Studies on the nature of anti-platelet aggregatory factors in the seeds of the Amazonian Herb Guarana (Paullinia cupana).

Guarana (Paullinia cupana) is a popular herb native to the Amazon Basin and used extensively in soft drinks in Brazil, other Latin American countries, and more recently in the United States. Extracts derived from the dried seeds of guarana possess strong anti-platelet aggregatory properties. In this study, an active fraction containing this activity was purified and analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS) techniques. It was noted that this fraction contains catechins, epicatechins, and their dimers, with a small amount of caffeine. It is suggested that complexes containing caffeine and catechins (and their dimers) might be responsible for anti-platelet aggregatory activity in guarana seeds and might offer health benefits towards decreasing risk of thrombosis and cardiovascular disease.

Decreased atherogenic response to dietary cholesterol in pigeons after stimulation of cholesterol catabolism in early life.

Cholesterol catabolism was stimulated in 6-wk-old White Carneau pigeons using a laboratory stock diet containing 1.3% cholestyramine resin. After 8 wk on this diet the animals were returned to control stock diet (no resin) for another 8-wk period. When subsequently challenged with a diet containing 0.5% cholesterol, cholestyramine-pretreated pigeons exhibited significantly lower serum cholesterol level when compared with controls and this "hyporesponder" behavior persisted throughout the study period. Furthermore, the aorta of cholestyramine-treated animals exhibited significantly (a) lower prevalence and severity of atherosclerosis and (b) lower cholesterol content. These studies demonstrate for the first time that enhancement of cholesterol catabolism in early life improves resistance to diet-induced atherosclerosis in later life in this avian model.

Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage.

The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu2+ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled OX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17 beta significantly decreased the formation of single and double strand breaks in DNA induced by H2O2 alone or with Cu2+. Equilin (an equine estrogen) was more effective than estradiol-17 beta at the doses tested. Arachidonic acid in the presence of Cu2+ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.

Cholest-3,5-dien-7-one formation in peroxidized human plasma as an indicator of lipoprotein cholesterol peroxidation potential.

Lipoprotein peroxidation susceptibility is routinely evaluated using products of unsaturated fatty acids as markers (e.g., malonaldehyde). The significance and factors influencing peroxidation of cholesterol moiety of lipoproteins are relatively unknown due to lack of a reliable marker product which can be measured easily. Under the influence of Cu2+ ions, the major product of lipoprotein cholesterol peroxidation (isolated after saponification) was cholest-3-5-dien-7-one (CSD). Apart from gas-liquid chromatography, this compound lends itself for measurement by alternative methods. Due to lack of the 3 beta-hydroxyl group, CSD was separated from the rest of the oxysterols and cholesterol by passing through digitonin-coated silica-gel G and its concentration was determined by absorption at 283 nm. The recovery of CSD by this method exceeded by 87%. The formation of CSD was also sensitive to vitamin E and therefore could be used as an index of lipoprotein cholesterol susceptibility to peroxidation.

Concentration changes of bile acids in sequential segments of pigeon intestine and their relation to bile acid absorption.

Changes in the concentration of bile acids in sequential segments of pigeon intestine were measured. It was found that the jejunum has the highest concentration of bile acids. When the ratio of bile acids to beta-sitosterol (a non-absorbable dietary marker) was examined in contents of various segments, it was found that the ratio showed a marked decrease (86.6%) from the upper to lower jejunum while the subsequent changes in other segments were not as striking. This indicates that the jejunum is the major site of bile acid absorption in the pigeon, unlike in mammals, where the ileum has been shown to be the major site of bile acid absorption. This notion was confirmed by studies of the effect of ileal bypass in these pigeons. This surgical procedure did not significantly change the fecal excretion of bile acids or neutral sterols indicating that the jejunum is capable of maintaining a normal enterohepatic circulation of bile acids in the absence of the ileum in the pigeons.

The characteristics and metabolism of a genetically hypercholesterolemic strain of rats (RICO).

