Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as antiatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-L-ascorbic acid (DHA) strongly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, as AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reducing capacity. Here we report that human LDL (0.4 mg protein/ml) incubated with 40 μM Cu²⁺ binds 28.0 ± 3.3 Cu ions per LDL particle (mean ± SD, n = 10). Co-incubation of LDL with AA or DHA led to the time- and concentration-dependent release of up to 70% of bound Cu, which was associated with the inhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also led to the time-dependent formation of 2-oxo-histidine, an oxidized derivative of histidine with a low affinity for Cu. Addition of free histidine prevented the formation of the LDL-Cu complexes and inhibited LDL oxidation, despite the fact that Cu remained redox-active. Interestingly, histidine was more effective than AA or DHA at limiting Cu binding to LDL, but at low concentrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu binding sites on LDL: those that bind Cu in a redox-active form critical for initiation of LDL oxidation, and those that bind Cu in a redox-inactive form not contributing to LDL oxidation. The former sites may be primarily histidine residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the unusual inhibitory effect of vitamin C on Cu-induced LDL oxidation.     

Browning and decomposed products of model orange juice

A model solution of orange juice containing sugars, ascorbic acid, and citric acid was prepared and its browning during storage was examined. The solution gradually turned brown. Ascorbic acid (AsA) most contributed to the browning. Citric acid and such amino acids as Arg and Pro promoted the browning. DTPA, a strong chelator, inhibited the browning. 3-Hydroxy-2-pyrone (3OH2P), 5-hydroxymethylfurfural (HMF), furfural, 5-hydroxymaltol, and 2-furoic acid were identified as decomposed products in the stored solution. When 3OH2P was stored, the solution turned slightly brown. Furfural solution added with amino acids turned yellow. 3OH2P showed a positive relation with the browning of retail orange juice during storage.     

Docosahexaenoic acid, a constituent of rodent fetal serum and fish oil diets, inhibits acquisition of macrophage tumoricidal function

Macrophage (M phi) activation is deficient in the fetus and neonate, at times when the serum concentration of docosahexaenoic acid (DHA; 22:6n3) is approximately 10-fold higher than in the adult. We tested the effects of highly purified DHA on M phi activation in vitro. M phi were stimulated with rIFN-gamma plus either of two second or "triggering" signals, LPS or heat-killed Listeria monocytogenes. M phi activation was assayed as the lysis of P815 mastocytoma cells, which are resistant to TNF-alpha. DNA inhibited the activation of peritoneal M phi and the M phi line RAW264.7 in a dose-dependent manner at concentrations between 20 and 160 microM. These concentrations are found in fetal and neonatal rodent sera. Another polyunsaturated fatty acid, arachidonic acid (20:4n6), was much less inhibitory. In contrast to its profound effect on tumoricidal activation, DHA did not inhibit phagocytosis and catabolism of 125I-heat-killed Listeria monocytogenes. Increasing the rIFN-gamma or second signals reduced the inhibition of tumoricidal activation by DHA but not M phi incorporation of 14C-DHA. When the rIFN-gamma and second signals were separated in time, DHA was far more inhibitory if delivered with the triggering signal than if delivered with the rIFN-gamma. However, the incorporation of 14C-DHA was the same under these two conditions. In M phi treated with DHA during LPS stimulation, the inhibition was time-dependent, requiring more than 2 h. Although DHA inhibits cyclooxygenase activity, its inhibition of M phi activation was not reversed with the following cyclooxygenase products: PGE2, a stable TXA2 analog (U-46, 619) or a stable PGI2 analog (Iloprost). Although DHA is metabolized by lipoxygenases, the inhibition was not reversed by the lipoxygenase inhibitors 5, 8, 11, 14-eicosatetraynoic acid and nordihydroguaiaretic acid. Altogether, the data indicate that DHA, at concentrations present in fetal and neonatal sera, inhibits M phi activation and may contribute to the previously observed deficits in M phi function in the fetus and neonate.     

L-ascorbic acid microencapsulated with polyacylglycerol monostearate for milk fortification

Efficiency was examined of microencapsulating L-ascorbic acid by polyglycerol monostearate (PGMS), and changes in the chemical and sensorial aspects of L-ascorbic acid and/or iron-fortified milk during storage were evaluated. The selected core materials were ferric ammonium sulfate and L-ascorbic acid. The highest efficiency (94.2%) of microencapsulation was found with the ratio of 5:1 as the coating to core material. The release of ascorbic acid from the microcapsules increased sharply from 1.6 to 6.7% up to 5 d of storage. The TBA value was the lowest in the milk sample with added encapsulated iron and unencapsulated L-ascorbic acid up to 5 d of storage in comparison with the other treated samples. A sensory analysis showed that most aspects were not significantly different between the control and fortified samples encapsulated with ascorbic acid after 5 d of storage. The results indicate that L-ascorbic acid microencapsulated with PGMS can be applied to fortify milk and acceptable milk products can be prepared with microencapsulated L-ascorbic acid and iron.     

