Isoflavonoids do not inhibit in vivo lipid peroxidation in subjects with high-normal blood pressure.

The isoflavonoids genistein and daidzein have been shown to have antioxidant activity in vitro, but their effects on in vivo oxidation have not been assessed. The newly described F2-isoprostanes are believed to currently represent the best available marker of in vivo lipid peroxidation. Therefore we have assessed the effects of a 55 mg daily isoflavonoid supplement on urinary F2-isoprostane concentrations in subjects with high-normal blood pressure (BP). A total of 59 subjects completed an 8-week parallel design, randomized, double blind, and placebo-controlled study. F2-isoprostanes, isoflavonoids and creatinine were measured in 24-h urine samples taken at baseline and at the end of the intervention. There were significant increases in urinary excretion of genistein (5.22+/-0.75 mg/day, P < 0.0001) and daidzein (2.53+/-0.43 mg/day, P < 0.0001) in the group taking the isoflavonoid supplement. Creatinine excretion was significantly correlated with F2-isoprostanes at baseline (r = 0.45, P < 0.01). After adjustment for baseline values, there was no significant difference between groups in creatinine adjusted post-intervention F2-isoprostane concentrations (P = 0.74). In addition, changes in genistein and daidzein excretion were not significantly correlated with changes in F2-isoprostanes in the isoflavonoid treatment group. These results are not consistent with the suggestion that the two soy derived isoflavonoids have in vivo antioxidant activity at a level of intake achievable by dietary means and in subjects with high-normal BP.     

Effects of isoflavonoids on blood pressure in subjects with high-normal ambulatory blood pressure levels: A randomized controlled trial

Vegetarian diets lower blood pressure (BP), but attempts to identify dietary components responsible have been unsuccessful. Isoflavonoids are commonly consumed as part of vegetarian diets. The objective of this study was to assess the effect of isoflavonoid supplementation on BP. Fifty-nine subjects with high-normal range systolic BP completed a randomized, double blind, placebo-controlled trial of two-way parallel design and 8 weeks duration. One tablet containing 55 mg of isoflavonoids, including 30 mg of genistein, 16 mg of biochanin A (a genistein precursor), 1 mg of daidzein, and 8 mg of formononetin (a daidzein precursor), or one placebo tablet, was taken daily with the evening meal. Significant increases in urinary excretion of genistein (5.22 mg/day, 95% CI: 3.72, 6.72) and daidzein (2.53 mg/day, 95% CI: 1.66, 3.40) were observed in the group taking the isoflavonoid supplement. There were no significant changes in isoflavonoid excretion in the placebo group. Clinic BP was measured at two visits, and ambulatory BP monitoring was performed over one 24-h period, at baseline and postintervention. There was no significant difference between groups, after adjustment for baseline values, in postintervention clinic supine BP (systolic 1.2 mm Hg, 95% CI: −2.3, 4.7; diastolic 0.6 mm Hg, 95% CI: −1.9, 2.5), clinic erect BP (systolic 1.7 mm Hg, 95% CI: −4.0, 8.4; diastolic 0.4 mm Hg, 95% CI: −2.4, 3.2), or 24-h ambulatory BP (systolic −1.4 mm Hg, 95% CI: −4.4, 1.6; diastolic −0.8 mm Hg, 95% CI: −2.3, 0.7). Adjustment for age, gender, and weight change did not alter the result. Therefore, these results do not support the hypothesis that isoflavonoids, and genistein in particular, are major contributors to the BP lowering effect of vegetarian diets.     

Low density lipoprotein oxidation, antioxidants, and atherosclerosis

Oxidized low density lipoproteins (LDLs) are believed to be the most atherogenic form of LDL. However, although a number of experimental data support this concept, the protective role of antioxidants that may prevent LDL oxidation in atherosclerosis is only partially confirmed by studies in humans. Observational and epidemiologic data as well as randomized trials failed to provide clear-cut indications because of mixed results on the protective role of antioxidants against cardiovascular diseases. In spite of the lack of a general consensus, recent data reinforce the concept that a regular intake of antioxidants present in food blocks the progression of atherosclerosis and that the reduced oxidisability of LDL may represent a good marker to follow the action of antioxidants. When it becomes possible to monitor the efficacy of any antioxidant therapy with validated markers of oxidation, the potential influence of vitamins and antioxidants on coronary artery disease will eventually be resolved.     

