Biomedical subjects

K D Croft

Publications and source records attributed to K D Croft.

At least 37 records · Page 2Linked to original sources

PubMed2000

Effect of omega 3 fatty acids on oxidative stress in humans: GC-MS measurement of urinary F2-isoprostane excretion.

Despite the reported benefits associated with omega3 fatty acids for cardiovascular disease, there remains concern that increased intake may lead to increased lipid peroxidation. To date, however, the data, particularly in vivo, are inconclusive. This report describes two interventions, one providing daily fish meals and the other eicosapentaenoic acid (EPA, 20:5 omega3) or docosahexaenoic acid (DHA, 22:6 omega3), the two principal omega3 fatty acids in marine oils, in which in vivo lipid peroxidation was assessed by measurement of urinary excretion of F2-isoprostanes. In both trials, urinary F2-isoprostanes were significantly reduced by 20-27%. Therefore, in contrast with previous reports in the literature, these results demonstrate that omega3 fatty acids reduce in vivo oxidant stress in humans.

Diabetes Mellitus, Type 2↗

PubMed2000

Cytochrome P450 metabolites of arachidonic acid may be important mediators in angiotensin II-induced vasoconstriction in the rat mesentery in vivo.

We have investigated the role of cytochrome P450 (CYP-450) metabolites of arachidonic acid in the modulation of vascular reactivity to angiotensin II in vivo using an in situ blood-perfused mesenteric preparation in anaesthetized spontaneously hypertensive rats (SHR). Miconazole, a non-selective inhibitor of CYP-450 that inhibits both hydroxylation and epoxidation, substantially suppressed mesenteric vasoconstrictor responses to angiotensin II in SHR, but had no effect on responses to noradrenaline or sympathetic nerve stimulation. In normotensive Wistar-Kyoto (WKY) rats, miconazole caused only a modest suppression of vasoconstrictor responses to angiotensin II. N-Methylsulphonyl-12, 12-dibromododec-11-enamide (DDMS), a new selective inhibitor of CYP-450 omega-hydroxylase activity, decreased mean intra-arterial blood pressure and significantly attenuated mesenteric angiotensin II-induced vasoconstrictor responses in SHR. Isolated mesenteric vessels were able to metabolize (14)C-labelled arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) in vitro, and this was substantially inhibited by DDMS. The results from the present studies combined with the existing evidence that angiotensin II stimulates the release of 20-HETE, a CYP-450 metabolite of arachidonic acid, suggest that CYP-450-derived HETEs may be important mediators in angiotensin II-induced vasoconstriction. However, the development of more sensitive assays for the detection in vivo of 20-HETE in mesenteric vessels would be required to confirm these findings.

Amides↗

PubMed1999

Measurement of urinary F(2)-isoprostanes as markers of in vivo lipid peroxidation-A comparison of enzyme immunoassay with gas chromatography/mass spectrometry.

This study aimed at comparing the two most commonly utilized methods for measuring urinary F(2)-isoprostanes, currently considered one of the best available markers of in vivo lipid peroxidation. The F(2)-isoprostanes were measured in 24-h urine samples from 14 male subjects using electron capture negative ionization gas chromatography-mass spectrometry (ECNI-GCMS) with 8-iso-PGF(2alpha)-d(4) as an internal standard and compared with levels obtained using an enzyme immunoassay (EIA, 8-iso-PGF(2alpha) kit, Cayman Chemical Co.). The methods were compared using Pearson correlation coefficients, and Bland-Altman plots were constructed for the difference in F(2)-isoprostane against the average F(2)-isoprostane measured by either method. Weighted least-products regression was used to determine fixed bias (where there is a consistent difference between the methods) and proportional bias (where one method gives values higher or lower than the other method by an amount proportional to the size of the measurement). The correlation between F(2)-isoprostane levels obtained using EIA and GCMS methods, although significant, was poor (r = 0.628, P < 0.02). Comparison of the methods using the Bland-Altman analysis showed that there were wide limits of agreement between the two methods with only 28% of the values falling within the 95% confidence limits for the difference. The GCMS gave higher values with a mean difference of 298.1 pM (636.6, -40.2; P = 0.079), and a near significant linear association between the differences and the mean F(2)-isoprostane level (r = -0.559, P = 0.05). Weighted least-product regression analysis confirmed the presence of both significant fixed and proportional bias with the EIA giving lower levels of F(2)-isoprostanes at low concentrations and higher levels at higher concentrations. The cross-reactivity in the EIA of 8-iso-15(R)-PGF(2alpha) and 9beta-PGF(2alpha) which coelute with the F(2)-isoprostane peak measured by GCMS was very low, 0.2 and 0.1%, respectively. The proportional bias observed between the methods may in part be due to differences in the relative amounts of 8-iso-15(R)-PGF(2alpha), 9beta-PGF(2alpha), and 8-iso-PGF(2alpha) with increasing lipid peroxidation. This study shows that the measurements of F(2)-isoprostanes by EIA and GCMS are not equivalent. Therefore, comparison of levels derived using a GCMS method which estimates concentration from a peak encompassing a number of F(2)-isoprostane isomers, and levels derived from enzyme immunoassay measuring a specific isoprostane, may be inappropriate.

