Robert J. Morin

Transport of cholesterol autoxidation products in rabbit lipoproteins

Radiolabeled pure [4-14C]cholesterol was kept at 60 degrees C under air to autoxidize for 5 weeks, after which approximately 12% cholesterol oxidation products were formed. The mixture, suspended in gelatin, was given to rabbits by gastric gavage. Rabbits were killed 4, 24, and 48 h after treatment. Cholesterol and its autoxidation products were separated by thin-layer chromatography into 5 fractions and radioactivities of each fraction were measured. Percentages of each fraction of cholesterol oxidation products and cholesterol in the original mixture before administration and in the rabbit sera after administration were similar, suggesting that the rates of absorption of cholesterol oxidation products are not significantly different from that of cholesterol. Lipoproteins were fractionated by ultracentrifugation into VLDL, LDL and HDL. Radioactivities of each fraction in lipoproteins separated by thin layer chromatography showed that fractions containing cholestane-3 beta,5 alpha,6 beta-triol, 7 alpha- and 7 beta-hydroxycholesterol and 7-ketocholesterol were more selectively transported in VLDL, whereas most of the 25-hydroxycholesterol was present in LDL. HDL contained only minute amounts of cholesterol oxidation products.

Changes in plasma carotenoid, alpha-tocopherol, and lipid peroxide levels in response to supplementation with concentrated fruit and vegetable extracts : A pilot study

Studies over the last two decades equating diet with chronic diseases have linked the highest consumption of mixed fruits and vegetables to a reduced risk of coronary heart disease (CHD), stroke, cataracts, and cancer at multiple sites. High levels of natural antioxidants, including the carotenoids, tocopherols, and ascorbic acid, appear to be responsible for these reductions in risk. However, long-term intervention studies to alter chronic disease outcomes have generally used a single nutrient such as beta-carotene at high doses, and results have been disappointing. Because antioxidants have multiple and synergistic interactions and also exhibit compartmentalization and tissue specificity, it appears desirable to use supplementation that increases blood levels while simulating combinations of these chemoprotective substances in amounts more closely approximating amounts of mixed diets. This study measured carotenoid and tocopherol levels in human plasma after supplementation with dehydrated fruit and vegetable extracts (JuicePlus+‘“). Serum lipid peroxides were also measured to assess the effectiveness of supplementation in modifying oxidative processes. Fifteen healthy adults (10 women, 5 men; age range, 18 to 53 years) consumed supplements twice daily with meals for 28 days, with fasting plasma and serum samples taken at baseline and 7, 14, and 28 days. After 28 days, plasma antioxidant levels increased significantly: beta-carotene, 510%; alpha-carotene, 119%; luteinl zeaxanthin, 44%; lycopene, 2046%; and alpha-tocopherol, 58%. Serum lipid peroxides decreased fourfold after 7 days and remained significantly lower than baseline at 28 days (baseline, 16.85 + 16.91 pmoll mL; 28 days, 4.22 rt 3.78 pmol/mL). Decreases in lipid peroxide levels were coincident with increases in carotenoids and alpha-tocopherol, and reflect functionally improved oxidative defense mechanisms. Because these bioactive compounds can act synergistically, the effect cannot be attributed to any one component, but it may reflect a combined mechanism of antioxidant defense. Marked increases in plasma

Effects of cholesterol oxidation derivatives on cholesterol esterifying and cholesteryl ester hydrolytic enzyme activity of cultured rabbit aortic smooth muscle cells

The effects of 5 μg/ml of 25-hydroxycholesterol; cholestane-3β, 5α,6β-triol; and cholesterol on acyl CoA cholesterol acyltransferase, acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase was studied in cultured rabbit aortic smooth muscle cells. After 1 hour incubation, 25-hydroxycholesterol resulted in a fourfold stimulation of acyl CoA cholesterol acyltrans-ferase activity. No stimulation by 25-hydroxycholesterol was noted before 15 minutes or after 5 hours of incubation. Neither cholestane-3β,5α,6β-triol nor cholesterol influenced acyl CoA cholesterol acyltransferase activity at any time interval. No significant effects of any of the sterols were noted on acid cholesteryl ester hydrolase or neutral cholesteryl ester hydrolase activity. The imbalance between acyl CoA cholesterol acyl trans-ferase and hydrolase activities induced by 25-hydroxycholesterol could result in cholesteryl ester accumulation by arterial smooth muscle cells, which may be associated with atherosclerosis.

Comparison of intraluminal and intravenous mediators of colonic response to eating

Eating a 1000-kcal mixed meal stimulates an increase in distal colonic motility. Fat is the dietary component which is the major stimulant of colonic spike activity. In this study the colonic spike activity increased similarly after the mixed meal [19.1±2.4 spike potentials (SP)/30 min] and after the fat meal (19.4±5.4 SP/30 min). Fat stimulated a concentration-dependent increase in colonic motility only when in contact with the gastroduodenal mucosa. Intravenous administration of Liposyn (100 kcal/hr) did not stimulate an increase in colonic spike activity (3.3±1.3 SP/30 min) despite greater increase in plasma total fatty acid levels than after the oral ingestion of fat. In contrast both the oral ingestion and the intravenous administration of an amino acid mixture (Aminosyn) inhibited the gastrocolonic response after the 1000-kcal mixed meal. Thus, these studies demonstrate: (1) fat stimulates colonic motility only through direct mucosal contact, and (2) a mixture of amino acid inhibits colonic motility through either mucosal contact or by circulating in the plasma. The exact neurohumoral mechanisms involved in both of these effects is unknown at present.

