Effects of dietary virgin olive oil phenols on low density lipoprotein oxidation in hyperlipidemic patients

The aim of this study was to assess the effects of the dietary intake of extra virgin olive oil on the oxidative susceptibility of low density lipoproteins (LDL) isolated from the plasma of hyperlipidemic patients. Ten patients with combined hyperlipidemia (mean plasma cholesterol 281 mg/dL, triglycerides 283 mg/dL) consumed a low-fat, low-cholesterol diet, with olive oil (20 g/d) as the only added fat, with no drug or vitamin supplementation for 6 wk. Then they were asked to replace the olive oil they usually consumed with extra virgin olive oil for 4 wk. LDL were isolated at the beginning, and after the 4 wk of dietary treatment. LDL susceptibility to CuSO₄-mediated oxidation was evaluated by measuring the extent of lipid peroxidation. We also determined fatty acid composition and vitamin E in plasma and LDL and plasma phenolic content. Extra virgin olive oil intake did not affect fatty acid composition of LDL but significantly reduced the copper-induced formation of LDL hydroperoxides and lipoperoxidation end products as well as the depletion of LDL linoleic and arachidonic acid. A significant increase in the lag phase of conjugated diene formation was observed after dietary treatment. These differences are statistically correlated with the increase in plasma phenolic content observed at the end of the treatment with extra virgin olive oil; they are not correlated with LDL fatty acid composition or vitamin E content, which both remained unmodified after the added fat change. This report suggests that the daily intake of extra virgin olive oil in hyperlipidemic patients could reduce the susceptibility of LDL to oxidation, not only because of its high monounsaturated fatty acid content but probably also because of the antioxidative activity of its phenolic compounds.     

Antioxidant capacity of extra-virgin olive oils

In this study, the oxygen radical absorbance capacity (ORAC) of vegetable oils was investigated using a spectrofluorometric method, which measures the protection of the phenolic substances of the oil on the β-phycoerythrin fluorescence decay in comparison with Trolox. More than 97% of the phenolic substances was extracted from the oil using methanol, and the methanolic extract was then used for the ORAC and the total phenolics assay. We found a significant correlation between ORAC values of different olive oils and the total amount of phenolics. For extra-virgin olive oils, maximal ORAC values reached 6.20±0.31 μmol Trolox equivalent/g, while refined and seed oils showed values in the 1–1.5 μmol Trolox equivalent/g range. Our method is useful to assess the quality of olive oils and to predict, in combination with the rancidity tests, their stability against oxidation.     

Protective effect of oleuropein, an olive oil biophenol, on low density lipoprotein oxidizability in rabbits

On the basis of the results obtained with pilot studies conducted in vitro on human low density lipoprotein (LDL) and on cell cultures (Caco-2), which had indicated the ability of certain molecules present in olive oil to inhibit prooxidative processes, an in vivo study was made of laboratory rabbits fed special diets. Three different diets were prepared: a standard diet for rabbits (diet A), a standard diet for rabbits modified by the addition of 10% (w/w) extra virgin olive oil (diet B), a modified standard diet for rabbits (diet C) differing from diet B only in the addition of 7 mg kg⁻¹ of oleuropein. A series of biochemical parameters was therefore identified, both in the rabbit plasma and the related isolated LDL, before and after Cu-induced oxidation. The following, in particular, were selected: (i) biophenols, vitamins E and C, uric acid, and total, free, and ester cholesterol in the plasma; (ii) proteins, triglycerides, phospholipids, and total, free, and ester cholesterol in the native LDL (for the latter, the dimensions were also measured); (iii) lipid hydroperoxides, aldehydes, conjugated dienes, and relative electrophoretic mobility (REM) in the oxidized LDL (ox-LDL). In an attempt to summarize the results obtained, it can be said that this investigation has not only verified the antioxidant efficacy of extra virgin olive oil biophenols and, in particular, of oleuropein, but has also revealed a series of thus far unknown effects of the latter on the plasmatic lipid situation. In fact, the addition of oleuropein in diet C increased the ability of LDL to resist oxidation (less conjugated diene formation) and, at the same time, reduced the plasmatic levels of total, free, and ester cholesterol (−15, −12, and −17%, respectively), giving rise to a redistribution of the lipidic components of LDL (greater phospholipid and cholesterol amounts) with an indirect effect on their dimesions (bigger by about 12%).     