Isoflavonoid Compounds Extracted from Pueraria lobata Suppress Alcohol Preference in a Pharmacogenetic Rat Model of Alcoholism

The extract from an edible vine, Pueraria lobata , has long been used in China to lessen alcohol intoxication. We have previously shown that daidzin, one of the major components from this plant extract, is efficacious in lowering blood alcohol levels and shortens sleep time induced by alcohol ingestion. This study was conducted to test the antidipsotropic effect of daidzin and two other major isoflavonoids, daidzein and puerarin, from Pueraria lobata administered by the oral route. An alcohol-preferring rat model, the selectively-bred P line of rats, was used for the study. All three isoflavonoid compounds were effective in suppressing voluntary alcohol consumption by the P rats. When given orally to P rats at a dose of 100 mg/kg/day, daidzein, daidzin, and puerarin decreased ethanol intake by 75%, 50%, and 40%, respectively. The decrease in alcohol consumption was accompanied by an increase in water intake, so that the total fluid volume consumed daily remained unchanged. The effects of these isoflavonoid compounds on alcohol and water intake were reversible. Suppression of alcohol consumption was evident after 1 day of administration and became maximal after 2 days. Similarly, alcohol preference returned to baseline levels 2 days after discontinuation of the isoflavonoids. Rats receiving the herbal extracts ate the same amounts of food as control animals, and they gained weight normally during the experiments. When administered orally, none of these compounds affected the activities of liver alcohol dehydrogenase and aldehyde dehydrogenase. Therefore, the reversal of alcohol preference produced by these compounds may be mediated via the CNS. Data demonstrate that isoflavonoid compounds extracted from Pueraria lobata is effective in suppressing the appetite for alcohol when taken orally, raising the possibility that other constituents of edible plants may exert similar and more potent actions.     

Lipid disorders in type 1 diabetes

Patients with type 1 diabetes (T1D) also present with lipid disorders. Quantitative abnormalities of lipoproteins are observed in T1D patients with poor glycaemic control (increased plasma triglycerides and low-density lipoprotein [LDL] cholesterol) or nephropathy (increased triglycerides and LDL cholesterol, low level of high density lipoprotein [HDL] cholesterol). In cases of T1D with optimal glycaemic control, plasma triglycerides and LDL cholesterol are normal or slightly decreased, while HDL cholesterol is normal or slightly increased. Several qualitative abnormalities of lipoproteins, which are potentially atherogenic, are observed in patients with T1D, even in those with good metabolic control. These abnormalities include increased cholesterol-to-triglyceride ratios within very low-density lipoprotein (VLDLs), increased triglycerides in LDLs and HDLs, compositional changes in the peripheral layer of lipoproteins, glycation of apolipoproteins, increased oxidation of LDLs and an increase in small, dense LDL particles. These qualitative changes in lipoproteins are likely to impair their function. In vitro, VLDLs and LDLs from patients with T1D induced abnormal responses in the cellular cholesterol metabolism of human macrophages. HDLs from patients with T1D are thought to be less effective in promoting cholesterol efflux from cells, and have been shown to have reduced antioxidative and vasorelaxant properties. These qualitative abnormalities are not fully explained by hyperglycaemia and may be partly due to peripheral hyperinsulinaemia associated with subcutaneous insulin administration. However, the precise consequences of these qualitative lipid changes on the development of cardiovascular disease in T1D are, as yet, unknown.     

Probucol selectively increases oxidation of atherogenic lipoproteins in cholesterol-fed mice and in Watanabe heritable hyperlipidemic rabbits