Adult↗

PubMed1999

An improved method for the measurement of urinary and plasma F2-isoprostanes using gas chromatography-mass spectrometry.

We have developed an improved method for the measurement of F2-isoprostanes using stable isotope dilution capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS). The F2-isoprostane family consists of a series of chemically stable prostaglandin F2 (PGF2)-like compounds generated during peroxidation of arachidonic acid in phospholipids. There is evidence that measurement of F2-isoprostanes represents a reliable and useful index of lipid peroxidation and oxidant stress in vivo. Furthermore, 8-epi-PGF2alpha, which is one of the more abundant F2-isoprostanes, is biologically active, being a potent mitogen and vasoconstrictor of rat and rabbit lung and kidney, as well as a partial agonist of platelet aggregation. Measurement of F2-isoprostanes in biological samples is complex and has involved methods which utilize multiple chromatographic steps, including separation by thin-layer chromatography, leading to poor sample recovery. We now present an improved method for the measurement of plasma and urinary F2-isoprostanes using a combination of silica and reverse-phase extraction cartridges, high-performance liquid chromatography (HPLC), and GC-ECNI-MS. Different approaches to the derivatization of the F2-isoprostanes prior to GC-ECNI-MS are also addressed. The overall recovery of F2-isoprostanes is improved (approx 70% for urine) and the within and between assay reproducibility is 6.7% (n = 23) and 3.7% (n = 3), respectively. The mean urinary excretion of F2-isoprostanes in eight healthy males was 365 +/- 5 pmol/mmol creatinine and in three smokers 981 +/- 138 pmol/mmol creatinine. The mean total (free + esterified) plasma F2-isoprostane concentration was 952 +/- 38 pmol/liter, with a within and between assay reproducibility of 8% (n = 13) and 5.6% (n = 3), respectively. This improved method for the measurement of F2-isoprostanes represents a significant advance in terms of the rapidity and yield in the purification of biological samples. The inclusion of HPLC separation enables improved analysis of F2-isoprostanes by GC-MS. This methodology will assist in defining the role of F2-isoprostanes as in vivo markers of oxidant stress in clinical and experimental settings.

Adult↗

PubMed1999

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.

Adult↗

PubMed1999

Effect of dietary fish and exercise training on urinary F2-isoprostane excretion in non-insulin-dependent diabetic patients.