A genetically hypercholesterolemic strain of rats was selectively bred, starting from an ordinary albino mutant of Rattus norvegicus. The new strain was given the designation RICO, standing for rats with increased cholesterol. In these animals, hypercholesterolemia is established, in both sexes, one day after weaning, and it increases progressively thereafter. It is due to elevated concentrations of LDL- and HDL-cholesterol. As in the ordinary rat, the HDL fraction makes up the main part of the serum cholesterol in the RICO rat. Metabolic studies revealed that in the RICO strain the overall rate of hepatic cholesterol synthesis is accelerated, as a result of higher than normal activity of 3-hydroxy-3-methylglutaryl-CoA reductase. The activity of cholesterol-7 alpha-hydroxylase is decreased in RICO rats, indicating a lower than normal rate of cholesterol catabolism. No difference was found between RICO and ordinary rats with respect to fecal excretion of bile acids and cholesterol.

Proteins and peptides bound to long-circulating liposomes.

Liposome formulations with prolonged circulation time have recently been developed as a potential sustained-release drug delivery system. Data shown in this report indicate that such formulations can also be used to prolong the circulation time of proteins and peptides by conjugating them to the surface of liposomes. Increase of the circulation halflife ranged from 2- to 150-fold depending on the protein/lipid ratio of the liposomal formulation, liposome size, and the lipid composition of liposomes. Since the proteins/peptides localize on the liposome surface, instead of being entrapped inside the liposomes, they are directly available for binding to its receptor molecules and express the biological activity. This strategy has been successfully applied to two proteins with known fast clearance rate, i.e. asialofetuin and ricin A-chain. The biological activities of both proteins are preserved when they are formulated in liposomes. Incorporation of a peptide, i.e. a-factor of the yeast Saccharomyces cerevisiae, into the liposome membrane also significantly enhanced the circulation time of the peptide.

Abnormal bile acid pool and composition in neonates of spontaneously diabetic Wistar BB rats and its change during development.

Streptozotocin-induced diabetes during pregnancy in rats causes a decrease in primary bile acid pool in neonates. To rule out direct drug effect on the fetus as the basis for this change, studies of bile acid pool and composition at birth and during subsequent development was carried out in neonates of spontaneously diabetic Wistar BB rats and compared to control neonates. The cholic acid pool in neonates of diabetic rats was lower when compared to control neonates at birth. The pool of secondary bile acids was markedly increased in neonates of diabetic rats, with increases in lithocholic and 3 beta,12 alpha-dihydroxycholanoic acid. With age, the cholic acid pool of neonates from diabetic rats was increased and at 3 months of age it was actually higher than in control neonates. The pool of chenodeoxycholic at diabetes onset age was lower in neonates of diabetic rats. HDL-cholesterol was lower in neonates of diabetic rats at 1 week, but this reversed at 3 months of age. These studies firmly establish that neonates of diabetic rats have abnormal bile acid pool and composition at birth which changes to adult diabetic pattern with age.

Human breast milk contains a factor which markedly stimulates aortic prostacyclin synthesis.

Human breast milk has been shown to contain a potent factor which markedly stimulates the synthesis of prostacyclin by rabbit aorta. The stimulatory effect of colostrum was modest when compared to mature milk. This factor appears to be protein in nature with a molecular weight of less than 10 000. The main site of action of the factor appears to be on the conversion of arachidonic acid into prostacyclin. This factor might have a role in the optimal development of prostacyclin synthetic pathways which are not fully developed at birth.

Cholestyramine treatment in early life of low-density lipoprotein receptor deficient Watanabe rabbits: decreased aortic cholesteryl ester accumulation and atherosclerosis in adult life.

Effect of cholestyramine treatment in early life of Watanabe heritable hyperlipidemic rabbits (an animal model lacking low-density lipoprotein receptor activity) on subsequent (6 months recovery) occurrence of natural atherosclerotic lesion and arterial cholesterol metabolism was investigated. Initial cholestyramine treatment decreased both plasma total cholesterol and HDL-cholesterol levels which normalized within 4 weeks after treatment was discontinued. At 9 months of age (age of occurrence of spontaneous atherosclerotic lesions), the extent of aortic atherosclerosis in cholestyramine pre-treated animals was modestly lower (P less than 0.05), as compared to controls, with a significant (P less than 0.05) decrease in aortic cholesteryl ester content. Furthermore, at the end of the recovery period aortic activity of acyl-CoA: cholesterol acyltransferase and neutral cholesterol esterase activity was significantly (P less than 0.05) lower in cholestyramine-pretreated animals. These studies show that early cholestyramine pre-treatment in a low-density lipoprotein receptor-deficient animal model causes persistent changes which might influence cholesteryl ester accumulation and atherogenesis in adult life, even after cholestyramine treatment is discontinued.

