soy based diets and fed to 9-week-old female chickens for 3 weeks to measure the inhibition of hepatic beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol 7 alpha-hydroxylase. Dose-related decreases in the activities of these two enzymes were obtained (2.5--15 ppm) of AMO 1618. Decreases in plasma total cholesterol, chol-HDL, and chol-LDL levels were observed, but the decreases in chol-LDL were substantially larger than those of chol-HDL in both chicken and rat. Assays of livers from rats fed 20 ppm AMO 1618 for 3 days had 24% less HMG-CoA reductase activity and 67% less cholesterol 7 alpha-hydroxylase activity than the controls. Plasma cholesterol in these animals was reduced 26%; the ratio of total cholesterol : chol-HDL was reduced from 3.27 to 2.67 and the chol-LDL : chol-HDL ratio was reduced from 1.96 to 1.14 as a result of the relatively brief treatment. Fatty acid synthetase (FAS) and other key lipogenic enzymes increased 1.5--4-fold in both the chicken and rat. The inhibition of HMG-CoA reductase and the induction of FAS by AMO 1618 were tested in vitro, using 10--100 micrograms (28--280 mumoles) for 15 min with isolated hepatocytes from chicken and rat. Linear responses in activity were dose-dependent and increased with duration of incubation (30 micrograms or 85 mumoles AMO 1618, 5--120 min) in both species. The results suggest the compound acts at the cellular level and AMO 1618 appears to possess several properties which recommend it for testing as a cholesterol-lowering agent in humans.

Inhibition of cholesterol and fatty acid biosynthesis in liver enzymes and chicken hepatocytes by polar fractions of garlic.

Different concentrations of polar fractions, methanol-soluble (MESF), or water-soluble (WASF), of 1-8% equivalent to fresh garlic paste were added to yellow corn-soybean based diets and fed to 5-week-old male broiler chickens for 3 weeks to measure the inhibition of hepatic beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7 alpha-hydroxylase (7 alpha-hydroxy) and fatty acid synthetase (FAS). Dose-related decreases in the activities of these enzymes were obtained. Decreases in serum total cholesterol and in low density lipoprotein (LDL) levels were also observed. There was no effect on the level of cholesterol in high density lipoprotein (HDL). The most effective dose for these decreases was found 0.54% (MESF) and 1.2% (WASF) equivalent to 6% of the fresh garlic. The inhibition of HMG-CoA reductase and FAS by 25-300 micrograms of MESF or WASF for 15 min was tested in vitro, in male and female chicken hepatocytes. Inhibitions of activity were dose-dependent and the degree of inhibition increased with duration of incubation (150 micrograms of MESF or WASF 5 to 60 min). Dietary supplementation of odorless WASF of garlic was found to be very effective in lowering the total and LDL cholesterol levels compared to control chickens.

Influence of culture filtrate of Trichoderma viride and barley on lipid metabolism of laying hens.

The suppression of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and cholesterol 7 alpha-hydroxylase, previously noted in studies of the influences of barley and the filtrate of Trichoderma viride culture (CF) on cholesterol metabolism in sexually immature birds, is shown in sexually mature birds. Barley, CF or both were fed in one study from the day of hatching, in another during the period of sexual maturation and, in a third study, CF was fed to mature layers. CF suppressed HMG CoA reductase by 30-50% and cholesterol 7 alpha-hydroxylase by 32-45% when added to the control diet. In birds fed barley rather than corn, the respective activities were 25-36% and 24-31% lower. These effects were expressed in the lowering of plasma cholesterol by 11-36%. Lipogenic activity based on the assays of 4 enzymes was increased 2-3 times by the treatments and plasma triglyceride elevated by 12-86%. The start of egg production by birds fed CF preceded the controls by 17 days. Birds fed barley trailed controls by 11-14 days. CF countered the barley-conditioned delay. Egg yolk cholesterol concentrations were lowered by both treatments. Eggs produced by hens fed barley were lower in weight; CF increased egg and yolk weights. Tissues from birds fed CF or barley for up to 30 weeks appeared to be normal.

Effects of sodium diacetate on the growth, feed efficiency, and intestinal microflora of broilers.