N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle

The positive effect of n-3 polyunsaturated fatty acids (FAs) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 polyunsaturated fatty acids) during IVM has an effect on oocyte maturation and in vitro embryo development in cattle. We reported that DHA (0, 1, 10, or 100 μM) had no effect on oocyte viability or maturation rate after 22-hour IVM. Incubation of oocyte-cumulus complexes with 1-μM DHA during IVM significantly increased (P < 0.05) oocyte cleavage rate as compared with control (86.1% vs. 78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (39.1% vs. 29.7%, respectively). Supplementation with 1 μM DHA during IVM also induced a significant increase in the blastocyst rate at Day 7 after IVF as compared with control (30.6% vs. 17.6%, respectively) and tended to increase the number of cells in the blastocysts (97.1 ± 4.9 vs. 81.2 ± 5.3, respectively; P = 0.08). On the contrary, 10-μM DHA had no effects, whereas 100-μM DHA significantly decreased the cleavage rate compared with control (69.5% vs.78.8%, respectively) and the greater than 4-cell embryo rate at Day 2 after parthenogenetic activation (19.5% vs. 29.7%). As was shown by real-time polymerase chain reaction, negative effects of 100-μM DHA were associated with significant increase of progesterone synthesis by oocyte-cumulus complexes, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CCs) of corresponding oocytes. Docosahexaenoic acid at 1 and 10 μM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1 μM) during IVM may have beneficial effects on oocyte developmental competence in vitro without affecting lipid metabolism gene expression in surrounding CCs, contrarily to 100 μM DHA which diminished oocyte quality associated with perturbation of lipid and steroid metabolism in CC.     

Seasonal variation in fatty acid composition of silver pomfrets,Pampus argenteus (Euphrasen), in Kuwait waters

Proximate and fatty acid composition of wild silver pomfrets, Pampus argenteus, were studied in Kuwait waters for a period of 1 year (November 2007–October 2008) to see whether there were any seasonal compositional differences between males and females. Ten adults (five males, five females) were sampled each month during (i) Pre‐spawning (March–May), (ii) Spawning (June–August), (iii) Post‐spawning (September–November), and (iv) Winter (December–February). Both sexes had significantly (P < 0.05) higher whole body moisture and lower crude protein and lipid contents in winter compared to the respective males and females sampled in other seasons. However, females had significantly higher (9.1%) lipid content during the pre‐spawning season than females in other seasons (7.0–8.2%). The most abundant fatty acid in whole body lipid in both sexes was C16 followed by C18:1n‐9, which accounted for about 31–35% and 22–24% of total lipids, respectively. Males in the pre‐spawning and spawning seasons had significantly higher total monosaturated fatty acids (MUFA) than males and females in post‐spawning and winter. Males had significantly higher total polyunsaturated fatty acids (PUFA) during post‐spawning seasons than females in pre‐spawning and winter seasons. However, there were no significant differences (P > 0.05) in total saturated fatty acids (SFA), PUFA, EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid) or n‐3/n‐6 ratios between respective males and females in different seasons. Livers in males had significantly (P < 0.05) higher MUFA, SFA, PUFA, EPA and DHA than respective females in all months during the spawning season. Female gonads had significantly (P < 0.05) higher MUFA and PUFA but lower SFA content than males in different months during the spawning season. In contrast to the liver, the gonad DHA content and n‐3/n‐6 ratios in females were significantly higher than in males. The gonads from both sexes contained more than double the amount of EPA present in liver; in the case of DHA this was more than three‐fold higher in female gonads, but not in males. Thus, the presence of higher proportions of PUFA, EPA and DHA in gonads, particularly in eggs of silver pomfret, indicates their need for these fatty acids, which may be used as a guideline for dietary essential n‐3 fatty acid requirements for feed formulation of this species. A higher content of DHA in eggs also indicates the higher requirement for DHA in the broodstock diet of silver pomfret.     

Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain.