Fourier transform infrared spectroscopy evaluation of low density lipoprotein oxidation in the presence of quercetin,catechin, and α-tocopherol

We investigated the changes in human LDL primary and secondary lipid oxidation products and modification of the apolipoprotein B-100 (apoB-100) secondary structures during Cu²⁺-mediated oxidation by FTIR spectroscopy in the presence of catechin, quercetin, and α-tocopherol at physiological concentrations. Catechin- and quercetin-containing samples had slower rates and longer lag phases for conjugated diene hydroperoxide (CD) formation than α-tocopherol-containing samples; however, all antioxidant-treated LDL samples generated similar CD levels (P<0.05). A lower maximum (98.4 nmol/mg LDL protein) of carbonyl compounds was produced in the quercetin- and catechin-treated samples than in α-tocopherol samples. Modification of the apoB-100 secondary structures corresponded closely to the formation of carbonyls and was hampered by the presence of antioxidants. Physiological concentrations of catechin and quercetin offered similar levels of protection against modification by carbonyls of the apoB-100 at advanced stages (carbonyls ∼96.0 nmol/mg LDL protein) but not at the intermediate stages (carbonyls ∼58.0 nmol/mg LDL protein) of LDL oxidation probably owing to differences in the protein-binding mechanisms of catechin and quercetin. Relationships between peroxide formation, carbonyl products, and LDL protein denaturation were shown by the FTIR approach. The FTIR technique provided a simple new tool for a comprehensive evaluation of antioxidant performance in protecting LDL during in vitro oxidation.     

Effect of homocysteine,folates, and cobalamin on endothelial cell- and copper-induced LDL oxidation

Oxidation of LDL contributes to endothelial dysfunction and atherosclerosis. This process could be associated with hyperhomocysteinemia, a condition that can be reduced after folic acid treatment. Because a reduction in LDL oxidation may improve endothelial function, we studied the effect of some vitamins (folic acid, 5-methyltetrahydrofolic acid, and vitamin B-12) on LDL oxidation, either in the presence or absence of homocysteine. For this purpose, two in vitro systems were used: an endothelial cell-catalyzed LDL oxidation system and a cell-free copper-initiated LDL oxidation system. The kinetics of coppercatalyzed LDL oxidation was determined by continuous monitoring of the production of conjugated dienes in the reaction medium. TBARS production, a parameter of lipid peroxidation, was also evaluated. In both in vitro systems, only 5-methyl-tetrahydrofolic acid was able to decrease TBARS production in a concentration-dependent manner, independently of the presence or absence of homocysteine. In the copper-induced LDL oxidation system, vitamin B-12 and 5-methyltetrahydrofolic acid increased the lag time of conjugated diene production by 25 and 47%, respectively, suggesting that both vitamins in this system had antioxidant properties. Folic acid was unable to show antioxidant properties when included in either in vitro system. The results demonstrate that 5-methyltetrahydrofolic acid and vitamin B-12 are important protective agents against LDL oxidative modifications.     

Fenofibrate protects lipoproteins from lipid peroxidation: Synergistic interaction with α-tocopherol

One of the earliest steps of atherosclerotic plaque formation is an increase of circulating apolipoprotein B-containing lipoproteins which, after infiltrating the subendothelial space, undergo oxidative modification. Fenofibrate is an effective cholesterol- and triglyceride-lowering agent which has been shown to be beneficial in the treatment of atherosclerosis. Vitamin E, or α-tocopherol, is a powerful antioxidant which has been shown in a variety of studies to prevent lipoprotein peroxidation. The purpose of the present study was to investigate the effect of fenofibrate treatment, either alone or in combination with α-tocopherol, in reducing the susceptibility of lipoproteins to oxidative modification. Rats fed a normal diet were treated for up to 27 d with fenofibrate, either alone or in combination with equimolar doses of α-tocopherol. Combined VLDL (very low density lipoproteins) and LDL (low density lipoproteins) isolated after fenofibrate treatment were more resistant to coppermediated oxidation, as assessed by conjugated diene formation. Lag time was prolonged up to 3.2-fold, while the maximal rate of diene production was significantly decreased by up to 2.2-fold. Treatment of rats with α-tocopherol alone at the selected dose had no significant effect on lag time, while the propagation rate was slightly decreased. Coadministration of fenofibrate with α-tocopherol prolonged the lag phase to a greater extent than fenofibrate alone, showing a synergistic interaction between the two compounds. Finally, the combination of fenofibrate and α-tocopherol was significantly more effective in modifying lipoprotein oxidation parameters than what was observed with α-tocopherol and bezafibrate or gemfibrozil. Thus, in addition to its well-established effects on lipoprotein concentrations and atherogenic parameters, fenofibrate reduces the susceptibility of VLDL and LDL to oxidative modification and exerts its action synergistically with α-tocopherol.     