Lipid peroxidation in the peritoneal fluid of infertile women with peritoneal endometriosis.

OBJECTIVE: To assess the level of lipid peroxidation in the peritoneal fluid of infertile women with peritoneal endometriosis and of fertile disease-free controls. STUDY DESIGN: Level of lipid peroxidation (malondialdeyde, malondialdeyde with copper addition, and cholest-3,5-dien-7-one) was measured in the peritoneal fluid obtained from 21 women with endometriosis-related infertility and from 21 fertile women having tubal ligation. RESULTS: : The level of lipid peroxidation did not differ significantly (P > 0.05) according to the stage of endometriosis. The level of lipid peroxidation (malondialdeyde, malondialdeyde with the addition of copper, and cholest-3,5-dien-7-one) did not differ significantly (P > 0.05) between patients with endometriosis-related infertility (0.07 nmol/ml, 0.34 nmol/ml, 0.24 microg/ml, respectively) and disease-free controls (0.04 nmol/ml, 0.21 nmol/ml, 0.25 microg/ml, respectively). CONCLUSION: The level of lipid peroxidation did not differ between women with endometriosis-related infertility and fertile disease-free controls, suggesting that increased reactive oxygen species may not be one of the factors responsible for compromised fertility in patients with endometriosis.

The role of human growth hormone in the regulation of cholesterol and bile acid metabolism.

Cholesterol and bile acid metabolism was studied in 16 children with human GH (hGH) deficiency (11 with isolated hGH deficiency and 5 with multiple trophic hormone deficiency) before and after 6 months of hGH therapy. We measured plasma lipid concentrations, biliary lipid composition, and cholesterol saturation indices; calculated the bile acid pool size measured by the isotopic dilution technique using the stable isotope chenodeoxycholic-[11,12-d2] acid; and measured cholesterol and bile acid synthetic rates by sterol balance techniques. In all 16 patients, plasmas lipid concentrations were unchanged after hGH therapy; total plasma cholesterol was 182 +/- 10 (+/-SEM) mg/dl before and 179 +/- 9 mg/dl after treatment, high density lipoprotein-cholesterol was 47 +/- 2 mg/dl before and 49 +/- 3 mg/dl after treatment, low density lipoprotein-cholesterol was 112 +/- 10 mg/dl before and 111 +/- 8 mg/dl after therapy, and triglyceride was 113 +/- 13 mg/dl before and 107 +/- 10 mg/dl after hGH therapy. Biliary lipid composition and cholesterol saturation in 10 patients were similar to those in controls and unchanged with hGH therapy. Cholesterol synthesis (n = 14) was unchanged (7.6 +/- 1.4 vs. 9.6 +/- 1.2 mg/kg X day); however, bile acid synthesis (n = 15) increased from 3.1 +/- 0.4 to 4.3 +/- 0.6 mg/kg X day (P less than 0.025) after therapy. The chenodeoxycholate pool size (n = 8) was significantly reduced (P less than 0.025) before hGH treatment (416 +/- 64 mg/m2) compared to that in controls (617 +/- 45 mg/m2) and increased to 620 +/- 72 mg/m2 after hGH therapy (P less than 0.05). Chenodeoxycholate pool size expansion during hGH therapy was, at least in part, caused by an increase in hepatic bile acid synthesis. These findings suggest that hGH may indirectly modulate cholesterol metabolism through regulation of hepatic cholesterol 7 alpha-hydroxylase activity, the rate-limiting enzyme of bile acid synthesis.

Antioxidant potential of specific estrogens on lipid peroxidation.

Coronary heart disease (CHD) is the leading cause of death in postmenopause. Estrogen administration in postmenopause lowers the risk of CHD by 50%. A variety of estrogen preparations are currently used in postmenopausal hormone replacement therapy. It is unknown, however, if structural differences in the estrogen molecule influence the cardioprotective effects of estrogens. In this communication we have shown that equine estrogens (especially equilin) exhibit higher antioxidant potency (as measured by fatty acids and sterols oxidation) when compared to estrone and estradiol-17 beta.

Human breast-milk factors influencing lipid metabolism by fetal rabbit aorta in organ culture.

