Influence of fish oil supplementation on in vivo and in vitro oxidation resistance of low-density lipoprotein in type 2 diabetes.

OBJECTIVE: Fish oil supplement has been proposed as a non-pharmacological strategy to correct the atherogenic lipid profile associated with type 2 diabetes mellitus. However, fish oil may have deleterious effects on lipid peroxidation and glycemic control. DESIGN: In this study, 44 type 2 diabetic patients were randomized to vitamin E standardized (53.6 mg/day) supplementation (capsules) with 4 g daily of either fish oil (n=23) or corn oil (n=21) for 8 weeks preceded by a 4 week run-in period of corn oil supplementation. LDL was isolated by density gradient ultracentrifugation and oxidized in vitro with Cu(2+). As a marker of in vivo oxidation malondialdehyde concentration in LDL (LDL-MDA) was measured. RESULTS: Fish oil reduced both mean lag time (before, 57.8; after, 48.8 min, P<0.001) and mean propagation rate (before, 0.018 DeltaOD/min; after, 0.015 DeltaOD/min, P<0.001), whereas corn oil had no influence on lag time and propagation rate. The changes in lag time and propagation rate differed significantly between fish oil and corn oil treatment. LDL-MDA changes differed borderline significantly between groups (FO, 110.4 pmol/mg protein; CO, 6.7 pmol/mg protein; P=0.057). Fish oil supplementation had no influence on glycemic control as assessed from HbA(1c) and fasting blood glucose. CONCLUSION: According to our findings, fish oil supplementation leads to increased in vivo oxidation and increased in vitro oxidation susceptibility of LDL particles. More studies are needed to clarify the clinical importance of this finding.

17beta-Estradiol but not the phytoestrogen naringenin attenuates aortic cholesterol accumulation in WHHL rabbits.

The effects of 17beta-estradiol (17beta-E(2)) or the phytoestrogen naringenin on spontaneous atherosclerosis were studied in 36 ovariectomized homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits receiving a semisynthetic control diet; this diet added 0.0040% 17beta-E(2;) or 0.20% naringenin, for 16 weeks. The uterine weight was increased (P < 0.001) and the concentration of estrogen receptor alpha was decreased (P < 0.001) in the 17beta-E(2) group compared with the controls. Total plasma cholesterol and triglycerides were not different from those in the controls. In lipoproteins, HDL cholesterol was increased (P < 0.01), and LDL triglyceride and IDL triglyceride were lowered (P < 0.05). The oxidation (as concentration of malondialdehyde) was increased in LDL (P < 0.05) but not in plasma. The cholesterol accumulation was decreased (P < 0.05) in the ascending aorta and in the total aorta but the ratio of intima to media and area of intima in ascending, thoracic, and abdominal aorta were not significantly different. In the naringenin group the only differences, compared with the control group, were increased HDL cholesterol (P < 0.001) and decreased activity of glutathione reductase (P < 0.05). In conclusion, 17beta-E(2), but not naringenin, attenuated aortic cholesterol accumulation independently of plasma and LDL cholesterol. Further, these results support previously suggested pro-oxidant ability of 17beta-E(2) toward LDL and a possible connection between the pro-oxidant nature of 17beta-E(2) and its antiatherogenic effect.

The effect of grape-skin extract on oxidative status.

Epidemiological studies indicate that moderate alcohol consumption, particularly wine, reduce the risk of CHD. The present study was designed to investigate the effect of grape-skin extract on markers of oxidative status. The study was designed as a randomised crossover. A diet with a low content of flavonoids was served with strict control of intake in two consecutive 1-week intervention periods to fifteen subjects (nine women, six men) divided randomly into two groups. During one of the weeks the subjects from either group consumed 200 ml grape-skin extract in water (1 mg extract/ml) at each of three daily meals (31.3 mg total phenolics, including 9.0 mg catechin). An increased activity of glutathione reductase and a borderline increase of glutathione peroxidase activity in erythrocytes were observed after grape-skin intervention, while the intervention had no significant effect on superoxide dismutase or catalase. Likewise, no effect was found on 2-aminoadipic semialdehyde (AAS) residues, a plasma protein oxidation product, or on malondialdehyde in plasma or in LDL, which are markers of lipoprotein oxidation. A marginal effect of grape-skin intervention was observed on plasma ascorbate levels. Intake of the experimental diet significantly reduced plasma vitamin C and plasma AAS in both groups. This effect was most pronounced in the particular week with no grape-skin extract addition. We speculate that grape-skin extract may have a sparing effect on vitamin C. The effects of the experimental diet may be partly ascribed to a low content of several fruit- and vegetable-related antioxidants like flavonoids and vitamin C and a relatively high content of carrot-derived antioxidants, such as carotenes.

Dose-response effects of lycopene on selected drug-metabolizing and antioxidant enzymes in the rat.