Antioxidant activity of grape extracts in a lecithin liposome system

Extracts of 14 different grapes were tested for their antioxidant activities in a copper-catalyzed lecithin liposome oxidation assay and analyzed for their phenolic components by high-performance liquid chromatography (HPLC). The total phenolic contents of the grape extracts varied from 176 to 1236 mg gallic acid equivalents (GAE)/L. Extracts of red wine grape varieties contained higher concentrations of phenolics than other varieties. When compared at the same 20 μM GAE basis, the grape extracts inhibited formation of conjugated diene hydroperoxides by 25.1 to 67.9%, and hexanal formation by 49.3 to 97.8%. Extracts of red table grape varieties Red Globe and Emperor and white wine grape varieties Chardonnay and Sauvignon Blanc gave the highest antioxidant activities. The relative percentage inhibition of conjugated dienes and hexanal correlated with total phenols (r=0.86 and 0.89). HPLC analyses showed that anthocyanins were the most abundant phenolic compounds in extracts of red grapes, and flavonols were most abundant in extracts of white grapes.     

Factors affecting resistance of low density lipoproteins to oxidation

Oxidation resistance (OR) of low density lipoproteins (LDL) is frequently determined by the conjugated diene (CD) assay, in which isolated LDL is exposed to Cu²⁺ as prooxidant in the range of 1–10 μM. A brief review on major findings obtained with this assay will be given. A consistent observation is that vitamin E supplements or oleic acid-rich diets increase OR. Oxidation indices measured by the CD assay and effects of antioxidants very significantly depend on the Cu²⁺ concentration used for LDL oxidation. For medium and high Cu²⁺ concentrations, the relationship between lag time and propagation rate can be described by a simple hyperbolic saturation function, which has the same mathematical form as the Michaelis-Menten equation. At medium and high Cu²⁺ concentrations (0.5 to 5 μM), vitamin E increases lag time in a dose-dependent manner. The increase is higher for 0.5 μM Cu²⁺ as compared to 5 μM. At low Cu²⁺ concentrations (0.5 μM or less), the mechanism of LDL oxidation changes. Significant oxidation occurs in a preoxidation phase, which commences shortly after addition of Cu²⁺. Preoxidation is not inhibited by vitamin E. It is concluded that much additional work is needed to validate the importance of oxidation indices derived from CD and similar assays.     

Antioxidant activity of red wine phenolic extracts towards oxidation of corn oil

The antioxidant activity of Xinomavro red wine phenolic extracts in corn oil stripped of tocopherols was evaluated. One wine extract, at 100 mg/L total phenolics, rich in phenolic acids and flavonols, inhibited the oxidation of corn oil stripped of tocopherols to a greater extent than butylated hydroxyanisole, at 200 mg/L. Moreover, another extract, at 100 mg/L total phenolics, rich in flavanols, flavonols and tyrosol, also exhibited high inhibitory action. The present results indicate that some red wine phenolics – such as phenolic acids, flavonols or flavanols – may be strong antioxidants in corn oil.     