The anti-atherogenic and cholesterol-lowering drug probucol (0.5–1%) or quercetin (1%), a natural antioxidant, was given to cholesterol-fed (1.5%) mice for a period of 6 weeks and to Watanabe heritable hyperlipidemic (WHHL) rabbits for a period of 8 weeks to investigate the oxidative changes in plasma and lipoproteins. Oxidation was measured as the total amount of malondialdehyde (nmol MDA/g protein) by a very specific MDA-HPLC method. A large and significant increase in MDA was seen in LDL from probucol treated WHHL rabbits (1778.7±585.5 nmol/g vs. 394.4±144.5 nmol/g, P<0.001) and cholesterol-fed mice (579.7±47.3 nmol/g vs. 408.1±85.8 nmol/g, P<0.05) as compared to controls while LDL cholesterol was lowered (WHHL rabbits: P<0.05; mice: P<0.01). In WHHL rabbits VLDL oxidation was determined additionally, and also revealed a large increase in the probucol group (2102.7±1156.1 nmol/g vs. 455.0±207.8 nmol/g, P<0.01). In contrast, the oxidation of plasma and HDL from probucol treated animals was not statistically significantly increased, implying that probucol mediates a selective oxidation of atherogenic cholesterol-transporting lipoproteins. Quercetin treated animals did not show increased oxidation of LDL (and VLDL in rabbits) and cholesterol levels were not decreased. Furthermore, no protective antioxidant effect of quercetin was seen. In conclusion, the results suggest that a prooxidant mechanism rather than antioxidative effects influences lipoprotein metabolism in these animals. It is hypothesized that the oxidation of lipoproteins might be a physiological mechanism performed by macrophages or other cells for uptake and degradation (by macrophages and liver) of excessive amounts of LDL or VLDL and that probucol oxidizes atherogenic lipoproteins and thereby leads to a decrease in cholesterol levels.     

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [¹⁴C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [¹⁴C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His⁴²⁶ because Cp_H426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.     

Effect of soy protein foods on low-density lipoprotein oxidation and ex vivo sex hormone receptor activity--a controlled crossover trial

Plant-derived estrogen analogs (phytoestrogens) may confer significant health advantages including cholesterol reduction, antioxidant activity, and possibly a reduced cancer risk. However, the concern has also been raised that phytoestrogens may be endocrine disrupters and major health hazards. We therefore assessed the effects of soy foods as a rich source of isoflavonoid phytoestrogens on LDL oxidation and sex hormone receptor activity. Thirty-one hyperlipidemic subjects underwent two 1-month low-fat metabolic diets in a randomized crossover study. The major differences between the test and control diets were an increase in soy protein foods (33 g/d soy protein) providing 86 mg isoflavones/2,000 kcal/d and a doubling of the soluble fiber intake. Fasting blood samples were obtained at the start and at weeks 2 and 4, with 24-hour urine collections at the end of each phase. Soy foods increased urinary isoflavone excretion on the test diet versus the control (3.8+/-0.7 v 0.0+/-0.0 mg/d, P < .001). The test diet decreased both oxidized LDL measured as conjugated dienes in the LDL fraction (56+/-3 v 63+/-3 micromol/L, P < .001) and the ratio of conjugated dienes to LDL cholesterol (15.0+/-1.0 v 15.7+/-0.9, P = .032), even in subjects already using vitamin E supplements (400 to 800 mg/d). No significant difference was detected in ex vivo sex hormone activity between urine samples from the test and control periods. In conclusion, consumption of high-isoflavone foods was associated with reduced levels of circulating oxidized LDL even in subjects taking vitamin E, with no evidence of increased urinary estrogenic activity. Soy consumption may reduce cardiovascular disease risk without increasing the risk for hormone-dependent cancers.     

Effects of an isoflavone-free soy diet on ovarian hormones in premenopausal women.