Despite the potential benefits of dietary treatment with marine omega3 fatty acids in cardiovascular disease, there remains concern with respect to their potential for increased lipid peroxidation. Thus far, data from in vivo studies are inconclusive. Increased lipid peroxidation has also been associated with acute exercise in some studies, but the methods have been nonspecific. The quantitation of F2-isoprostanes provides a more reliable and useful assessment of in vivo lipid peroxidation. We therefore aimed to assess the independent and combined effects of dietary omega3 fatty acids and aerobic exercise training on urinary F2-isoprostane levels in dyslipidemic non-insulin-dependent diabetic (NIDDM) patients. In a randomized controlled trial, 55 untrained, sedentary, dyslipidemic NIDDM patients were randomly assigned to a low-fat diet (30% of daily energy) with or without one daily fish meal (3.6 g omega3 fatty acids per day) and further randomized to either a moderate (55% to 65% maximal oxygen consumption [VO2max]) or light (heart rate <100 bpm) exercise training program for 8 weeks. Twenty-four-hour urine samples from 49 subjects were collected for measurement of urinary F2-isoprostanes by gas chromatography-mass spectrometry before and after intervention. The fish diets reduced urinary F2-isoprostanes by 830+/-321 pmol/24 h (20%, P = .013) relative to the low-fat diet alone. This effect was independent of age, gender, and body weight change. Moderate exercise training did not alter F2-isoprostanes. These findings show that, at least in the short-term, exercise had no effect, whereas the inclusion of regular fish meals as part of a low-fat diet reduced in vivo lipid peroxidation in dyslipidemic NIDDM patients. This response could further complement the known benefits of omega3 fatty acids and exercise favoring a reduced cardiovascular risk in diabetic patients.

Adult↗

PubMed1999

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.

Adult↗

PubMed1999

Alcohol, stroke and coronary heart disease. Are there anti-oxidants and pro-oxidants in alcoholic beverages that might influence the development of atherosclerotic cardiovascular disease?

Anti-oxidant effects of polyphenolic flavonoid compounds found in alcoholic beverages, especially red wine, have been proposed to mediate, at least in part, protective effects of regular light-to-moderate alcohol use against stroke and coronary artery disease. The proposed mechanism is through the quenching of free radicals decreasing the oxidative modification of low-density lipoprotein cholesterol particles and hence reducing their atherogenicity. In this review, the extent and limitations of the evidence in support of such a hypothesis are outlined. In particular, the paucity of epidemiological evidence linking dietary flavonoids to stroke and coronary artery disease is highlighted. The competing notion that alcohol itself has direct and indirect pro-oxidant and pro-atherogenic effects is canvassed, and the limitations of the in vitro rather than in vivo nature of much of the evidence linking red wine polyphenolics to reduced lipid peroxidation and other relevant biological effects is discussed. Within this framework of current epidemiological evidence together with the results of basic laboratory studies, the conclusion at present is that while we may continue to speculate that there are anti-oxidants and pro-oxidants in alcoholic beverages that influence the development of atherosclerotic cardiovascular disease, this cannot yet be considered as an established scientific fact.

Alcohol Drinking↗

PubMed1998

The chemistry and biological effects of flavonoids and phenolic acids.

Flavonoids and phenolic acids are widely distributed in higher plants and form part of the human diet. Recent interest in these substances has been stimulated by the potential health benefits arising from the antioxidant activity of these polyphenolic compounds. This review outlines the basic chemistry, biosynthesis, and structure-activity relationships of these compounds with respect to their antioxidant activity. Although there is considerable in vitro evidence establishing antioxidant activity for polyphenolics found in the diet, there are few studies in humans on the absorption and bioavailability of these compounds. The possible in vivo antioxidant effects of the flavonoids is even less well understood. For example, controlled human intervention studies with beverages, such as red wine, that are rich in polyphenolic compounds, have yielded conflicting results. Our own work and that of others suggests that the final effects of such beverages may be a balance between the well-described prooxidant effects of alcohol and its metabolism and the antioxidant effects of the polyphenolic constituents. There is a need for further studies to increase our understanding of the absorption and in vivo biological effects of this family of compounds.

Antioxidants↗

PubMed1998

Alcohol, free radicals and antioxidants.

