Furostanol saponins from Tribulus terrestris.

An HPLC-ELSD-ESI-MS method has been developed for the analysis of the steroidal saponins in the aerial parts of Tribulus terrestris. Protodioscin, a new saponin (5,6-dihydroprotodioscin, neoprotodioscin) and their respective sulfates were detected. The structure of the new compound was elucidated on the basis of NMR and ESI-MS spectral analysis.

Identification of Ruscus steroidal saponins by HPLC-MS analysis.

A novel HPLC-UV method has been developed for the fingerprint analysis of the steroidal saponins in the rhizomes of three Ruscus species (Ruscus aculeatus, Ruscus hypoglossum and Ruscus colchicus). Saponins were identified by HPLC-ESI-MS. During the study a new major saponin was detected in the rhizomes of R. hypoglossum and R. colchicus. The structure of the new compound was defined as 1-O-[alpha-L-rhamnopyranosyl-(1-->2)-6-O-acetyl-beta-D-galactopyranosyl]-1beta,3beta,22xi,26-tetrahydroxy-furost-5(6)-en-26-O-beta-D-glucopyranoside (8) by spectral analysis.

Soy proteins and cardiovascular disease.

The soybean diet is the most potent dietary tool for hypercholesterolemia. The United States Food and Drug Administration recently approved the health claim for its role in reducing the risk of coronary disease. The hypocholesterolemic effect is directly correlated to the patient's cholesterolemia, with minimal or no reductions occurring at cholesterol of 6 mmol/L or less, and the most benefit occurring in patients with cholesterol of greater than 7 mmol/L. Hypotheses on the mechanism of action include soy fiber, isoflavones (phytoestrogens), and the protein itself. Although there is no evidence for the effect of fiber, studies with ethanol-extracted soy (devoid of isoflavones) indicated a loss of effect, but the extract itself (isoflavone rich) has no hypocholesterolemic activity. In humans, soy protein activates the low-density lipoprotein (LDL) receptor pathway. Recent data suggest that soy protein subunits, particularly 7S, directly activiate LDL receptors in the human liver, thus providing a novel mechanism of plasma cholesterol reduction different from currently available diets and hypolipidemic drugs.

Soy protein peptides regulate cholesterol homeostasis in Hep G2 cells.

The activation of LDL receptors was described recently in a human hepatoma cell line (Hep G2) exposed both to alpha + alpha' subunits from 7S soy globulin and to Croksoy(R)70, a commercial isoflavone-poor soy concentrate. To assess the final identity of the peptide(s) putatively responsible for the biochemical effect, experiments were performed in Hep G2 cells, exposed either to synthetic peptides corresponding to specific sequences of 7S soy globulin or to peptides from the in vitro digestion of Croksoy(R)70. Moreover, the ability of the whole 7S globulin, its subunits and whole Croksoy(R)70 to interfere in the apolipoprotein B (apo B) secretion in the medium as well as in sterol biosynthesis was evaluated in the same model. Increased (125)I-LDL uptake and degradation vs. controls were shown after Hep G2 incubation with a synthetic peptide (10(-)(4) mol/L, MW 2271 Da) corresponding to positions 127-150 of the 7S globulin. Cells exposed to Croksoy(R)70 enzyme digestion products showed a more marked up-regulation of LDL receptors vs. controls, compared with vs. Hep G2 cells incubated with undigested Croksoy(R)70. Among soy-derived products, only the 7S globulin inhibited apo B secretion and (14)C-acetate incorporation when tested in Hep G2 cells at a concentration of 1.0 g/L. These findings support the hypothesis that if one or more peptides can reach the liver after intestinal digestion, they may elicit a cholesterol-lowering effect. Moreover, the protein moiety, devoid of isoflavone components, is likely to be responsible for this major biochemical effect of soy protein.

Proteins of rat serum: I. Establishing a reference two-dimensional electrophoresis map by immunodetection and microbore high performance liquid chromatography-electrospray mass spectrometry.

In the present investigation, we have identified 56 major spots, or spot rows, corresponding to 22 proteins, in the 2-DE pattern of adult male rats. This was done mainly by applying two complementary techniques, namely immunoblotting and high performance liquid chromatography-mass spectrometry (HPLC-MS) peptide mapping. Glycoproteins were characterized by affinity blotting with six lectins. We have also detailed how rat serum differs from human serum in two main respects: (i) relative abundance of individual proteins, which amounts in some cases to a complete absence in either sample, and (ii) varying molecular parameters for homologous proteins. It was thus possible to establish a first-generation reference map of rat serum proteins, which can be accessed through http://weber.u.washington.edu/ruedilab/aebersold++ +.html. We hope the present database will be a useful reference for the evaluation of changes in serum protein distribution in the course of pharmacological and toxicological studies. The recognition of species-specific proteins appears of special relevance in this respect.

