Microbiological oxidation of sterols by Coriolus hirstus

Fermentation of cholesterol with a culture of Coriolus hirstus yielded a mixture containing 7-oxo-cholesterol and hydroxylated-cholesterol derivatives. Preparation of a possible precursor of antheridiol, 7-oxofucosterol, by this fungus was also examined.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Modulation of LDL oxidation by 7,8-dihydroneopterin

Human macrophages stimulated with interferon-γ generate neopterin and 7,8-dihydroneopterin which interfere with reactive species involved in LDL oxidation. While neopterin was found to have pro-oxidative effects on copper-mediated LDL oxidation, the influence of 7,8-dihydroneopterin is more complex. This study provides detailed information that 7,8-dihydroneopterin reveals both pro-oxidative and anti-oxidative effects on copper mediated LDL oxidation. 7,8-dihydroneopterin inhibited the oxidation of native LDL effectively monitored by (i) formation of conjugated dienes, (ii) relative electrophoretic mobility (EM) and (iii) specific oxidized epitopes. Using minimally oxidized LDL (mi-LDL) or moderately oxidized LDL (mo-LDL) 7,8-dihydroneopterin changed its antioxidative behavior to a strongly pro-oxidative. Incubation of 7,8-dihydroneopterin with native LDL, mi-LDL or mo-LDL in the absence of copper ions showed that formation of conjugated dienes was more increased in mo-LDL than in mi-LDL while no diene formation was observed with native LDL.

We suggest that 7,8-dihydroneopterin is a modulator for LDL oxidation in the presence of copper ions depending on the “oxidative status” of this lipoprotein.     

Free radicals generated during oxidation of green tea polyphenols: Electron paramagnetic resonance spectroscopy combined with density functional theory calculations

Electron paramagnetic resonance spectroscopy and density functional theory calculations have been used to investigate the redox properties of the green tea polyphenols (GTPs) (?)-epigallocatechin gallate (EGCG), (?)-epigallocatechin (EGC), and (?)-epicatechin gallate (ECG). Aqueous extracts of green tea and these individual phenols were autoxidized at alkaline pH and oxidized by superoxide anion (O₂^?) radicals in dimethyl sulfoxide. Several new aspects of the free radical chemistry of GTPs were revealed. EGCG can be oxidized on both the B and the D ring. The B ring was the main oxidation site during autoxidation, but the D ring was the preferred site for O₂^? oxidation. Oxidation of the D ring was followed by structural degradation, leading to generation of a radical identical to that of oxidized gallic acid. Alkaline autoxidation of green tea extracts produced four radicals that were related to products of the oxidation of EGCG, EGC, ECG, and gallic acid, whereas the spectra from O₂^? oxidation could be explained solely by radicals generated from EGCG. Assignments of hyperfine coupling constants were made by DFT calculations, allowing the identities of the radicals observed to be confirmed.     

Factors Affecting Events During Oxidation of Low Density Lipoprotein: Correlation of Multiple Parameters of Oxidation

The present study shows that copper oxidation of LDL is a tightly-ordered process which can be finely controlled by appropriate selection of duration of oxidation and of concentrations of LDL and copper. Oxidation of LDL (0.1-2.0 mg LDL protein/ml) was carried out by copper catalysis (in the ratio of 2.5 μM Cu²⁺ to 0.1 mg LDL protein/ml) in phosphate-buffered saline, and was monitored by agarose gel electro-phoresis, gas chromatography (GC), anion exchange fast protein liquid chromatography (FPLC), fluorescence spectroscopy and dynamic light scattering. Analysis of the data showed strong cross correlations between many of the parameters of oxidation. Oxidation was more rapid for lower concentrations than for higher concentrations of LDL, despite the same ratio of copper to LDL being employed. Chemical kinetics analysis of the GC data suggested that 7β-hydroxycholesterol formation occurred as a first order (or pseudo first order) consecutive reaction to the oxidation of linoleate. The first order rate constants for decomposition of lioleate and production of 7β-hydroxycholesterol correlated closely with the theoretically-calculated times between collision of LDL particles. LDL particle diameter, measured by dynamic light scattering, increased by ca. 50% over 24 h oxidation, suggesting unfolding of apo B-100.