Three levels of sodium diacetate (.625, 1.25, and 3.75 g/kg diet) were incorporated into the diets of day-old broiler chicks. A control ration and a 20 mg/kg aureomycin ration were fed to additional groups of chicks. Each treatment contained 40 chickens at 3 weeks; the number was reduced to 20 chickens at 5 and 8 weeks. At 3 and 8 weeks of age, the large and small intestines of 5 chickens from each treatment were examined for selected microorganisms. Chick growth and feed efficiency were also recorded. The entire experiment was run twice, but in Trial 2 the lowest level of sodium diacetate treatment was replaced by a combination of sodium diacetate (.625 g/kg diet) and aureomycin (20 mg/kg diet). No improvement in the rate of growth was found in the sodium diacetate-treated groups, although additional weight gain was detected in aureomycin-fed groups at 2 weeks of age. Improved feed efficiency in the treated groups (sodium diacetate and aureomycin) was observed in both trials at 2 weeks and at 3 weeks of age in Trial 1. No feed efficiency effects were observed after that time. The sodium diacetate-fed groups showed an increased lactobacilli population in the small intestine along with a concurrent decrease in streptococci. The effect of sodium diacetate in reducing total coliforms in the large intestine was more obvious at 3 weeks than at 8 weeks. Aureomycin appeared to suppress the population of lactobacilli and total coliforms in this study. A combination of sodium diacetate and aureomycin failed to exhibit a synergistic effect either on the growth rate or on the intestinal microflora.

Effect of pH on solubility and ionic state of lipopolysaccharide obtained from the deep rough mutant of Escherichia coli.

The dissociation of the highly aggregated form of lipopolysaccharide (LPS) from Gram-negative bacteria to the monomeric (or soluble) form is though to be the initial step in the activation of responding cells (macrophages, B-cells, neutrophils, monocytes, and endothelial cells) by LPS. This process is presently not adequately understood. Using the equilibrium dialysis apparatus and a highly purified and well-characterized radiolabeled deep rough chemotype LPS ([14C]ReLPS) from Escherichia coli D31m4, we have examined the effect of pH on its solubility (CT) and ionic states in aqueous media. The solubility range of [14C]ReLPS suspended in 50 mM Tris-HCl-100 mM KCl buffer (or 50 mM MES-100 mM KCl buffer at pH 6.5) was determined to be from (2.91 +/- 0.01) x 10(-8) to (4.55 +/- 0.07) x 10(-8) M over a pH range of 6.50-8.20, respectively. These experimental data satisfactorily fitted the curve generated by the solubility equation CT = S0(1 + K5/[H+])/([H+]/K4' + 1), where S0 is the concentration of the tetraanionic ReLPS, K5 is the dissociation constant of the tetraanionic ReLPS in solution, and K4' is the dissociation constant of the trianionic ReLPS at the surface of the solid particles in suspension. The increase in solubility of ReLPS with increase in pH from 7.00 to 8.20 is primarily caused by the formation of the pentaanionic form from the tetraanions. The pK5 (primarily the second dissociation of the 1-phosphate) of ReLPS was determined to be 8.58 from experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)

Physicochemical properties of the lipopolysaccharide unit that activates B lymphocytes.