Proteins of rat serum: II. Influence of some biological parameters of the two-dimensional electrophoresis pattern.

This report complements the database already detailed for serum proteins of healthy adult male rats (P. Haynes et al., Electrophoresis 1998, 19, 1484-1492). The influence on the two-dimensional electrophoresis (2-DE) pattern of certain physiological conditions (sex, age) was studied as well as of changes in thyroid metabolism. We have extended the information about the major components of rat serum by identifying the proteins typical for the response to acute inflammation. Analyzing 27 spots, six proteins not found in control sera could be recognized; migration at overlapping or close positions with five already characterized proteins was observed for some. A compilation of all our rat data can be accessed through: http://weber.u.washington.edu/ruedilab/ aebersold.html.

7S globulin from soybean is metabolized in human cell cultures by a specific uptake and degradation system.

We examined the biological fate of 7S globulin from soybean in a hepatoma cell line (Hep G2) and in human skin fibroblasts (HSF) to gain new insights into the 7S globulin cell process, the final effect of which is an enhanced expression of the LDL-receptor. The ability of 7S globulin to bind and to be internalized and degraded by both cell types was investigated under different experimental conditions. In all cases, specific uptake (binding + internalization) and degradation of 125I-7S globulin were curvilinear functions of substrate concentration at 37 degrees C. The two processes were saturated at around 80 mg/L, a concentration at which an up-regulation of LDL-receptor was previously reported. The specific uptake of 125I-7S globulin at 37 degrees C was a curvilinear function of time, and achieved equilibrium after 6 and 12 h in HSF and Hep G2 cells, respectively. Binding experiments, conducted at 4 degrees C in Hep G2 cells, showed a specific and saturable association of 7S globulin to the cell membrane. Linear Scatchard analysis demonstrated a single population of binding sites. The amount of 7S globulin bound at saturation (Bmax) was about 2.73 mg/L, with an apparent Kd of 21 micromol/L, assuming 175 kDa as the 7S globulin molecular weight. SDS-PAGE of Hep G2 membrane proteins incubated with 125I-7S globulin revealed a specific interaction of 7S globulin with a cell protein component with molecular weight between 14 and 21 kDa. Further studies are needed to ascertain whether this interaction is directly or indirectly related to the observed stimulation of the LDL-receptor.

Effects of sub-chronic exposure to SO2 on lipid and carbohydrate metabolism in rats.

Sulfur dioxide (SO2) is a ubiquitous air pollutant, present in low concentrations in the urban air, and in higher concentrations in the working environment. While toxicological reports on SO2 have extensively dealt with the pulmonary system, essentially no data are available on the effects of chronic exposure to this pollutant on intermediary metabolism, although some biochemical changes in lipid metabolism have been detected. The present investigation was aimed at evaluating the effects of sub-chronic exposure to SO2 on concentrations of serum lipids/lipoproteins and on glucose metabolism, in animal models of hypercholesterolemia and diabetes. A specially designed control-inert atmosphere chamber was used, where male Sprague-Dawley rats fed on either standard or cholesterol enriched (HC) diets, as well as streptozotocin diabetics, were exposed to SO2 at 5 and 10 ppm, 24 h per day for 14 days. In rats, both on a standard diet and on a HC regimen, SO2 exposure determined a significant dose-dependent increase in plasma triglycerides, up to +363% in the 10 ppm HC exposed animals. This same gas concentration significantly reduced HDL cholesterol levels. In contrast, exposure of diabetic animals to 10 ppm SO2 resulted in a fall (-41%) of plasma and liver triglycerides and in a concomitant increase (+62%) of plasma HDL cholesterol. This discrepancy could apparently be related to diverging effects of SO2 exposure on plasma insulin levels in the different animal groups. Kinetic analyses of triglyceride synthesis carried out in rats on a standard diet revealed, in exposed animals, a significant reduction in the secretory rate, in spite of the concomitant hypertriglyceridemia. These findings suggest that SO2 exposure can markedly modify major lipid and glycemic indices, also indicating a differential response in normo/hyperlipidemic versus diabetic animals.

Effect of gemfibrozil treatment in hypercholesterolemia on low density lipoprotein (LDL) subclass distribution and LDL-cell interaction.