Effects of human or rabbit milk on cholesterol content, incorporation of [14C]oleate, and DNA synthesis were investigated in organ cultures of aorta from fetal and suckling rabbits. Human skim milk (50 mL/L) in organ culture decreased content (mumol/g protein) of aortic total cholesterol (control: 162.9 +/- 24.6 milk: 117.6 +/- 4.9) with significant decrease in cholesteryl esters (control: 16.5 +/- 5.1, milk: 2.3 +/- 0.5). The effect was observed in lipoprotein (total cholesterol: 120.2 +/- 8.8) and lipoprotein-free (total cholesterol: 85.4 +/- 5.1) fractions with molecular weight greater than 100,000. The human milk fraction with molecular weight greater than 100,000 was also the most active in promoting [3H]thymidine incorporation into DNA in fetal aorta. Although milk stimulated the incorporation of [14C] oleate into triglycerides (control: 38.8 +/- 2.5%, milk: 82.1 +/- 4.2%), decreased incorporation to phospholipids (control: 55.0 +/- 3.0%, milk: 11.3 +/- 1.8%) was observed. These studies suggest that milk contains factors influencing aortic lipid metabolism during development.

Short-term exposure to high dietary cholesterol in early life: arterial changes and response after normalization of plasma cholesterol.

Elevated serum cholesterol is an established risk factor for the development of atherosclerosis but the effect of high dietary cholesterol in early life on subsequent arterial response to atherogenic diet in adult life is unknown. Weanling rabbits were exposed for 6 wk to a diet containing 0.25% cholesterol, allowed to recover for 9 wk (at least 3 wk after normalization of plasma cholesterol), and subsequently rechallenged with cholesterol to determine atherogenic response. Enhanced activity of acyl-CoA-cholesterol-acyl-transferase in aorta induced by cholesterol feeding persisted even after normalization of serum cholesterol. When rechallenged with cholesterol for 3 mo, these animals displayed significantly (p less than 0.05) increased development of aortic atherosclerosis and accumulation of cholesterol esters when compared with control animals. Exposure to cholesterol in early life appears to cause persistent changes in cholesterol ester synthetic enzyme activity in aorta after normalization of plasma cholesterol and these residual effects might increase aortic response to subsequent cholesterol challenge in adult life.

Bile acid metabolism in ascorbic acid-deficient guinea pigs.

Sterol balance techniques have been used to determine the effect of short-term ascorbic acid (AA) deprivation on bile acid excretion in the guinea pig. The effects of a brief (2-week) AA deficiency on bile acid pool sizes and the activity of the rate controlling enzyme in bile acid biosynthesis have been determined. It was found that, while food intake and body weight were not affected by the short-term AA deficiency, liver AA levels had fallen to 25% of control levels. At the same time, the rate of excretion of bile acids and the size of the bile acid pool were both reduced by about 50% in guinea pigs deficient in AA. These results were supported by a decrease in the activity of cholesterol 7 alpha-hydroxylase in the deficient animals. It is concluded that an AA deficiency will significantly impair bile acid metabolism independent of any side effects of clinical scurvy.

Effect of enhancement of cholesterol catabolism in guinea pigs after weaning on subsequent response to dietary cholesterol.

Cholesterol catabolism was stimulated (by cholestyramine) in post-weaned guinea pigs for a 4-wk period. The animals were then switched to regular Chow diet for another 4 wk. When subsequently challenged with 0.25% cholesterol-containing Chow, plasma cholesterol level was significantly (p less than 0.05) lower in cholestyramine pretreated guinea pigs when compared to control guinea pigs and this "hyporesponder" pattern was maintained throughout the study period. Cholestyramine pretreated animals continued to excrete significantly (p less than 0.05) higher bile acids even at 4 wk after switching to regular Chow diet. This study demonstrates that stimulation of cholesterol catabolism even at postweaning stages can still be as effective in improving subsequent cholesterol handling capacity as noted previously after manipulation at the neonatal stage.

Effect of sucrose polyester on fecal bile acid excretion and composition in normal men.