The administration of lycopene to female rats at doses ranging from 0.001 to 0.1 g/kg b.w. per day for 2 weeks was found to alter the drug-metabolizing capacity and antioxidant status of the exposed animals. An investigation of four cytochrome P450-dependent enzymes revealed that benzyloxyresorufin O-dealkylase activity in the liver was significantly induced in a dose-dependent fashion at all lycopene doses investigated. Likewise, ethoxyresorufin O-dealkylase activity was induced, although only at the two highest lycopene concentrations tested. An investigation of selected phase 2 detoxification enzymes provided evidence that lycopene was capable of inducing hepatic quinone reductase, approximately two-fold, at doses between 0.001 and 0.05 g/kg b.w. per day, whereas no effect was observed at the remaining doses tested. Glutathione transferase, using the two substrates, 2,4-dichloronitrobenzene and 1-chloro-2, 4-dinitrobenzene, was significantly induced at the 0.1 g/kg b.w. per day dose, whereas no effect was observed at the remaining lycopene doses. Analysis of the antioxidant status of the blood compartment revealed that three out of four antioxidant enzymes were affected by lycopene treatment. The activity of superoxide dismutase was thus significantly induced at lycopene doses of 0.005 and 0.05 g/kg b.w, whereas glutathione reductase and glutathione peroxidase was only induced at the 0.005 g/kg b.w. per day dose. For all antioxidant enzymes investigated, the activities seemed to return to the control level after exerting peak induction at doses between 0.005 and 0.05 g/kg b.w. per day. The explanation for this remains unknown. The plasma concentration of lycopene at dietary levels of 0.001, 0.005, 0.05 and 0.1 g/kg b.w. per day was estimated to be 16, 32, 71 and 67 nM, which is barely within the lower range of the mean human plasma concentration of lycopene, which ranges from 70-1790 nM. Oxidative stress induced by the heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), and investigated by analyzing for malondialdehyde in plasma, was not found to be affected by prior lycopene exposure. The level of PhIP-DNA adducts in the liver or colon was likewise not affected by lycopene at any dose. Overall, the present study provides evidence that lycopene administered in the diet of young female rats exerts minor modifying effects toward antioxidant and drug-metabolizing enzymes involved in the protection against oxidative stress and cancer. The fact that these enzymatic activities are induced at all of these very low plasma levels, could be taken to suggest that modulation of antioxidant and drug-metabolizing enzymes may indeed be relevant to humans, which in general exhibit a plasma lycopene level several fold above the effective levels observed in this study.

[Polyphenolic antioxidants in fruit juice. Urinary excretion and effects on biological markers for antioxidative status]. / Polyfenoliske antioxidanter i frugtjuice. Udskillelse i urin samt indflydelse på biomarkører for antioxidativ status.

This intervention study was designed as cross-over (four women, one man) with three doses of black currant/apple (1:1) juice (750, 1000, and 1500 mL) for one week corresponding to an intake of 4.8, 6.4, and 9.6 mg quercetin per day. Urinary excretion of quercetin increased significantly with dose and with time. The fraction excreted in urine was constant 0.29-0.47%. Plasma quercetin did not change with juice intervention. Plasma ascorbate increased during intervention due to ascorbate from the juice. Total plasma malondialdehyde decreased with time during 1500 mL juice intervention. Plasma protein 2-adipic semialdehyde residues, increased with time and dose, and glutathione peroxidase increased with juice dose, whereas other selected markers of oxidative status did not change. These effects might be related to several components of the juice and cannot be attributed solely to its quercetin content.

beta-carotene does not change markers of enzymatic and nonenzymatic antioxidant activity in human blood.

In vitamin A-replete populations, increased concentrations of serum carotenoids have been associated with a decreased risk of degenerative diseases. The mechanism of action of carotenoids in determining antioxidant activity is largely unknown. The aim of the study was to examine the effect of carotenoid supplementation and spinach intake on erythrocyte enzyme antioxidant activities, serum or plasma nonenzymatic antioxidant concentrations, and concentrations of oxidatively damaged amino acids in plasma. Subjects received for 3 wk a basic diet (n = 10), a basic diet with a carotenoid supplement (n = 12) or with a spinach product (n = 12 per group), i.e., whole-leaf, minced, liquefied or liquefied spinach plus added dietary fiber. After 3 wk of dietary intervention, changes in serum or plasma concentrations of ascorbic acid, alpha-tocopherol, FRAP (ferric reducing ability of plasma) and uric acid and erythrocyte enzyme activities were assessed, and differences among experimental groups were tested. Consumption of spinach resulted in greater (P < 0.01) erythrocyte glutathione reductase activity and lower (P < 0.05) erythrocyte catalase activity and serum alpha-tocopherol concentration compared with the control group. Consumption of the carotenoid supplement led to lower alpha-tocopherol responses (P = 0.02) compared with the basic diet only. Our data suggest that the short-term changes in erythrocyte glutathione reductase activity and serum alpha-tocopherol concentration can be attributed to an increased carotenoid (lutein and zeaxanthin) intake, but beta-carotene is unlikely to be a causative factor. Lower erythrocyte catalase activity after intervention with spinach products may be related to other constituents in spinach such as flavonoids.