Antioxidant Phenolics of Millet Control Lipid Peroxidation in Human LDL Cholesterol and Food Systems

Phenolic extracts from seven millet varieties, namely kodo, finger (Ravi), finger (local), proso, foxtail, little and pearl were evaluated for their inhibitory effects on lipid peroxidation in in-vitro copper-mediated human LDL cholesterol oxidation and several food model systems, namely cooked comminuted pork, stripped corn oil, and linoleic acid emulsion. The total phenolic content (TPC) and free radical scavenging activities were measured. The TPC ranged from 146 to 1156 μmol ferulic acid equiv (FAE)/g crude extract and the corresponding values based on defatted weight of grain ranged from 8.6 to 32.4 μmol FAE/g. At a final concentration of 0.05 mg/mL, millet extracts inhibited LDL cholesterol oxidation by 1–41%. All seven varieties exhibited effective inhibition of lipid oxidation in food systems used in this study and kodo millet exhibited superior inhibition of lipid peroxidation, similar to butylated hydroxyanisole at 200 ppm. Thus, millets may serve as a natural source of antioxidants in food applications and as a nutraceutical and functional food ingredient in health promotion and disease risk reduction.     

Antioxidative and synergistic effects of bene kernel and hull oils during oxidation of virgin olive oil

Total amounts of conjugated diene hydroperoxides and carbonyl compounds of a virgin olive oil (VOO) and its purified form as affected by 0.1–6% w/w bene kernel (BKO) and hull (BHO) oils were monitored during 16 h heating at 180°C. The VOO was more prone to the production of off‐flavour carbonyl compounds than to the formation of conjugated diene hydroperoxides. The VOOs oxidative stability decreased significantly due to the removal of the indigenous antioxidative compounds. Oxidative stability, especially regarding the secondary oxidation, significantly improved with increasing concentrations of the BKO than with those of the BHO.     

Postprandial and short-term effects of dietary virgin olive oil on oxidant/antioxidant status

It is generally believed that virgin olive oil consumption has beneficial effects, but little is known about its effects postprandially on oxidant/antioxidant status. The aim of this study was to determine changes in oxidative stress biomarkers and lipid profile after a single dose of virgin olive oil and after 1 wk of daily consumption. Sixteen subjects (9 men, 7 women) ingested 50 mL of virgin olive oil in a single dose. Blood samples were collected from 0 to 24 h. Thereafter, 14 participants (8 men, 6 women) followed a 1-wk 25 mg/d virgin olive oil dietary intervention. Blood samples were collected at the end of this period. Serum TAG (P=0.016), plasma FA (P<0.001) and lipid peroxidation products in plasma (P<0.001) and VLDL (P=0.007) increased, reaching a peak at 4–6 h, and returning to baseline values at 24 h after oil ingestion. The opposite changes were observed in plasma glutathione peroxidase (P=0.001) and glutathione reductase (GR) (P=0.042). No changes in LDL lipid peroxidation or resistance to oxidation were observed postprandially. At 24 h, plasma oleic acid remained increased (P<0.05) and resistance of LDL to oxidation improved (P<0.05). After 1 wk of virgin olive oil consumption, plasma oleic acid (P=0.031), resistance of LDL to oxidation (P<0.05), and plasma GR activity (P=0.005) increased. These results indicate that changes in oxidant/antioxidant status occur after oral virgin olive oil. Virgin olive oil consumption could provide short-term benefits for LDL resistance to oxidation and in glutathione-related enzyme activities.     

Oxidative stability of oils containing olive leaf extracts obtained by pressure,supercritical and solvent‐extraction

The effect of the addition of olive leaf (Olea europaea, cv. Arbequina) extracts, i.e. hydroalcoholic (ethanol–water 1:1; OHE), juice (OJ) and supercritical fluid‐CO₂ (OSFE) on the oxidative stability of vegetable oils with different unsaturation, such as soybean oil (SBO), canola oil (CO) and high oleic sunflower oil (HOSO), were studied at two concentrations (250 and 630 mg/kg oil, expressed as caffeic acid equivalent (CAE)). The extracts were characterized by the total phenolic content (Folin–Ciocalteau method), phenol chromatographic profiles (LC‐MS) and antioxidant activity (DPPH). OHE showed the highest phenol content (7.7 mg CAE/mL) while OJ and OSFE showed values of 5.4 and 2.2 mg CAE/mL, respectively. Oleuropein and its derivatives were the major phenolic compounds identified in OHE. The addition of 630 mg CAE/kg oil of OHE and OSFE to HOSO, SBO and CO showed an antioxidant effect, increasing significantly the induction time (IT) (p<0.05). That effect was highest when the system was more monounsaturated. In contrast, OJ showed a pro‐oxidant effect for all oils systems for both concentration studied. This behaviour could be attributed to the diphenol oxidase (PPO) activity.     