Gemfibrozil, a widely used fibric acid derivative, corrects hypercholesterolemia in a non-negligible fraction of patients. To investigate the mechanism of the cholesterol-lowering activity of fibric acids, a study was performed in 12 type IIa hyperlipidemic patients treated with gemfibrozil for 12 weeks. Changes in low density lipoprotein (LDL) structure and composition, agonist capacity of LDL against the LDL-receptor in human skin fibroblasts, LDL-receptor activity in mononuclear cells, lecithin:cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) activity, were evaluated. Plasma total and LDL cholesterol levels decreased by 17% and 20% after 12 weeks of treatment, the reduction being directly correlated with the baseline levels (r = 0.75 and 0.78, respectively). The mean LDL diameter increased significantly, from 25.5 to 26.1 nm, while the relative content of small LDL particles (< 25.1 nm) increased from 23.4% to 32.8% of total LDL. Neither the apolipoprotein (apo) B secondary structure nor the affinity of LDL for the LDL-receptor of fibroblasts were affected. The LDL-receptor activity in patients' mononuclear cells increased 3-fold, the rise being unrelated to the plasma cholesterol reduction. LCAT activity did not change, while CETP activity was reduced by 25% (P = 0.13) after treatment. These findings indicate that gemfibrozil causes significant changes in LDL structure that do not, however, affect the LDL interaction with peripheral cells.

Soy and cholesterol reduction: clinical experience.

A role of vegetable proteins in reducing coronary artery disease risk was postulated as long ago as 1909 in Russia by Ignatowski. The protein hypothesis of atherosclerosis was pursued by many investigators, who studied the possible role of animal vs. vegetable protein in modifying concentrations of plasma lipids and thus cardiovascular disease risk. Over the past 20 y, our research group has examined the potential of a diet based on vegetable protein (in most cases, textured vegetable protein, or TVP) to modify plasma lipid concentrations. Textured products allow administration of a large percentage of protein (up to 50-60% in the product) and are available in a variety of food items. We studied > 1000 patients. An extensive review of the literature indicates that similar findings have been reported by others when administering TVP or TVP-like items to subjects with well-characterized hypercholesterolemia (Fredrickson type II). Data are less consistent for treatment of patients with marginal hypercholesterolemia or hypercholesterolemia already corrected by a standard diet before administration of soy products. The TVP diet, is, however, effective when normolipidemic individuals are made hypercholesterolemic by dietary cholesterol administration. These and other findings suggest that, in man, similar to experimental animals, soy protein may in some way up-regulate LDL receptors depressed by hypercholesterolemia or by dietary cholesterol administration.

Low density lipoprotein receptor activity is modulated by soybean globulins in cell culture.

The effects of major storage globulins from soybean on cholesterol homeostasis were investigated in vitro and in vivo systems. The low density lipoprotein (LDL) uptake and degradation was studied both in human skin fibroblasts (HSF) and in a human hepatoma cell line (Hep G2). In Hep G2 cells a dose-dependent increase of both uptake and degradation of 125I-LDL was induced by the 7S globulin, whereas the 11S globulin exerted a lesser effect that was not dose-related. In HSF cells the 11S globulin increased the uptake of 125I-LDL to a greater extent than did 7S globulin; in this cell line, LDL degradation was not stimulated by either of the globulins. Rats fed a casein-cholesterol diet were treated daily with the 11S or 7S globulins for 2 wk. The administration of soybean globulins significantly reduced cholesterolemia (-35 and -34% with 7S and 11S globulins, respectively, vs. controls). Liver membrane preparations from the casein-cholesterol-fed rats showed a nonsignificant increase in the maximal binding of labeled cholesterol-rich lipoprotein fraction (beta-VLDL) to high affinity receptors.

Hyperresponsiveness to dietary cholesterol in inbred rabbits is not associated with enhanced reduction in binding of beta-VLDL to liver membranes.

In two inbred strains of rabbits with high or low response of serum cholesterol to dietary cholesterol, binding of rabbit beta-VLDL to hepatic membrane preparations was determined. The objective was to test the hypothesis that after cholesterol feeding, hyperresponders show a more dramatic reduction in hepatic apolipoprotein (apo) B/E receptors, which may explain the development of the high degree of cholesterolemia in these animals. The number of hepatic high affinity receptors for beta-VLDL in hyperresponders fed a diet without added cholesterol was, on average, 20% lower than that in hyporesponders. After the addition of increasing amounts of cholesterol to the diet, liver cholesterol concentrations were elevated to a greater extent in hyper- than in hyporesponsive rabbits. Liver free cholesterol concentrations were negatively associated with maximal binding of beta-VLDL to liver membranes. With increasing liver free cholesterol concentrations, maximal binding was less effectively depressed in hyper- than hyporesponders. We conclude that in the inbred strains of rabbits, hyperresponsiveness to dietary cholesterol is not caused by enhanced depression of hepatic apo B/E receptors.

Mechanisms of lipid-lowering agents.