Prolonged oxidation of LDL at low concentration suggested that the radical chain reaction was able to propagate, albeit slowly, on cholesterol after all the polyunsaturated fatty acid was consumed. For higher concentrations of LDL, prolonged oxidation resulted in partial aggregation. These findings are applicable to preparing oxidised LDL with different degrees of oxidation, under controlled conditions, for studying its biological properties.     

In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma

Recently we identified four conjugated glucuronide metabolites of epicatechin, (-)-epicatechin-3'-O-glucuronide (E3'G), 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide (4'ME3'G), (-)-epicatechin-7-O-glucuronide (E7G) and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide (3'ME7G) from plasma and urine. E3'G and 4'ME3'G were isolated from human urine, while E7G and 3'ME7G were isolated from rats that had received oral administration of (-)-epicatechin (Natsume et al. (2003), Free Radic. Biol. Med. 34, 840-849). It has been suggested that these metabolites possess considerable in vivo activity, and therefore we carried out a study to compare the antioxidant activities of the metabolites with that of the parent compound. This was achieved by measuring superoxide scavenging activity, reduction of plasma TBARS production and reduced susceptibility of low-density-lipoprotein (LDL) to oxidation. (-)-Epicatechin was found to have more potent antioxidant activity than the conjugated glucuronide metabolites. Both (-)-epicatechin and E7G had marked antioxidative properties with respect to superoxide radical scavenging activity, plasma oxidation induced by 2,2'-azobis-(2-aminopropane) dihydrochloride (AAPH) and LDL oxidation induced by copper ions or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). In contrast, the other metabolites had light antioxidative activities over the range of physiological concentrations found in plasma.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

Assays for cholesterol 7 alpha-hydroxylase and 12 alpha-hydroxylase using high performance liquid chromatography

Rapid and accurate assay methods for cholesterol:NADPH oxidoreductase (EC 1.14.13.17, 7 alpha-hydroxylating) and 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase (enzyme not yet registered) are described. 7 alpha-Hydroxylase utilizes the endogenous cholesterol of liver microsomes as substrate. The reaction products were separated by high performance liquid chromatography monitored at 214 nm. Much higher activity was obtained with the method compared to literature values, which were obtained using externally added radioactive cholesterol as the substrate. The 12 alpha-hydroxylase activity was measured using non-radioactive steroid as the substrate. The reaction products were separated by the chromatography and detected at 240 nm. Comparable activities were obtained by this method compared to those that were obtained using radioactive substrate.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

The production of hydroxyl radical from hydrogen peroxide

The formation of hydroxyl radical (OH·) from the oxidation of glutathione, ascorbic acid, NADPH, hydroquinone, catechol, and riboflavin by hydrogen peroxide was studied using a range of enzymes and copper and iron complexes as possible catalysts. Copper-1,10-phenanthroline appears to catalyze the production of OH· from hydrogen peroxide without superoxide radical being formed as an intermediate, and without the involvement of a catalyzed Haber-Weiss (Fenton) reaction. Superoxide radical is involved, however, in the Cu²⁺ -catalyzed decomposition of hydrogen peroxide, and in the oxidation of glutathione by atmospheric oxygen. For this latter oxidation, copper-4,7-dimethyl-1,10-phenanthroline was found to be a much more effective catalyst than the copper complex of 1,10-phenanthroline, which is normally used. Mechanisms for these reactions are proposed, and the toxicological significance of the ability of a variety of biological reductants to provide a prolific source of OH· when oxidized by hydrogen peroxide is discussed.     

Persistent enhancement of bile acid synthesis in guinea pigs following stimulation of cholesterol catabolism in neonatal life

Cholesterol catabolism to bile acids was stimulated in neonatal guinea pigs by feeding 1,11% cholestyramine (CT)-containing diet for 8 weeks. The animals were then switched to standard laboratory diet for an additional 4 weeks. At the end of the laboratory diet period: a) CT-pre-treated guinea pigs continued to excrete significantly higher (p<0.05) amounts of bile acids, b) the activity of hepatic 7α-hydroxylase was significantly elevated (p<0.01) in CT-pre-treated animals, and c) isolated hepatocytes from CT-pre-treated guinea pigs secreted significantly higher (p<0.05) amounts of bile acid when compared to controls during a 4-hour incubation. These data provide biochemical support for our contention that stimulation of cholesterol catabolism during neonatal life can have effects that persist into adult life.     