Plasma and lipoprotein lipid peroxidation in humans on sunflower and rapessed oil diets

The effects of natural mixed diets on lipid peroxidation were investigated in humans. In the first study, 59 subjects were fed a rapeseed oil-based diet rich in monounsaturated fatty acids (MUFA) and a sunflower oil-based diet rich in polyunsaturated fatty acids (PUFA) in a cross-over manner for three and a half weeks. The lipid peroxidation products in plasma were determined by measuring conjugated dienes and malondialdehyde (MDA). In a second study, plasma thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides, and the susceptibility of very low density lipoprotein + low-density lipoprotein (LDL) toin vitro oxidation were measured from subjects fed similar MUFA and PUFA diets for six week diets. No significant differences in plasma MDA or conjugated diene concentrations were found after the rapeseed oil diet or the sunflower oil diet in Study 1. In the second study, a small but significant decrease (P<0.05) in both lipid hydroperoxides and TBARS was observed in the LDL fraction after the sunflower oil diet. Thein vitro oxidation gave opposite results, showing increased oxidation after the sunflower oil diet. Despite a high intake of α-tocopherol during the oil peroids, no increase in plasma α-tocopherol was noticed in either study. The results suggest that moderate changes in the fatty acid composition in the Western-type diet may be adequate to affect lipoprotein susceptibility to oxidationin vitro, but there is considerable disparity with some indices ofin vivo lipid peroxidation.     

Oxidative Modification and Poor Protective Activity of HDL on LDL Oxidation in Thalassemia

Oxidative modification of low-density lipoprotein (LDL) has been reported in thalassemia, which is a consequence of oxidative stress. However, the levels of oxidized high-density lipoprotein (HDL) in thalassemia have not been evaluated and it is unclear whether HDL oxidation may be linked to LDL oxidation. In this study, the levels of total cholesterol, iron, protein, conjugated diene (CD), lipid hydroperoxide (LOOH), and thiobarbituric acid reactive substances (TBARs) were determined in HDL from healthy volunteers and patients with β-thalassemia intermedia with hemoglobin E (β-thal/Hb E). The protective activity of thalassemic HDL on LDL oxidation was also investigated. The iron content of HDL₂ and HDL₃ from β-thal/HbE patients was higher while the cholesterol content was lower than those in healthy volunteers. Thalassemic HDL₂ and HDL₃ had increased levels of lipid peroxidation markers i.e., conjugated diene, LOOH, and TBARs. Thalassemic HDL had lower peroxidase activity than control HDL and was unable to protect LDL from oxidation induced by CuSO₄. Our findings highlight the oxidative modification and poor protective activity of thalassemic HDL on LDL oxidation which may contribute to cardiovascular complications in thalassemia.     

Bene hull oil as a highly stable and antioxidative vegetable oil

Fatty acid composition, peroxide value, acid value, iodine value, saponification number, unsaponifiable matter content, total tocopherols and phenolics contents, and wax content of Bene hull oil (BHO) were determined and compared to those of Bene kernel oil (BKO) and extra‐virgin olive oil (EVOLO). Considering the fatty acid composition and total tocopherols and phenolics contents, the resistance to the production of conjugated diene hydroperoxides and carbonyl compounds during the heating process at 170 °C for BHO was about 4.2 and 7.3 times and about 1.7 and 2.0 times those of BKO and EVOLO, respectively. The antioxidant activity of BHO was exactly the same as that of tert‐butylhydroquinone at low concentrations (100 ppm).     

Quantitative determination of low density lipoprotein oxidation by FTIR and chemometric analysis

This study was conducted to develop a quantitative FTIR spectroscopy method to measure LDL lipid oxidation products and determine the effect of oxidation on LDL lipid and protein. In vitro LDL oxidation at 37°C for 1 h produced a range of conjugated diene (CD) (0.14–0.26 mM/mg protein) and carbonyl contents (0.9–3.8 μg/g protein) that were used to produce calibration sets. Spectra were collected from the calibration set and partial least squares regression was used to develop calibration models from spectral regions 4000-650, 3750-3000, 1720-1500, and 1180-935 cm⁻¹ to predict CD and carbonyl contents. The optimal models were selected based on their standard error of prediction (SEP), and the selected models were performance-tested with an additional set of LDL spectra. The best models for CD prediction were derived from spectral regions 4000-650 and 1180-935 cm⁻¹ with the lowest SEP of 0.013 and 0.013 mM/mg protein, respectively. The peaks at 1745 (cholesterol and TAG ester C=O stretch), 1710 (carbonyl C-O stretch), and 1621 cm⁻¹ (peptide C=O stretch) positively correlated with LDL oxidation. FTIR and chemometrics revealed protein conformation changes during LDL oxidation and provided a simple technique that has potential for rapidly observing structural changes in human LDL during oxidation and for measuring primary and secondary oxidation products.     