Soy intakes have been associated with reduced rates of breast cancer in some Asian populations. The isoflavones daidzein and genistein and other components of soybeans may modulate endocrine function and lead to beneficial health effects. This study determined the effects of a soy diet containing minimum amounts of isoflavones on circulating levels of ovarian hormones and gonadotropins. Nine healthy, regularly cycling women consumed a constant soya-containing diet on a metabolic unit starting on day 2 of a menstrual cycle until day 2 of the next cycle. The soy diet was calculated to maintain constant body weight and included a 36-oz portion of soymilk that provided 334 kilocalories and less than 5 mg/day of total isoflavones. The energy distribution of the soy diet was 35.9% fat, 14.0% protein, and 49.8% carbohydrate whereas the home diets averaged 39% fat, 16.6% protein, and 42.5% carbohydrate. For the group, the soya diet provided more carbohydrate (P = 0.002) and less protein (P = 0.005) than the home diets. Daily consumption of the soya diet reduced daily circulating levels of 17beta-estradiol over the entire menstrual cycle by 20% (P < 0.01, paired t test, two-tailed) and progesterone by 33% (P < 0.0001) compared with levels during the home diet period, but had no effect on LH, FSH, or sex hormone-binding globulin. The decreases in follicular phase 17beta-estradiol during the soy diet can be accounted for by changes in energy intakes, nutrient density, and fiber intake, whereas changes in luteal phase 17beta-estradiol were most strongly associated with differences in fiber intake. Changes in progesterone levels were most strongly associated with changes in protein intake and much less with other nutrients. Isoflavones were not detectable in plasma and urine during either the soy or home diet periods. These results suggest that at least under the conditions of this study, a soy diet with low levels of isoflavones and low energy intake from protein can reduce circulating ovarian steroids without altering gonadotropins. Our results are consistent with previous studies showing decreased ovarian hormone levels and decreased risk of breast cancer in populations consuming soya diets and an inverse relationship between animal protein intake and breast cancer risk and, therefore, may have implications for breast cancer prevention.     

Effect of genistein against copper-induced lipid peroxidation of human high density lipoproteins (HDL)

Several studies have demonstrated that the isoflavone genistein exerts a protective effect against lipid peroxidation of low density lipoproteins (LDL). Aim of our study was to investigate whether genistein protects high density lipoproteins (HDL), isolated from normolipemic subjects, against Cu(++)-induced lipid peroxidation. Our results demonstrated that genistein exerts an inhibitory effect against Cu(++)-induced lipid peroxidation of HDL, as shown by the lower increase in the levels of conjugated dienes in lipoproteins oxidized after preincubation with different concentrations of genistein (0.5-2.5microM). Moreover the analysis of fluorescence emission spectra of tryptophan (Trp) and Laurdan (6-dodecanoyl-2-dimethyl-aminonaphthalene) demonstrated that genistein prevents the alterations of apoprotein structure and physico-chemical properties, associated with Cu(++)-triggered lipid peroxidation of lipoproteins. The protective effect exerted by genistein against oxidative damage of lipoproteins was realized at concentrations similar to those observed in plasma of human subjects consuming a traditional soy diet or receiving a soy supplement. Therefore, we suggested that antioxidant activity exerted by genistein against lipid peroxidation of HDL in vitro could be of physiological relevance.     

Induction of Bradyrhizobium japonicum common nod genes by isoflavones isolated from Glycine max

The early events in legume nodulation by Rhizobium spp. involve a conserved gene cluster known as the common nod region. A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium japonicum nodDABC-lacZ translational fusion was constructed and used to monitor nod gene expression in response to soybean root extract. Two inducing compounds were isolated and identified. Analysis using ultraviolet absorption spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 4′,7-dihydroxyisoflavone (daidzein) and 4′,5,7-trihydroxyisoflavone (genistein). Induction was also seen with some, but not all, of the flavonoid compounds that induce nod genes in fast-growing Rhizobium strains that nodulate clover, alfalfa, or peas. When pEA2-21 was introduced into Rhizobium trifolii, it was inducible by flavones but not by daidzein and genistein. In Rhizobium fredii, pEA2-21 was induced by isoflavones and flavones. Thus, the specificity of induction appears to be influenced by the host-strain genome.     

Effects of isolated isoflavonoids on lipids, lipoproteins, insulin sensitivity, and ghrelin in postmenopausal women

The low cardiovascular risk in Asian women has been thought to result from high isoflavonoid intake. In a double-blind, randomized, placebo-controlled trial, we studied the effects of isolated isoflavonoids (114 mg/d) on lipids, lipoproteins, insulin sensitivity, and ghrelin in 56 nondiabetic postmenopausal women with a history of breast cancer. Isoflavonoid or placebo tablets were given for 3 months, and the treatment regimens crossed over after a 2-month washout period.The concentrations of total cholesterol, high- and low-density lipoprotein cholesterol, triglycerides, apolipoproteins B and A1, and lipoprotein (a) were not affected by isoflavonoids. However, during the isoflavonoid regimen, women with low-density lipoprotein cholesterol level above the median (4.20 mmol/liter) showed a rise [0.65 +/- 0.60 (sd) mmol/liter], which was statistically different from the fall during the placebo regimen (-0.45 +/- 0.67 mmol/liter, P = 0.009).Isoflavonoids did not affect insulin sensitivity as assessed by an oral 2-h glucose tolerance test (75 g). Changes in ghrelin levels differed (P = 0.048) during the isoflavonoid (-7.1 +/- 151 micromol/liter) and placebo regimens (+47.9 +/- 198 micromol/liter).In conclusion, we found no effects of isolated isoflavonoids on lipids, lipoproteins, or insulin sensitivity in postmenopausal women, implying no vascular benefit. Isoflavonoids may reduce ghrelin levels and thus hunger and weight.     