Enhanced oxidation of flavan-3-ols and proanthocyanidin accumulation in water-stressed tea plants

(-)-Epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two major tea flavan-3-ols, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, flavan-3-ols serve as proanthocyanidin (PA) building blocks, and although both monomeric flavan-3-ols and PAs show antioxidant activity in vitro, their antioxidant function in vivo remains unclear. In the present study, EC quinone (ECQ) and EGCG quinone (EGCGQ), the oxidation products of EC and EGCG, increased up to 100- and 30-fold, respectively, in tea plants exposed to 19 days of water deficit. Oxidation of EC and EGCG preceded PAs accumulation in leaves, which increased from 35 to 53 mg gDW(-1) after 26 days of water deficit. Aside from the role monomeric flavan-3-ols may play in PAs biosynthesis, formation of ECQ and EGCGQ strongly negatively correlated with the extent of lipid peroxidation in leaves, thus supporting a protective role for these compounds in drought-stressed plants. Besides demonstrating flavonoid accumulation in drought-stressed tea plants, we show for the first time that EC and EGCG are oxidized to their respective quinones in plants in vivo.     

Copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate act as inhibitors of Ribonuclease A

Green tea polyphenols, which have the ability to inhibit angiogenesis, form complexes with Cu(II), a known potent stimulator of blood vessel proliferation. Copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate were found to inhibit the enzymatic activity of Ribonuclease A (RNase A) as revealed by an agarose gel based assay and urea denatured gel electrophoresis. The copper complexes were found to be non-competitive inhibitors of RNase A with inhibition constants in the micromolar range. Changes in the secondary structure of the protein are found to occur due to the interaction as revealed from Fourier transform infrared and circular dichroism studies.     

Methoxylation of methyl 3α,7α-dihydroxychol-4-en-24-oate and its 3β-epimer. —a contribution to chenodeoxycholic acid biogenesis

By the conventional methods of gas liquid chromatography (GLC) as well as mass spectrometry, 3β,7α-dihydroxychol-5-en-24-oic acid (Δ⁵-acid), a key intermediate of chenodeoxycholic acid biogenesis and its metabolic by-product, 3α,7α-dihydroxychol-4-en-24-oic acid (Δ⁴-acid) have not yet been identified as such probably due to thermal decomposition. However, taking advantage of the observation that they are readily methoxylated in methanoi containing a trace of acids, their individual methoxy-compounds were easily prepared and proved to be useful for their identification, even though they are present in minimal amounts as was the case with the human or hen bile. The present paper reported physical as well as spectral properties of the methoxy-compounds derived from methyl 3α,7α-dihydroxychol-4-en-24-oate, compared with those of its 3β-epimer     

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts: II. Effects of sterol biosynthesis inhibitors on cell division

The relationships between cholesterogenesis and cell division were studied by using two inhibitors of hydroxymethylglutaryl-CoA reductase activity — 25-hydroxycholesterol and compactin. The effects of both compounds on DNA synthesis were compared in synchronized rat fibroblasts cultured in a cholesterol-containing medium. Compactin did not inhibit DNA synthesis, except after a long time of contact and at high and almost cytotoxic concentrations. 25-Hydroxycholesterol inhibited DNA synthesis (without cytotoxic effects) after only 9–16 h of contact, depending on the phase of the cell cycle at which this compound was added to the culture medium. Sensitivity of cells to 25-hydroxycholesterol was maximal at the end of the S phase/beginning of the G₂M phase. The rapid effect of 25-hydroxycholesterol on DNA synthesis appears to be separate from the inhibitory effect on sterol or non-sterol mevalonate-derived compound synthesis. Indeed, under our experimental conditions, the suppression of cholesterol biosynthesis is compensated by the presence of cholesterol in the culture medium, as demonstrated by the lack of effect of compactin on DNA synthesis; moreover, addition of mevalonolactone to the culture medium did not reverse the effect of 25-hydroxycholesterol. 25-Hydroxycholesterol could inhibit DNA synthesis by a direct action on the nucleus, after transfer by the intermediary of a specific hydroxysterol-binding protein.     