Low concentrations of L-ascorbic acid caused release of acetylcholine from isolated synaptic vesicles (rat, guinea-pig and rabbit) in the presence of 2mM ATP, 2 mM MgCl₂ and 10^?5 M CaCl. The half maximum effect was obtained with about 2 to 2.5 ωM L-ascorbic acid, and the effect was inhibited by addition of 1mM EGTA. The release of noradrenaline from rat synaptic vesicles was also enhanced by L-ascorbic acid, but the concentration for half maximal stimulation was about 20 ωM, indicating that noradrenaline release was less sensitive to L-ascorbic acid than acetylcholine release. The physiological function of L-ascorbic acid in the brain is discussed in relation to release of transmitters.     

Formation of 3-Keto-4-deoxypentosone and 3-Hydroxy-2-pyrone by the Degradation of Dehydro-l-ascorbic Acid

A crystalline phenylhydrazone was obtained when a heated solution of dehydro-l-ascorbic acid (DHA) was treated with phenylhydrazine-HCl. Its molecular formula was C₁₇H₁₈N₄O₂, and the structure was determined to be 1,2-bis(phenylhydrazone) of 3-keto-4-deoxypentosone, a new tricarbonyl compound which was considered to be one of the possible intermediates of the browning reaction of DHA. 3-Hydroxy-2-pyrone was also isolated from the ether extract of the heated DHA solution as a main aroma compound produced from DHA. Possible formation mechanisms of these compounds were discussed.     

Ebselen has dehydroascorbate reductase and thioltransferase-like activities

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a seleno-organic compound, has been reported to mimic glutathione peroxidase (GPX). Since bovine erythrocyte GPX showed dehydroascorbic acid (DHA) reductase and thioltransferase (TTase) activities, ebselen was also examined for DHA reductase and TTase-like activities. Evidence is reported that, in the presence of GSH, ebselen catalyzed the in vitro reduction of DHA to L-ascorbic acid in a dose-dependent manner. Using S-sulfocysteine and GSH as co-substrates, ebselen catalyzed the in vitro formation of glutathione disulfide in a dose-dependent manner, thereby acting as a TTase mimic. 1-Chloro-2,4-dinitrobezene (CDNB), a co-substrate with GSH for glutathione S-transferase, was used to measure rates of adduct formation with ebselen pretreated with GSH and compared with GSH alone. The reaction rate was proportional to ebselen, and ebselen was about 250 times more reactive than GSH on an equimolar basis. The DHA reductase and TTase-like activities, in addition to the powerful nucleophilic reactivity of ebselen selenol, may contribute to ebselen's significant anti-inflammatory and anti-oxidative properties in vivo.     

Isolation and characterization of a new advanced glycation endproduct of dehydroascorbic acid and lysine

Proteins are subject of posttranslational modification by sugars and their degradation products in vivo. The process is often referred as glycation. L-Dehydroascorbic acid (DHA), an oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. A new product of modification of lysine epsilon -amino group by DHA was discovered as a result of the interaction between Boc-Lys and dehydroascorbic acid. The chromatographic and spectral analyses revealed that the structure of the product was 1-(5-ammonio-5-carboxypentyl)-3-oxido-4-(hydroxymethyl)pyridinium. The same compound was isolated from DHA modified calf lens protein after hydrolysis and chromatographic separation. The study confirmed that L-erythrulose is an important intermediate of modification of proteins by DHA. The structure of the reported product and in vitro experiments suggested that L-erythrulose could further transform to L-threose, L-erythrose and glycolaldehyde under conditions similar to physiological. The present study revealed that the modification of epsilon -amino groups of lysine residues by DHA is a complex process and could involve a number of reactive carbonyl species.     

Incorporation and Accumulation of Docosahexaenoic Acid from the Medium by Pichia methanolica HA-32

Yeast species were screened for the incorporation and accumulation of docosahexaenoic acid (DHA) with a yeast-malt medium containing 0.5% free fatty acid prepared from fish oil (DHA, 28% of total fatty acids in fish oil). The most suitable strain was Pichia methanolica HA-32. The optimum cultivation conditions for the accumulation of lipids and incorporation of DHA were as follows: 5% glucose, 20% yeast extract, and 3% free fatty acid in the medium, at pH 6.0 and with incubated at 25°C for 3 days. Under these conditions, about 200 mg of total lipids and 60 mg of DHA were recovered from 1 g of dry cells. The accumulation of DHA in cells increased in conjunction with the amount of yeast extract added to the medium. Vitamin B groups and minerals also had an effect on the accumulation of DHA. Choline and K₂HPO₄, which caused browning of the medium, promoted the accumulation of DHA in cells.     