Ghrelin interacts with human plasma lipoproteins

Ghrelin, a peptide hormone produced predominantly by the stomach, stimulates food intake and GH secretion. The Ser(3) residue of ghrelin is mainly modified by a n-octanoic acid. In the human bloodstream, ghrelin circulates in two forms: octanoylated and desacylated. We previously demonstrated that ghrelin is desoctanoylated in human serum by butyrylcholinesterase (EC 3.1.1.8) and other esterase(s), whereas in rat serum, only carboxylesterase (EC 3.1.1.1) is involved. The aims of this study were to determine the role of lipoprotein-associated enzymes in ghrelin desoctanoylation and the role of lipoproteins in the transport of circulating ghrelin. Our results show that ghrelin desoctanoylation mostly occurred in contact with low-density lipoproteins (LDLs) and lipoprotein-poor plasma subfractions. Butyrylcholinesterase and platelet-activating factor acetylhydrolase (EC 3.1.1.47) were responsible for the ghrelin hydrolytic activity of the lipoprotein-poor plasma and LDL subfractions, respectively. Moreover, we observed that ghrelin is associated with triglyceride-rich lipoproteins (TRLs), high-density lipoproteins (HDLs), very high-density lipoproteins (VHDLs), and to some extent LDLs. In conclusion, we report that the presence of the acyl group is necessary for ghrelin interaction with TRLs and LDLs but not HDLs and VHDLs. Ghrelin interacts via its N- and C-terminal parts with HDLs and VHDLs. This suggests that, whereas TRLs mostly transport acylated ghrelin, HDLs and VHDLs transport both ghrelin and des-acyl ghrelin.     

The effect of plasma low density lipoprotein apheresis on the hepatic secretion of biliary lipids in humans

Background—The liver is a key organ in themetabolism of cholesterol in humans. It is the only organ by whichsubstantial amounts of cholesterol are excreted from the body, eitherdirectly as free cholesterol into the bile or after conversion to bile acids. The major part of cholesterol synthesis in the body occurs inthe liver. Cholesterol is also taken up by the liver from plasma lipoproteins. The relative contributions of newly synthesised cholesterol and plasma lipoprotein cholesterol to bile acid synthesis and biliary cholesterol secretion, respectively, are not known in detail.
Aims—To determine how a rapid lowering of plasmalow density lipoprotein (LDL) and very low density lipoprotein (VLDL)cholesterol influences the biliary secretion rates of cholesterol andbile acids in patients with cholesterol gallstones and complete biliary drainage. In this model with a completely interrupted enterohepatic circulation, the secretion of bile acids equals the new synthesis ofbile acids in the liver.
Patients—Eight patients with common bileduct stones of cholesterol type undergoing conventional cholecystectomyand choledocholithotomy.
Methods—At operation a balloon occludable Foleycatheter attached to a T tube was inserted into the bile duct with theballoon placed just past the distal limb of the T tube. The T tube was allowed to drain the bile externally. One week after the operation theFoley catheter balloon was inflated, creating complete biliary drainage. Twelve hours following the inflation plasma LDL apheresis wascarried out for two hours. Bile was collected for 15 minute periodsstarting one hour before the apheresis and ending two hours after itstermination. During the collection of bile, plasma lipids were analysedon several occasions.
Results—The plasma level of LDL cholesteroldecreased by 26% from (mean (SEM)) 2.19 (0.29) to 1.63 (0.17) mmol/lduring the LDL apheresis while high density lipoprotein (HDL)cholesterol in plasma was unaffected. During LDL apheresisapolipoprotein B containing lipoproteins bind to the column, causing asignificant decrease of not only plasma LDL but also of VLDLcholesterol. The secretion rate of bile acids decreased significantlyby 31% from 131 (38) to 90 (16) µmol/15 minutes (p=0.045). Theoutput of phospholipids also decreased by 19%. The biliary secretion rate of cholesterol was not, however, affected by the plasma LDL apheresis.
Conclusions—The results suggest that, inpatients with cholesterol gallstones and complete biliary drainage,lowering of plasma LDL and VLDL cholesterol reduces the biliarysecretion rate—synthesis—of bile acids without affecting the biliarysecretion rate of cholesterol.