Cholesterol oxidation and the behavior of 5-α-hydroperoxycholesterol at the air/water interface

The oxidation of cholesterol and the behavior of an oxidized sterol, 5-α-hydroperoxy-cholesterol (5-AHC), have been investigated. It is demonstrated that previous work is correct in observing that cholesterol oxidation does take place at the air/water interface, but predicts initial effects and rates that are much too large. The oxidation of cholesterol is found to be autocatalytic as long as the oxidized sterol compounds (OSC) remain miscible with the cholesterol. The OSC are postulated to adopt tilted conformations with respect to the air/water interface when oxidized at or about the sterol-5,6-positions, and to segregate out when saturation OSC levels in cholesterol are reached. Pure films of 5-AHC are found to be more expanded, more compressible and less stable than those of cholesterol. In mixed films with other selected lipids, 5-AHC behaves as a greater impurity than does cholesterol when the second component is more condensed, and as a poorer condensing agent when the second component is more expanded.     

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C₇ having a relatively weak carbon–hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen (¹ΔgO₂) and ozone (O₃) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ_5,6 –double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.     

A sensitive chemiluminescence method to measure the lipoxygenase catalyzed oxygenation of complex substrates

Oxidative modification of low-density lipoprotein (LDL) has been implicated as a patho-physiological process in early atherogenesis and 15-lipoxygenases (15-LOX) may be involved. While studying the in vitro kinetics of the 15-LOX/LDL interaction, we found that the conventional spectrophotometric assays failed in the range of substrate saturation owing to the high optical density of concentrated LDL solutions. Therefore, we developed a much more sensitive assay system which was based on peroxide induced isoluminol enhanced chemiluminescence. With this method reliable kinetic data were obtained at LDL concentrations of up to 1 mg/ml. To validate this luminometric method the kinetic parameters of 15-LOX catalyzed oxygenation of linoleic acid (K_m=3.7 μM, k_cat=17 s^?1) were determined and we observed a good agreement with previously published data obtained with a spectrophotometric assay. Moreover, we found that the kinetic constants of 15-LOX catalyzed LDL oxidation (K_m=0.64 μM, k_cat=0.15 s^?1) are quite different from those of free fatty acid oxygenation and that the cholesterol esters are preferentially oxidized during 15-LOX/LDL interaction. Vitamin E depletion does not reduce the rate of LDL oxidation and analysis of the structure of the oxygenation products suggests that the majority of the products were formed via direct LOX catalyzed oxidation of LDL ester lipids. The luminometric method described here is not restricted to the measurement of LOX catalyzed LDL oxidation, but may also be used to determine kinetic constants for the oxidation of other complex substrates such as biomembranes or liposomes.     

The role of antioxidants in the long-term glycation of low density lipoprotein and its Cu2+-catalyzed oxidation

In the present study we investigated the influence of antioxidants such as EDTA, α-tocopherol, troglitazone and acetylsalicylic acid on the long-term-glycation of LDL and its copper ion-catalyzed oxidation. We observed that (a) all antioxidants inhibited AGE-formation, while Amadori product formation was only diminished by extreme concentrations of acetylsalicylic acid, (b) glycated LDL was more susceptible to coppercatalyzed oxidation than unglycated LDL, and (c) the oxidation of native LDL was more dramatically inhibited by the antioxidants than that of glycated LDL. The observed differences may be a consequence of the significantly higher endogenous content in hydroperoxides of glycated LDL as compared to native LDL. Therapeutic implications of these findings regarding vitamin E, which is supposed to slow atherogenesis and the development of microvascular complications in diabetes, are obvious: Vitamin E-monotherapy, while blocking oxidative and AGE-modification of LDL, is unable to inhibit its AP-formation. As a consequence, tocopherol is susceptible to increased consumption by AP-associated radical production in hyperglycemic patients, which could be checked in part by the tocopherol-protecting agent troglitazone and/or by acetylsalicylic acid.     

In vitro inhibition of rat liver cholest-5en-3 beta-ol (cholesterol) biosynthesis by non-mercurial sulfhydryl reagents.

In vitro conversion of 2-14C-mevalonate to cholest-5en-3 beta-ol (cholesterol) in rat liver homogenates is inhibited by arsenite, beta-mercaptoethanol, dithiothreitol and ethanethiol. Two sterols containing 20 carbon atoms accumulate under these conditions. One of these is identified as 4,4 dimethyl-5alpha-cholest-8en-3beta-ol and the other tentatively identified as 4,4 dimethyl-5alpha-cholest-8,24-dien-3beta-ol. Based on these observations, these non-mercurial sulfhydryl reagents do not inhibit 5alpha-lanosta-8,24-dien-3beta-ol 14alpha demethylase.