Antioxidants in alcoholic beverages, especially polyphenolic compounds in red wine, have been proposed as an important contributory factor to the protective effect of regular alcohol use against atherosclerotic cardiovascular disease. The postulated mechanism involves quenching of free radicals with decreased oxidative damage to low density lipoprotein (LDL) hence reducing its potential atherogenicity. There is definitive in vitro evidence that extracts of red wine, white wine, grape juice and beer can inhibit the oxidation of LDL, the degree of inhibition being directly proportional to beverage polyphenolic content and able to be abolished by prior stripping of the polyphenolics from the alcoholic beverage. These in vitro antioxidant effects have not been reliably reproduced in vivo after acute or short-term administration of alcoholic beverages. In fact, in some studies where white wine or beer have been given over 2-4 week periods, enhanced oxidizability of LDL cholesterol has been reported. Such findings are consistent with the possibility that, depending on the beverage, a predominant pro-oxidant effect of alcohol itself may outweigh any antioxidant effect of beverage polyphenolics. Increased oxidant stress and enhanced lipid peroxidation with alcohol have several biologically plausible explanations and have been reported as possible mechanisms for alcohol-related toxicity and injury in various tissues. Therefore, before the promotion of any particular benefits of ingestion of polyphenolics from alcoholic beverages (especially red wine) for prevention of atherosclerotic cardiovascular disease, the balance of redox effects in vivo will need careful further clinical and laboratory evaluation.

Alcohol Drinking↗

PubMed1998

Hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 studied with a stable isotope technique in men with visceral obesity.

OBJECTIVE: To test the hypothesis that the hepatic secretion of very-low-density lipoprotein (VLDL) apolipoprotein B-100 (apoB) is increased in men with visceral obesity and to examine whether the oversecretion of this apolipoprotein is related to insulin resistance and increased hepatic availability of lipid substrates. SUBJECTS: 16 obese men (body mass index (BMI) > 30 kg/m2, waist circumference > 100 cm) and 16 non-obese, age matched men, were studied. MEASUREMENTS: The hepatic secretion of VLDL apoB was measured using a primed (1 mg/kg), constant (1 mg/kg/h), intravenous infusion of 1-[13C]-leucine. Isotopic enrichment of VLDL apoB was determined using gas-chromatography mass spectrometry and a multi-compartmental model (SAAM-II) was used to estimate the fractional turnover rate of VLDL apoB. RESULTS: Plasma concentrations of total cholesterol, triglyceride, glucose, insulin, mevalonic acid and lathosterol, as well as dietary fat intake, were significantly higher (P < 0.05) in obese than control subjects. The obese subjects had significantly lower high-density-lipoprotein cholesterol (P < 0.01). VLDL apoB pool size and hepatic secretion rate (mg/kg fat free mass/d) were significantly higher in the obese than non-obese subjects (P < 0.02). The fractional catabolic rate of VLDL apoB was lower in the obese subjects compared with controls, but the difference did not attain conventional significance (P=0.053). In pooled analysis, there was a significant positive association (P < 0.05) between VLDL apoB secretion rate (mg/kg fat free mass/d) and waist-to-hip ratio (WHR), waist circumference, and fasting plasma triglyceride, insulin and glucose concentrations. In multiple linear regression analysis, the association between VLDL apoB secretion and fasting insulin concentration was independent of age, apolipoprotein E (apoE) genotype, mevalonic acid concentration, free fatty acid concentration and fat intake. CONCLUSION: Our findings are consistent with the hypothesis that in visceral obesity, insulin resistance and the associated increased lipid substrate supply to the liver contribute to hepatic oversecretion of apoB; expansion in the VLDL apoB pool size may also be due to a catabolic defect related to insulin resistance.

Adult↗

PubMed1998

Effects of beta-blockers on the concentration and oxidizability of plasma lipids.

1. beta-blockers improve morbidity and mortality after myocardial infarction, probably by several mechanisms. We investigated potentially relevant effects of beta-blockers in vivo and in vitro on plasma lipid oxidizability. Forty-two healthy men were randomized to receive placebo (13), metoprolol (14) or propranolol (15). 2. At 4 weeks, the effects on heart rate, blood pressure and lipids appeared similar and subjects taking a beta-blocker were combined. Compared with placebo, those on a beta-blocker gained 0.5 kg in weight (P = 0.04), heart rate fell from 63 to 52 beats/min (P < 0.0001) and blood pressure fell from 116/74 to 113/69 mmHg (P < 0.005); high-density lipoprotein (HDL)-cholesterol fell from 1.26 to 1.11 mmol/l (P = 0.005), there being no change in the ratio of free to esterified cholesterol in HDL, and there was an apparent rise in serum triacylglycerols from 1.18 to 1.43 mmol/l (P = 0.15 when adjusted for weight gain). Low-density lipoprotein (LDL)-cholesterol and lipoprotein (a) did not change. In this study, the oxidizability of LDL was unaffected by beta-blocker therapy. beta-blockade was not associated with any change in LDL fatty acid profile, or beta-carotene or alpha-tocopherol content which might account for the reduced LDL oxidizability previously reported in patients treated with beta-blockers. Furthermore, neither atenolol nor propranolol, at concentrations up to 100 mumol/l, had any effect on in vitro oxidizability of LDL obtained from healthy volunteers. 3. In contrast to the favourable haemodynamic effects conferred by beta-blockers, the effects on weight and serum triacylglycerols and HDL-cholesterol appear to be adverse and we did not demonstrate any changes in lipid oxidizability which might be relevant to the protective effects of beta-blockers in patients with coronary disease.