Lipid-lowering agents are used with the purpose of ameliorating hyperlipoproteinemias, in order to prevent arterial disease. Lipid-lowering drugs can be classified into absorbable agents and into nonabsorbable compounds, acting within the gastrointestinal lumen. Absorbable drugs (fibric acids, nicotinic acid, probucol, HMG-CoA reductase inhibitors) reduce plasma very-low-density lipoproteins (VLDL) and/or low-density lipoproteins (LDL) by a variety of mechanisms. Fibric acids, in particular, act by stimulating the catabolism of VLDL and also, as a consequence, improving LDL delipidation, thus favoring receptor uptake. Nicotinic acid and acipimox interfere with the biosynthesis of LDL and can also improve the clearance of VLDL/LDL. Probucol acts by a newly described mechanism, i.e. accelerating reverse transport of cholesteryl esters from high-density lipoproteins to lower-density lipoproteins. Finally, HMG-CoA reductase inhibitors, interfering with the biosynthesis of cholesterol, can induce an increased expression of liver high-affinity lipoprotein receptors. Nonabsorbable agents (anion-exchange resins, neomycin, beta-sitosterol) interrupt the recirculation of bile acids and/or reduce the absorption of cholesterol with the gut. They display a selective activity on hypercholesterolemia, again by increasing LDL receptor expression. The choice of one or more lipid-lowering agents will depend upon the patient's phenotype, determining responsiveness to the pharmacological treatment.

Differential effects of beclobrate on lipid/lipoprotein distribution in normal and hypercholesterolemic rats.

Beclobrate, a new fibric acid derivative, displays remarkable lipid lowering activity in rodents. In order to evaluate changes in the distribution and liver handling of lipoproteins, beclobrate was tested in rats fed on a normal or hypercholesterolemic diet. On the normal diet, beclobrate lowered total plasma cholesterol by 22-33.4% (10-50 mg/kg); the cholesterol reduction occurred mainly in high density lipoproteins (HDL) (by 24-45% with the three tested doses). The metabolic clearance of 125I-labelled beta-very low density lipoproteins (beta-VLDL) injected into these animals almost doubled (0.20 1/h vs. 0.13 1/h in controls) after treatment with 20 mg/kg of beclobrate. In addition, beclobrate administration dramatically increased the activity of the high-affinity receptors for beta-VLDL in isolated liver membranes (Bmax: 208 +/- 17.6 vs. 146 +/- 2.6 ng/mg of protein for controls). On the hypercholesterolemic diet, beclobrate treatment (50 mg/kg) was associated with a 25% reduction in total cholesterol accompanied, however, by a 166% rise in HDL cholesterol. In these animals, the composition of VLDL, typically cholesterol-enriched, became close to normal. The increased HDL was characterized by a remarkable enrichment with particles containing apolipoprotein E (apo E), which is compatible with either an improved peripheral cholesterol removal or an enhanced direct secretion of apo E. The two models offer different opportunities for evaluating the mechanism of action of this new lipid lowering agent. Lipoprotein catabolism and receptor-mediated clearance were characteristically improved in normolipidemic rats whereas major effects on HDL metabolism could be demonstrated in hypercholesterolemic rats.

Dietary animal proteins and cholesterol metabolism in rabbits.

The effect in rabbits of giving isonitrogenous purified diets containing casein, ovalbumin, fish protein, milk-whey protein and soya-bean protein were compared. The diets were balanced for cholesterol and for the amount and type of fat. When incorporated into low-cholesterol diets (0.08 g cholesterol/kg), casein, ovalbumin and soya-bean protein produced similar levels of serum cholesterol. With a high background of dietary cholesterol (1.5 g/kg), serum cholesterol concentrations increased with soya-bean protein, whey protein, casein and fish protein, in that order. Thus, the hypercholesterolaemic effect of casein in carefully balanced diets was only seen against a high-cholesterol background. The development of hypercholesterolaemia produced by giving fish protein was different from that produced by casein. First, less cholesterol accumulated in the very-low-density-lipoprotein fractions and more in the lipoproteins of higher density with fish protein than with casein. Second, fish protein, unlike casein, did not increase liver cholesterol. Third, transfer of rabbits from a diet containing soya-bean protein to one containing casein resulted in an immediate marked depression in neutral steroid and bile acid excretion in faeces. However, when rabbits were fed on the diet with fish protein after the diet with soya-bean protein, there was no significant depression in neutral steroid output and the depression in bile acid output was delayed. The present study suggests that different animal proteins cause hypercholesterolaemia by different mechanisms.

Dietary animal proteins and cholesterol metabolism in rats.

The effects on cholesterol metabolism in rats of diets containing various animal proteins or soy protein were studied. The animal proteins tested were casein, whey protein, fish protein, hemoglobin, plasma proteins, ovalbumin, egg-yolk protein, beef protein and chicken-meat protein. The semi-purified diets were isonitrogenous and balanced for residual fat and cholesterol in the protein preparations. The nature of the dietary protein had no effect on serum cholesterol concentration. Group mean liver cholesterol concentration was increased and fecal excretion of bile acids was decreased by all animal proteins when compared with soy protein. This study suggests that carefully balancing diets for components other than protein in the protein preparations prevents protein effects on serum cholesterol in rats but not on liver cholesterol and bile acid excretion.