ESTERIFICATION OF CHOLESTEROL BY SUBCELLULAR FRACTIONS FROM SWINE ARTERIES, AND INHIBITION BY AMPHIPATHIC AND POLYANIONIC COMPOUNDS

Abstract Cholesterol esterification in swine arteries in vitro was catalyzed by an ATP and CoA dependent acyltransferase, with optimal activity at pH 7.4, and a noncofactor dependent esterifying enzyme, with optimal pH at 5.6. Activity of the former enzyme was highest in the microsomal fraction, whereas the low pH enzyme activity was highest in the mitochondrial fraction. Palmitoyl CoA was esterified by the microsomal fraction at a much faster rate than was palmitate with added ATP and CoA. Microsomal esterifying activity was inhibited by 10 −5 to 10 −3 M concentrations of compounds with ionic amphipathic properties, such as SKF 525-A, SQ 10,591, sodium taurocholate and sodium decyl sulfate, and also by the non-ionic surface active agents, Tween 20 and 80. Compounds with polyanionic properties, such as polyphloretin phosphate, heparin and dextran sulfate also effectively inhibited esterification at concentrations down to 0.1 μg/ml.

Effects of cholesterol autoxidation derivatives on hexose transport in cultured aortic smooth muscle cells

Several cholesterol autoxidation derivatives known to be cytotoxic to arterial smooth muscle cells both in vivo and in vitro were shown to inhibit hexose transport in these cells in culture. Cholestane-3 beta, 5 alpha, 6 beta-triol was the most potent inhibitory sterol. The rapid onset of inhibition (15 min) and the reversibility of the effect upon removal of the sterol from the tissue culture medium suggests that the effect may be due to incorporation of the sterol into the plasma membrane. 25-Hydroxycholesterol, a potent inhibitor of sterol biosynthesis in cultured arterial smooth muscle cells, did not affect hexose transport up to 8 hr of incubation. The cytotoxic effect of 25-hydroxycholesterol, therefore, may be a consequence of the reduced sterol biosynthesis caused by this sterol.

Platelet adhesion to collagen in normal and von Willebrand's disease subjects.

Abstract A rapid and sensitive method for quantitation of platelet adhesion was developed, utilizing a combination of optical density monitoring and interval platelet counting after addition of partially purified rat tail tendon collagen in a stirred cell system. EDTA inhibited aggregation but did not affect adhesion. The rate of platelet adhesion was significantly diminished in von Willebrands disease and by decreasing normal plasma concentrations to less than 20%. Addition of dipyridamole to normal platelet rich plasma resulted in inhibition of adhesion, whereas acetylsalicylic acid and sulfinpyrazone had no significant effects on adhesion.

EFFECT OF HEMODIALYSIS WITH ACETATE VS BICARBONATE ON PLASMA LIPID AND LIPOPROTEIN LEVELS IN UREMIC PATIENTS

A group of 18 stable hemodialysis patients were dialyzed alternately for 4 week periods against acetate or bicarbonate solutions. Total plasma cholesterol and triglyceride concentrations and the content of these lipids in the VLDL, LDL, and HDL lipoprotein fractions were not significantly different when these dialysis periods were compared. When patients were subgrouped according to whether they were normo or hypertriglyceridemic or whether they had an adequate or deficient lipolytic response to heparin infusion, there were also no differences apparent between the acetate vs. bicarbonate dialysis periods. The data indicates, therefore, that acetate is probably not a contributory factor to the hypertriglyceridemias observed in some chronic hemodialysis patients.

β-Carotene and α-tocopherol in healthy overweight adults; depletion kinetics are correlated with adiposity

Healthy overweight subjects (24 males, 68 females; mean age=48.8 years; body mass index=27.1±4.9) participated in a randomized, double-blind, placebo-controlled crossover study with two periods of 28-day supplementation using a nutritional product composed primarily of dehydrated juice concentrates from mixed fruits and vegetables (JuicePlus +®). Compared with placebo, supplementation for 28 days increased concentrations of serum β-carotene by 264% (P <0.001) and α-tocopherol by 14% (P < 0.01). After crossover of the active group to placebo, β-carotene and α-tocopherol declined via first-order kinetics, with serum half-lives (t1/2) for β-carotene and α-tocopherol determined to be 22.8±3.1 and 4.6±2.3 days, respectively. Depletion rates for β-carotene correlated with adiposity (quartile 1, body mass index=21.96, t1/2=17.6 days vs. quartile 4, body mass index=37.87, t1/2=26.3 days; P < 0.05). In conclusion, the supplementation period resulted in significantly elevated levels of β-carotene and α-tocopherol, indicating bioavailability. These increased levels persisted 2–4 weeks after supplementation was discontinued, and the rates of depletion were correlated with the levels of general adiposity.

Inhibition of rat liver sterol formation by isoprenoid and conjugated ene compounds

Summary The isoprenoid compounds dolichol, citral, α-tocopherol, ubiquinone, geraniol, and phytol and the diene pentachloropentadienoic acid, when introduced in sonicated liposomes with phosphatidyl serine, inhibited the conversion of squalene to sterols by the reaction mixture. Other compounds tested and found to have no effect on this reaction were lycopene (an isoprenoid), eleostearic acid, 4′ methoxychalcone and 5(α-methyl vanillidene) rhodanine (ene compounds). Only citral, α-tocopherol, squalene and pentachloropentadienoic acid were found to inhibit the incorporation of 3 H-mevalonic acid into sterols.