We have examined the physical state of highly purified deep rough chemotype lipopolysaccharide (ReLPS) from Escherichia coli D31m4 as an aqueous suspension and as complexes with bovine serum albumin min (BSA). The ReLPS suspension showed large ellipsoidal particles 12-38 nm wide and 40-100 nm long. The solubility of this form of ReLPS was determined by equilibrium dialysis experiments to be 3.3 x 10(-8) M at 22 degrees C and 2.8 x 10(-8) M at 37 degrees C in 150 mM Tris-KCl, pH 7.5; 3.0 x 10(-8) M at 37 degrees C in 0.75 mM Tris-KCl, pH 7.5. The BSA-ReLPS complexes were fractionated on a Sephacryl S-200 column to yield peaks I and II with apparent masses of about 240 and 70 kDa, respectively. Peak II was a BSA monomer with estimated BSA:ReLPS molar ratios of 1:1-1:7. The ReLPS suspension and the two complexes were compared as antigens in enzyme-linked immunosorbent assays using three select monoclonal antibodies to lipopolysaccharide. The results were consistent with the high state of disaggregation of the ReLPS in both peaks I and II. Since the ReLPS in these complexes were not visible by electron microscopy, they did not contain vesicles or large particles. All forms of ReLPS tested were capable of stimulating 70Z/3, a lipopolysaccharide-responsive murine pre-B cell line. However, peak II was consistently more stimulatory at very low concentrations than the other preparations. The maximally stimulatory concentration of ReLPS for 70Z/3 cells was 40 ng/ml (1.6 x 10(-8) M) for peak II and 70 ng/ml (2.8 x 10(-8) M) for the ReLPS suspension. As expected, the above concentrations were at or below the solubility of the ReLPS. These results suggested that the highly disaggregated form of ReLPS (possibly the monomer) is the active unit that stimulates the cellular response in 70Z/3 cells.

Suppression of avian hepatic lipid metabolism by solvent extracts of garlic: impact on serum lipids.

The effects of garlic on lipid metabolism were examined in White Leghorn pullets that had been fed for 4 weeks either a control diet based on corn and soybean meal or an experimental diet containing either 3.8% garlic paste, a solvent extract (petroleum ether, methanol and water in sequence) of garlic paste, the residue or commercial garlic oil. Significant decreases in hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (79-83%), cholesterol 7 alpha-hydroxylase (43-51%), fatty acid synthetase (17-29%) and in representative pentose-phosphate pathway (23-39%) activities accompanied the feeding of the petroleum ether-, methanol- and water-soluble fractions of garlic. Garlic paste and oil also suppressed these activities. Significant decreases in serum lipids occurred in response to the feeding of these garlic fractions: serum total cholesterol by 20-25%, low density lipoprotein cholesterol by 28-41% and triglycerides by 10-26%; but high density lipoprotein cholesterol failed to respond to these treatments. The residue remaining after solvent fractionation had little influence on these parameters. These findings were substantiated by a second study in which pullets of a commercial broiler line were fed the garlic fractions. The results confirm that garlic oil and odorous components of garlic lower cholesterol levels. An odorless water-soluble component of garlic also has this effect. The mechanism of the hypocholesterolemic action is at the level of the suppression of cholesterol biosynthesis.

Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages.

Using the equilibrium dialysis apparatus, an aqueous suspension of predominantly aggregated Re lipopolysaccharide (ReLPS) from Escherichia coli D31 m4 (99.9% at 82.5 microM) can be processed to yield a solution of monomeric ReLPS at a saturation concentration of 77 ng/ml (3.4 x 10(-8) M). We compared the in vitro biological activities of these two physically distinct types of ReLPS preparations in two select assays, reaction in the Limulus amebocyte lysate (LAL) assay and induction of Egr-1 mRNA in macrophages. These assays were chosen for their rapid response times and relatively short incubation periods. The monomeric ReLPS was 179- and 1000-fold more active than the aggregated ReLPS preparation in the LAL assay and induction of Egr-1 mRNA by thioglycollate-elicited murine peritoneal macrophages, respectively. These results clearly showed that the monomeric ReLPS is the more active form. The lower biological activities of the aggregated ReLPS preparation might be due to the presence of a small amount of monomeric ReLPS (0.01-0.6%) produced during its preparation and the incubation periods in the biological assays. Thus, aggregated ReLPS may be relatively inactive.

Effect of NADH-X on cytosolic glycerol-3-phosphate dehydrogenase.

At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase purified from muscle cytosol contains tightly bound NADH-X, NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a Ki, in the reaction with NADPH at pH 7.05, in the range of 0.2 microM with respect to the total concentration of ligands ([ADP-ribose] + [NAD] + [NADH-X] = 0. 2 microM). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a Ki considerably higher than 0.2 microM. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bound NADH-X, NADH-X might become attached to the enzyme in vivo where it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.

Ability of cytosolic malate dehydrogenase and lactate dehydrogenase to increase the ratio of NADPH to NADH oxidation by cytosolic glycerol-3-phosphate dehydrogenase.

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.