Keywords:bile acids; biliary lipids; cholesterol; lipoproteins; plasma apheresis

     

Cationic peptides neutralize Ox-LDL,prevent its uptake by macrophages,and attenuate inflammatory response

Objective

Apolipoprotein A1 (ApoA1) and apolipoprotein E (ApoE) mimetic peptides have attracted attention due to their ability to reduce atherosclerosis and exhibit antioxidant, anti-inflammatory, and hypolipidemic properties. In this study, we tested whether three distinct and unrelated cationic peptides would inhibit the oxidation of lipoproteins and whether they would counteract and neutralize the negatively charged modified lipoproteins, inhibit their uptake and inflammation by macrophages.

Methods and results

5F-mimetic peptide of ApoA1, LL27 derived from the anti-microbial peptide hCAP, and a human glycodelin derived peptide were commercially synthesized. We noted that these three distinct cationic lysine-rich peptides, two of which were unrelated to any known apolipoproteins, inhibited copper-mediated oxidation of lipoproteins and reduced lipid peroxides in a lysine dependent manner. The peptides also retarded the electrophoretic mobility of previously oxidized LDL and acetylated LDL by virtue of their net positive charge. Pre-incubation of peptides with modified lipoproteins reduced the uptake of the latter by macrophages, thus preventing the formation of foam cells. The cationic peptides inhibited oxidized LDL (Ox-LDL)-induced inflammatory response both in vitro and in vivo.

Conclusion

Based on these results, we suggest that in addition to the well known mimetic peptides, other suitable cationic peptides may be of use for controlling Ox-LDL mediated inflammation and atherosclerotic progression.     

Inhibition of receptor-mediated clearance of lysine and arginine modified lipoproteins from the plasma of rats and monkeys

Reductive methylation of at least 30% of the lysine residues or 1,2-cyclohexanedione modification of 45% of the arginine residues prevented low density lipoproteins (LDL) from binding to cell surface receptors of fibroblasts in vitro, without significantly altering other physical or chemical properties of the LDL. When rat or human LDL with more than 30% of the lysine residues methylated were injected intravenously into rats, the clearance of these lipoproteins from the plasma was slowed considerably. The half-life of the reductively methylated LDL was approximately twice that obtained for control (unmodified) LDL, and the value for the fractional catabolic rate was approximately half that of the control. Furthermore, when human LDL modified by reductive methylation were injected into rhesus monkeys, the rate of clearance was similarly retarded, and the value for the fractional catabolic rate was reduced by approximately 50% as compared with the value for control LDL. A dual isotope labeling technique (¹²⁵I and ¹³¹I) was used to compare the disappearance of the control and modified LDL in the same animal. It was demonstrated that not only modification of lysine residues but also modification of the arginine residues with 1,2-cyclohexanedione retarded the plasma clearance of the rat LDL. However, the cyclohexanedione modification was spontaneously reversible at 37°C, whereas reductive methylation of the lysine residues was stable. It is concluded that the selective chemical modification of lysine or arginine residues of LDL interferes with the normal uptake of these lipoproteins in vivo as well as by fibroblasts in vitro. These data provide an estimation of the level of receptor-mediated clearance of LDL from the plasma, a value that may be as high as 50% in rats and monkeys.     