Adrenergic beta-Antagonists↗

PubMed1998

Supplementation with isoflavonoid phytoestrogens does not alter serum lipid concentrations: a randomized controlled trial in humans.

Isoflavonoids are a class of flavonoids that are derived in the human diet mainly from soybean-based foods. The major dietary isoflavonoids, genistein and daidzein, have estrogen-like activity and are classed as phytoestrogens. Because estrogens can lower serum LDL cholesterol and raise HDL cholesterol, the objective of this study was to determine if isoflavonoids could improve serum lipids in healthy subjects. Forty-six men and 13 postmenopausal women not receiving hormone replacement therapy completed a randomized, double-blind, placebo-controlled trial of two-way parallel design and 8 wk duration. One tablet containing 55 mg of isoflavonoids (predominantly in the form of genistein) or one placebo tablet was taken daily with the evening meal. Subjects maintained their usual diet and physical activity, which were unchanged throughout the intervention. Measurement of isoflavonoids and their metabolites in 24-h urine samples provided an assessment of compliance and of isoflavonoid metabolism. Serum total, LDL, HDL and HDL subclass cholesterol, triglycerides and lipoprotein (a) were assessed at baseline and during the last week of intervention. After adjustment for baseline values, no significant differences in postintervention serum lipid and lipoprotein (a) concentrations between groups were identified. Further adjustment for age, gender and weight change did not alter the results. In addition, changes in urinary isoflavonoids were not significantly correlated with changes in serum lipids and lipoprotein (a). Therefore, this study does not support the hypothesis that isoflavonoid phytoestrogens can improve the serum lipids, at least in subjects with average serum cholesterol concentrations.

Adult↗

PubMed1998

Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to oxidation and increase aortic cholesterol concentration of rabbits.

Early fatty streaks and advanced lesions are characterized by the deposition of cholesterol and cholesterol oxidation products (oxysterols). Oxysterols have been shown to be cytotoxic and pro-atherogenic compared to cholesterol and are found in cholesterol-rich processed foods. The consumption of dietary oxysterols may be significant in the onset and development of vascular disease. In order to study the short term effects of low levels of ingested dietary oxysterols on lipoprotein and aortic cholesterol and oxysterol levels, rabbits were fed either standard chow, chow supplemented with 1.0% oxidized cholesterol (containing 6% oxysterols), or 1.0% purified cholesterol (control). To determine the distribution and uptake of oxysterols after a 2-week dietary period, triglyceride-rich plasma lipoproteins, low density lipoproteins and aorta were analyzed by GC-MS. The concentration of 7beta-hydroxycholesterol was similar in all groups but the oxidized cholesterol-fed animals showed five times the concentration of 5alpha,6alpha-epoxycholesterol and double the level of 7-ketocholesterol in triglyceride-rich lipoproteins compared to the purified cholesterol-fed animals. The presence of 7-ketocholesterol in LDL was exclusive to animals fed the oxidized cholesterol diet. In addition, oxidation of triglyceride-rich lipoproteins was significantly greater in rabbits fed oxidized cholesterol compared to the pure cholesterol-fed animals. The oxidized cholesterol-fed animals also had a 64% increase in total aortic cholesterol, despite lower plasma cholesterol levels compared to the pure cholesterol control animals. Taken together these results suggest that dietary oxysterols may substantially increase the atherogenicity of lipoproteins.