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.

Effect of fruit juice intake on urinary quercetin excretion and biomarkers of antioxidative status.

BACKGROUND: Epidemiologic studies suggest that foods rich in flavonoids might reduce the risk of cardiovascular disease. OBJECTIVE: Our objective was to investigate the effect of intake of flavonoid-containing black currant and apple juice on urinary excretion of quercetin and on markers of oxidative status. DESIGN: This was a crossover study with 3 doses of juice (750, 1000, and 1500 mL) consumed for 1 wk by 4 women and 1 man corresponding to an intake of 4.8, 6.4, and 9.6 mg quercetin/d. RESULTS: Urinary excretion of quercetin increased significantly with dose and with time. The fraction excreted in urine was 0.29-0.47%. Plasma quercetin did not change with juice intervention. Plasma ascorbate increased during intervention because of the ascorbate in the juice. Total plasma malondialdehyde decreased with time during the 1500-mL juice intervention, indicating reduced lipid oxidation in plasma. Plasma 2-amino-adipic semialdehyde residues increased with time and dose, indicating a prooxidant effect of the juice, whereas erythrocyte 2-aminoadipic semialdehyde and gamma-glutamyl semialdehyde concentrations, Trolox-equivalent antioxidant capacity, and ferric reducing ability of plasma did not change. Glutathione peroxidase activity increased significantly with juice dose. CONCLUSIONS: Urinary excretion of quercetin seemed to be a small but constant function of quercetin intake. Short-term, high intake of black currant and apple juices had a prooxidant effect on plasma proteins and increased glutathione peroxidase activity, whereas lipid oxidation in plasma seemed to decrease. These effects might be related to several components of the juice and cannot be attributed solely to its quercetin content.

Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat.

1. Gavage administration of the natural flavonoids tangeretin, chrysin, apigenin, naringenin, genistein and quercetin for 2 consecutive weeks to the female rat resulted in differential effects on selected phase 1 and 2 enzymes in liver, colon and heart as well as antioxidant enzymes in red blood cells (RBC). 2. Glutathione transferase (GST) activity assayed by use of the substrate 1-chloro-2,4-dinitrobenzene was significantly induced by apigenin, genistein and tangeretin in the heart but not in colon or liver. 3. In RBC chrysin, quercetin and genistein significantly decreased the activity of glutathione reductase (GR), catalase (CAT) and glutathione peroxidase (GPx), whereas superoxide dismutase (SOD) was only significantly decreased by genistein. 4. The oxidative status of the animal, measured as plasma malondialdehyde, revealed that chrysin, quercetin, genistein, and beta-naphthoflavone (BNF) significantly protected against, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced oxidative stress. Hepatic PhIP-DNA adduct formation was not affected by any of the administered flavonoids, whereas PhIP-DNA adduct formation in colon was slightly, but significantly, inhibited by quercetin, genistein, tangeretin and BNF. 5. The observed effects of chrysin, quercetin and genistein on antioxidant enzymes, concurrently with a protection against oxidative stress, suggest a feedback mechanism on the antioxidant enzymes triggered by the flavonoid antioxidants. 6. Despite the use of high flavonoid doses, which by far exceed the human exposure levels, the effect on drug metabolizing and antioxidant enzymes was still very minor. The role of singly administered flavonoids in the protection against cancer and heart disease is thus expected to be limited.

Effect of parsley (Petroselinum crispum) intake on urinary apigenin excretion, blood antioxidant enzymes and biomarkers for oxidative stress in human subjects.

Seven men and seven women participated in a randomized crossover trial to study the effect of intake of parsley (Petroselinum crispum), containing high levels of the flavone apigenin, on the urinary excretion of flavones and on biomarkers for oxidative stress. The subjects received a strictly controlled diet low in flavones and other naturally occurring antioxidants during the 2 weeks of intervention. This basic diet was supplemented with parsley providing 3.73-4.49 mg apigenin/MJ in one of the intervention weeks. Urinary excretion of apigenin was 1.59-409.09 micrograms/MJ per 24 h during intervention with parsley and 0-112.27 micrograms/MJ per 24 h on the basic diet (P < 0.05). The fraction of apigenin intake excreted in the urine was 0.58 (SE 0.16)% during parsley intervention. Erythrocyte glutathione reductase (EC 1.6.4.1; GR) and superoxide dismutase (EC 1.15.1.1; SOD) activities increased during intervention with parsley (P < 0.005) as compared with the levels on the basic diet, whereas erythrocyte catalase (EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.9) activities did not change. No significant changes were observed in plasma protein 2-adipic semialdehyde residues, a biomarker of plasma protein oxidation. In this short-term investigation, an overall decreasing trend in the activity of antioxidant enzymes was observed during the 2-week study. The decreased activity of SOD was strongly correlated at the individual level with an increased oxidative damage to plasma proteins. However, the intervention with parsley seemed, partly, to overcome this decrease and resulted in increased levels of GR and SOD.