Antiatherogenic properties of high-density lipoproteins from arterial plasma are attenuated as compared to their counterparts of venous origin

Background and aimsHigh-density lipoprotein (HDL) particles play atheroprotective roles by their ability to efflux cholesterol from foam cells and to protect low-density lipoproteins (LDLs) from oxidative damage in the arterial intima. We hypothesized that antioxidative properties of HDLs can be attenuated in the oxygen-rich prooxidative arterial environment, contributing to the development of atherosclerosis. To evaluate this hypothesis, we compared antioxidative activity of HDLs from arterial and venous plasmas.Methods and resultsArterial and venous blood samples were simultaneously obtained from 16 patients (age 68 ± 10 years; 75% males) presenting with ischemic or valvular heart disease. Major HDL subfractions and total HDLs were isolated by density gradient ultracentrifugation and their chemical composition and the capacity to protect LDLs from in vitro oxidation were evaluated. HDL-cholesterol, triglycerides and apolipoprotein (apo) B-100 levels were slightly but significantly reduced by −4 to −8% (p < 0.01) in the arterial vs. venous samples. Total mass of HDL subpopulations was similar and HDL subpopulations did not reveal marked compositional differences between the arterial and venous circulation. Potent antioxidative activity of the small, dense HDL3c subpopulation was significantly reduced in the particles of arterial origin vs. their counterparts from venous plasma (increase of +21% in the propagation rate of LDL oxidation, p < 0.05). Interestingly, antioxidative properties of venous HDLs were enhanced in statin-treated patients relative to untreated subjects.ConclusionAntioxidative properties of small, dense HDLs from arterial plasma are attenuated as compared to the particles of venous origin, consistent with the development of atherosclerosis in the arterial wall.     

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion

[PSI⁺], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI⁺]. However, another non-Mendelian determinant, [PIN⁺], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI⁺] by Sup35p overproduction in [PIN⁺] strains, but has no effect on the propagation of [PSI⁺] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI⁺] strains. We argue that excess Sup45p inhibits [PSI⁺] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.     

Phytoestrogens and lipoproteins in women

OBJECTIVES: We undertook a study to evaluate relationships among blood phytoestrogen levels, lipoprotein levels, estrogen levels, and angiographically defined coronary artery disease in women. BACKGROUND: Evidence for a beneficial role and the potential mechanism(s) of plant estrogens (phytoestrogens) on blood lipoproteins in humans is controversial. METHODS: We evaluated 483 women enrolled in the National Heart, Lung, and Blood Institute-sponsored Women's Ischemia Syndrome Evaluation with coronary risk factors undergoing coronary angiography for evaluation for suspected ischemia for blood phytoestrogen levels (daidzein and genistein), lipoprotein levels [total cholesterol, triglycerides, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol (HDL-C)], estrogen levels (estradiol, bioavailable estradiol, estrone), and angiographic coronary artery disease using core laboratories. RESULTS: Higher blood levels of the phytoestrogen daidzein were associated with lower triglycerides (P = 0.01), higher HDL-C (P = 0.05) levels, and a beneficial total cholesterol to HDL-C ratio (P = 0.02). This beneficial association was evident among the subgroup of women with low [<184 pmol/liter (<50 pg/ml)] blood estradiol levels, regardless of age and lipoprotein levels. The phytoestrogen associations with lipoproteins were incrementally related to the magnitude of daidzein level and independent of other lipoprotein modulators. There were no detectable relationships between the phytoestrogen levels and angiographic coronary artery disease. CONCLUSIONS: Higher blood phytoestrogen daidzein levels are associated with beneficial lipoprotein levels in women with low estrogen levels, possibly by an estrogen receptor mechanism. These results suggest a potential explanation for the variable lipoprotein results observed in prior randomized controlled trials and call for investigation regarding subgroups of subjects who may preferentially benefit from dietary intake of food products, such as soy.     

The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. Ure2p^1–65 is the prion domain of Ure2p, sufficient to propagate [URE3] in vivo. We show that full length Ure2p–green fluorescent protein (GFP) or a Ure2p^1–65-GFP fusion protein is aggregated in cells carrying [URE3] but is evenly distributed in cells lacking the [URE3] prion. This indicates that [URE3] involves a self-propagating aggregation of Ure2p. Overexpression of Ure2p^1–65 induces the de novo appearance of [URE3] by 1,000-fold in a strain initially [ure-o], but cures [URE3] from a strain initially carrying the [URE3] prion. Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] “crystal” of Ure2p poisons its propagation.