Animals↗

PubMed1997

Absorption of dietary cholesterol oxidation products and incorporation into rat lymph chylomicrons.

Cholesterol oxidation products (oxysterols) induce macrophage lipid loading and accumulate in early arterial fatty streaks. The origin of lesion oxysterols has not been elucidated. The absorption of oxysterols from the diet and transport to the arterial wall by postprandial lipoprotein remnants may be a significant source. This study aimed to investigate the extent of oxysterol absorption and the effect on chylomicron composition. Cholesterol was heat-treated, causing 30% oxidation; the major oxidation products were 7 beta-hydroxycholesterol, 7-keto-cholesterol, 5 alpha,6 alpha-epoxycholesterol, and 5 beta,6 beta-epoxycholesterol. Conscious lymph-cannulated rats were given a bolus gastric infusion of 50 mg oxidized cholesterol or 50 mg purified cholesterol in a vehicle of triglyceride. In the rats given the oxidized cholesterol, 6% of the oxysterol load was absorbed and incorporated into lymph chylomicrons. Rats given pure cholesterol had no increase in oxysterols above baseline levels. The incorporation of oxysterols into lymph chylomicrons differed over time with 7 beta-hydroxycholesterol, having peak absorption at 3 h, followed by 7-ketocholesterol at 4 h and 5 alpha,6 alpha-epoxy-cholesterol at 5 h. The absorption of oxysterols in animals given the oxidized cholesterol gastric infusate was associated with lymph chylomicron compositional changes at 2-4 h. The oxidized cholesterol-treated group had a twofold increase in the cholesterol (890 +/- 84 micrograms vs. 440 +/- 83 microgram at 3 h) and triglyceride content (19.76 +/- 3.4 micrograms vs. 8.49 +/- 3.8 micrograms at 3 h). This led to a doubling of chylomicron size over this postprandial period, with particles having a mean diameter of 294 nm in the oxidized cholesterol-treated animals, compared to 179 nm in the purified cholesterol group. In conclusion, dietary oxysterols appear to influence postprandial lipoprotein particle size and composition. These changes may have effects on the clearance of chylomicrons from plasma, arterial delivery of oxysterols, and possible deposition in arterial lesions.

Animals↗

PubMed1997

Direct association between the hepatic secretion of very-low-density lipoprotein apolipoprotein B-100 and plasma mevalonic acid and lathosterol concentrations in man.

Apolipoprotein B-100 (apo B) is the principal structural and functional protein of the pro-atherogenic lipoproteins, but its homeostasis in man has not been clearly established. The hepatic availability of cholesterol substrate may be a determining factor. We examined whether there was a direct correlation between plasma concentrations of mevalonic acid (MVA) and lathosterol (indices of in vivo cholesterol synthesis) and hepatic secretion of very-low-density lipoprotein (VLDL) apo B in 13 normolipidaemic, healthy male subjects. The secretion of VLDL apo B was measured using a primed constant intravenous infusion of 1-[13C]-leucine (1 mg/kg per h) over 8 h. Gas-chromatography mass spectrometry (GCMS) was used to derive isotopic enrichment of apo B and fractional turnover rate was calculated using a monoexponential function. There was a highly significant positive correlation between the absolute secretion rate (ASR) of VLDL apo B and the plasma concentrations of mevalonic acid (r = 0.72, P = 0.005) and lathosterol (r = 0.81, P = 0.001) and the lathosterol:cholesterol ratio (r = 0.79, P = 0.001). In multiple regression analysis, these correlations remained significant after adjusting for waist circumference, age, apolipoprotein E genotype and dietary fat intake. The data further support the notion that the availability of cholesterol substrate regulates the hepatic secretion rate of apo B.

Adult↗

PubMed1997

The role of copper reduction by alpha-tocopherol in low-density lipoprotein oxidation.