The effects of sucrose polyester (SPE) (a nonabsorbable mixture of hex-a, hepta,- and octa-fatty acid esters of sucrose with physical properties similar to those of common dietary fats) on fecal bile acid excretion and composition were assessed in 24 healthy, nonobese, normolipemic male volunteers, in a 40-day, inpatient, metabolic balance study. Isocaloric diets provided either 800, 300, or less than 50 mg of cholesterol/day (P/S ratios respectively 0.4, 1.0, and 1.5). After diet-only perids of 10 days (for the 800 and 300 mg cholesterol regimens), and 21 days (for the 50 mg diet), the diets were continued for 30 days, with addition of SPE to diets over three successive treatment periods of 10 days each, with 8, 16, and 35 g of liquid SPE/day, or 15, 30, and 50 g SPE/day in a SPE-hydrogenated palm oil mix. On both the liquid SPE and SPE-hydrogenated palm oil mix, there were no significant changes in fecal bile acid excretion as a function of dietary SPE, at any level of cholesterol intake, P > 0.1. In most subjects SPE changed fecal bile acid composition; lithocholic acid was decreased, and in most instances this was accompanied by the appearance and increase in chenodeoxycholic acid. In one subject, both deoxycholic and 3 beta, 12 alpha-dihydroxycholanic acid were reduced, with an accompanying increase in cholic acid. The hypocholesterolemic effect of SPE appears to be mediated through its reduction of intestinal absorption of cholesterol, not through effects on bile acid excretion.

Prostaglandin E2 biosynthesis and effect in pigeon aorta. Possible role in atherogenesis.

This study for the first time examines the biosynthesis and effect of prostaglandin E2 (PGE2) in aorta during genetic atherosclerosis. Biosynthesis of PGE2 from [1-14C]arachidonic acid was investigated in the aorta of spontaneously atherosclerosis-susceptible White Carneau pigeons and was compared with that of the atherosclerosis-resistant Show Racer breed. Most of the PGE2 synthetase activity was located in the microsomes. The synthesis was linear up to an hour and was optimal at pH 7.4. The formation of PGE2 in the aorta in the White Carneau pigeons was significantly higher (P less than 0.01) than that in age-matched Show Racer pigeons. In vitro PGE2 strongly inhibited the cholesteryl ester hydrolase activity (51.6% inhibition at 4 X 10(-7) M concentration) in the supernatant fraction of the aorta. On the basis of (1) the increased formation of PGE2 in the aorta of atherosclerosis-susceptible pigeons and (2) its effect on specific enzymes that control cholesteryl ester concentration in aorta, it is hypothesized that PGE2 synthesized at a higher rate in damaged aorta has a significant role in cholesteryl ester accumulation during atherogenesis.

Studies on aorta during development. I. Fetal rabbit aorta under ex vivo and in vitro conditions: rapid changes in smooth muscle cell phenotype, cell proliferation and cholesterol content with organ culture.

The structural development of the already well defined fetal rabbit aortic wall from 22 to 31 days of gestation in vivo consists of increasing aortic wall thickness, elastic lamina continuities, extracellular matrix deposition, and maturing of the fine structure of the medial smooth muscle cells. In vivo at term (31 days), the mature aortic smooth muscle cells demonstrated the characteristic thin, thick and intermediate filaments, dense plaques, endoplasmic reticulum, golgi, plasmalemma vesicles and an incomplete basal lamina. The fetal aorta rapidly responded to organ culture with various changes. Fetal smooth muscle cells modified their phenotype to the synthetic state when cultured in both serum-supplemented and serum-free media. This smooth muscle cell modification occurred after 3 days of culture in fetal explants. The synthetic type smooth muscle cells (fetal) began to proliferate after 6 days of culture. This proliferation resulted in a peripheral outgrowth after 9 days of 10-20 layers in fetal cultures from serum-supplemented media and of 2-4 layers in serum-free media. The orderly arrangement of the internal elastic lamina and alternating medial layers of smooth muscle cells and elastic lamina seen in vivo was disrupted along with increased matrix after 9 days of fetal explant culture. Significant numbers of 'modified' synthetic phenotype smooth muscle cells were not observed in adult aortic explants until after 15 days in culture in serum supplemented media. The mature contractile phenotype smooth muscle cell predominated in adult explants cultured in serum-free media. Significant synthetic phenotype smooth muscle cell proliferation only occurred in adult explants after 15 days culture in serum-supplemented media. When compared to aorta in vivo evidence for increases in cholesterol esterification were observed in both fetal (9 days) and adult (15 days) explants cultured in both serum-supplemented and serum-free media. The fetal aorta in organ culture appeared to be more susceptible than the adult aorta to (a) phenotypic modulation of smooth muscle cells to the synthetic state, (b) smooth muscle cell proliferation, and (c) early cholesteryl ester accumulation.