Antioxidative activity of 3,4-dihydroxyphenylacetic acid and caffeic acid in rat plasma

The purpose of the present paper is to study and compare in vitro the inhibitory effect of 3,4-dihydroxyphenylacetic acid (DOPAC) and caffeic acid (CA) on lipid peroxidation in rat plasma. Rat plasma was oxidized at 37°C by the radical initiators 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) or 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). The consumption of endogenous α-tocopherol (α-TOH) and the accumulation of conjugated diene hydroperoxides were measured by high-performance liquid chromatography and by ultraviolet spectroscopy, respectively. α-TOH was consumed at the same rate in the presence of 20 mM AAPH or 2 mM MeO-AMVN. DOPAC and CA suppressed the α-TOH consumption in a dose-dependent manner. A concentration of 50 μM of both phenolic acids was sufficient to induce a lag phase and to delay the rate of α-TOH consumption. The effect was more pronounced in rat plasma oxidation by AAPH than by MeO-AMVN. CA spared vitamin E more effectively than DOPAC in both oxidations. DOPAC and CA suppressed the formation of conjugated diene hydroperoxides. DOPAC and CA at concentration 50 μM suppressed α-TOH consumption during oxidation of soybean phosphatidylcholine (2.8 mM) multilamellar vesicles containing 15 μM α-TOH, in which the lipophilic initiator 2,2′-azobis (2,4-dimethylvaleronitrile) (6 mM) was incorporated. In conclusion, we demonstrated that DOPAC and CA in micromolar concentrations have antioxidant activity in rat plasma, a medium very close to the conditions in vivo, suggesting that supplementation with the phenolic acids will provide significant antioxidant protection.     

Influence of ascorbic acid and ascorbyl palmitate on the aroma composition of an oxidized vegetable oil and its emulsion

The formation of conjugated diene hydroperoxides and hexanal was compared to the development of aroma profiles during initial lipid oxidation of a vegetable oil and its 40% oil-in-water emulsion at 60°C. The aroma profiles of the oil and the emulsion with and without addition of ascorbic acid or ascorbyl palmitate were compared. The aroma compounds were isolated under a model mouth system and analyzed by gas chromatography/sniffing port analysis. Detectable odors were found and corresponded to 11 and 14 volatile compounds in the oil and the emulsion, respectively. The emulsion had higher lipid oxidation rates than the oil. Addition of ascorbic acid and ascorbyl palmitate had little influence on the aroma composition of the oil. In the emulsion, addition of these compounds resulted in diminished generation of odor active compounds. Results of measurements of conjugated diene hydroperoxides and headspace hexanal corresponded to that of the lipid oxidation rate in general, but predicted insufficiently the alterations in the aroma compositions by antioxidants.     

Olive oil phenolics protect LDL and spare vitamin E in the hamster

An animal feeding trial was conducted to investigate whether olive oil phenolics can act as functional antioxidants in vivo. To this end, hamsters were exposed for a period of 5 wk to a dietary regime with either a phenol-rich extra virgin olive oil or extra virgin olive oil from which phenols were removed by ethanol/water-washing. The original oil used in the high olive phenol diet was also used for the preparation of the low phenol diet in order to keep the FA compositions exactly the same. In addition, the vitamin E content was kept identical in both diets. This careful preparation of the diets was undertaken in order to prevent these factors from influencing the antioxidative status in plasma and LDL. Removal of olive oil phenols was shown to reduce both the vitamin E level in plasma and the resistance of LDL to ex vivo oxidation. The results of this study support the idea that extra virgin olive oil phenols improve the antioxidant defense system in plasma by sparing the consumption of vitamin E under normal physiological circumstances.