The oxidation of lipoproteins is thought to be an important early step in atherogenesis. The measurement of lipid peroxidation in low-density lipoprotein (LDL) challenged with Cu2+ has become a widespread test to determine the "susceptibility" of LDL to oxidation. The determination of lag time to oxidation is thought to be a measure of the total antioxidant capacity of the LDL. However, we and others have failed to observe any correlation between lag time and the LDL content of its major lipid antioxidant, alpha-tocopherol. In fact, several studies now suggest a pro-oxidant role for tocopherol under some conditions of LDL oxidation. In the present study we sought to determine if there was a relationship between Cu2+ reduction by LDL and kinetic parameters of LDL oxidation. LDL (0.3 mmol/l cholesterol, approximately 0.1 mg protein/ml) was incubated at 30 degrees C with 2 microM Cu2+ and the formation of conjugated dienes measured over a 4-h period. Using neocuproine, an indicator molecule that specifically complexes Cu+ but not Cu2+, the reduction of Cu2+ by LDL was monitored. The final Cu concentration in these assays was 100 microM and neocuproine 750 microM. Cu+ formation was measured by absorbance at 454 nm. A strong negative correlation was observed between copper reduction by LDL and lag time to oxidation (r = -0.66, p < .005, n = 16). Further experiments showed that (1) LDL was able to reduce Cu2+ to Cu+ in a time and concentration-dependent manner; (2) blocking of free -SH groups on LDL apoprotein B by preincubation with dithionitrobenzoic acid (DTNB) had no significant effect on the rate and extent of Cu2+ reduction; (3) consumption of tocopherol in LDL undergoing oxidation with Cu was very rapid (rate = 6 x 10(-10) M s(-1)). When Cu+ formed during incubation with LDL was complexed with neocuproine, there was significant inhibition of LDL oxidation, as indicated by lipid peroxide formation and mobility on agarose gel electrophoresis. Surprisingly, tocopherol consumption was even more rapid in the presence of neocuproine, consistent with a shift in Cu2+/Cu+ equilibrium and faster reduction of Cu2+ by alpha-tocopherol. These results indicate that under these conditions tocopherol is a major reducing agent in LDL, converting Cu2+ to Cu+, and therefore, may play an important role in promoting LDL oxidation. However, there was no correlation between LDL tocopherol content and reduction of Cu2+. Examination of the time course of Cu2+ reduction in tocopherol enriched and depleted LDL indicates that tocopherol may determine Cu reduction at early time points but that the eventual capacity of LDL to reduce Cu may depend on more complex interactions between tocopherol and other LDL components.

Cations, Divalent↗

PubMed1997

Unexpected dose response of copper concentration on lipoprotein oxidation in serum: discovery of a unique peroxidase-like activity of urate/albumin in the presence of high copper concentrations.

Oxidative modification of low-density lipoprotein (LDL) may be an important factor in atherogenesis. The susceptibility of LDL to oxidation is usually determined in isolation by exposing LDL to oxidative stress induced by Cu ions or a free radical initiator. In these cases oxidation is carried out in the absence of water-soluble vitamins or serum proteins that may be present at the site of oxidation in vivo. We have examined the Cu2+-induced oxidation of lipoproteins in diluted serum. When oxidizing isolated LDL, there is a decrease in lag time with increasing concentration of Cu2+ until a minimum "lag time" is reached at a Cu:LDL ratio of about 50:1. In serum, we have shown an initial decrease in "lag time" with increasing Cu concentration up to 12.5 microM. However, with higher Cu concentrations "lag time" to oxidation increases, contrary to expectation, until a maximum is reached at about 50 microM Cu. This dose response observed for Cu oxidation of diluted serum was highly reproducible in a number of individual subjects. When serum was gel-filtered to remove low molecular weight compounds, the resulting filtrate behaved the same as isolated LDL. Uric acid was found to be an important component of the low molecular weight fraction responsible for the paradoxical effect of Cu concentration on serum oxidation. The same paradoxical effect was found when isolated LDL was incubated with uric acid in the presence of human serum albumin (HSA) and Cu. The incubation of HSA with reducing agents such as uric acid or bilirubin in the presence of high Cu concentrations, produces a "peroxidase-like" activity, capable of breaking down hydrogen peroxide as well as lipid hydroperoxides. The decomposition of lipid peroxides is a likely explanation for the longer serum oxidation lag times seen at higher Cu concentrations. Our study highlights the possible importance of interactions between uric acid and serum proteins in the presence of high metal ion concentrations.

Copper Sulfate↗