Closely related colon cancer cell lines display different sensitivity to polyunsaturated fatty acids, accumulate different lipid classes and downregulate sterol regulatory element-binding protein 1

N-6 polyunsaturated fatty acids (PUFAs) may be associated with increased risk of colon cancer, whereas n-3 PUFAs may have a protective effect. We examined the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid and arachidonic acid on the colon carcinoma cell lines SW480 derived from a primary tumour, and SW620 derived from a metastasis of the same tumour. DHA had the strongest growth-inhibitory effect on both cell lines. SW620 was relatively more growth-inhibited than SW480, but SW620 also had the highest growth rate in the absence of PUFAs. Flow cytometry revealed an increase in the fraction of cells in the G2/M phase of the cell cycle, particularly for SW620 cells. Growth inhibition was apparently not caused by increased lipid peroxidation, reduced glutathione or low activity of glutathione peroxidase. Transmission electron microscopy revealed formation of cytoplasmic lipid droplets after DHA treatment. In SW620 cells an eightfold increase in total cholesteryl esters and a 190-fold increase in DHA-containing cholesteryl esters were observed after DHA treatment. In contrast, SW480 cells accumulated DHA-enriched triglycerides. Arachidonic acid accumulated in a similar manner, whereas the nontoxic oleic acid was mainly incorporated in triglycerides in both cell lines. Interestingly, nuclear sterol regulatory element-binding protein 1 (nSREBP1), recently associated with cell growth regulation, was downregulated after DHA treatment in both cell lines. Our results demonstrate cell-specific mechanisms for the processing and storage of cytotoxic PUFAs in closely related cell lines, and suggest downregulation of nSREBP1 as a possible contributor to the growth inhibitory effect of DHA.     

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.     

Red Pigment Produced by the Reaction of Dehydro-l-ascorbic Acid with α-Amino Acid

At the initial stage of the browning reaction of dehydro-l-ascorbic acid (DHA) with α-amino acid, a kind of red pigment was produced. The pigment was isolated as very hygroscopic red powder from non-aqueous reaction system, and its characterization was made. It was revealed that it had the same structure with that of the red pigment produced by the oxidation of l-scorbamic acid, an intermediate amino-reductone expected to be produced by Strecker degradation. Formation mechanism of the pigment which was considered to be an intermediate of browning reaction of DHA with α-amino acid was also discussed.     

Docosahexaenoic acid supplementation of primary rat hippocampal neurons attenuates the neurotoxicity induced by aggregated amyloid beta protein42 and up-regulates cytoskeletal protein expression

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by extracellular deposits of fibrillar aggregates of amyloid-β peptide (Aβ). Levels of docosahexaenoic acid (DHA, 22:6n-3), the major fatty acid component of the neuronal membrane, are reduced in the AD hippocampus. We hypothesized that hippocampal neurons with reduced DHA levels would be more susceptible to aggregated Aβ-induced death and that this might be overcome by increasing hippocampal neuronal DHA levels. Embryonic Day 18 rat hippocampal cells were cultured in neurobasal medium with B27 supplemented with 0–100 μM DHA for 8 days, then were treated with 5 μM aggregated Aβ₄₂ for 1 day. We found that supplementation with 5–10 μM DHA, which resulted in hippocampal neuron DHA levels of 12–16% of total fatty acids, was optimal for primary hippocampal neuronal survival, whereas supplementation with 5 or 25 μM DHA attenuated aggregated Aβ₄₂-induced neurotoxicity and protected hippocampal neurons, with 25 μM DHA being more effective. DHA supplementation also resulted in significant up-regulation of expression of tyrosine tubulin and acetylated tubulin. We suggest that hippocampal neuronal DHA levels may be critical for AD prevention by attenuating the neurotoxicity induced by Aβ and in maintaining hippocampal neuron survival.     

Evaluation of liquid residues from beer and potato processing for the production of docosahexaenoic acid (C22:6n-3, DHA) by native thraustochytrid strains

Liquid residues from beer (RB) and potato (RP) processing were evaluated as carbon sources for the production of docosahexaenoic acid (C22:6n-3, DHA) by two native Thraustochytriidae sp., M12-X1 and C41, in shaking flask experiments. Results were compared with those obtained in the fermentations of glucose, maltose, soluble starch and ethanol. Both strains produced the highest biomass concentration (2.3 g/L) in the fermentation of RB supplemented with nitrogen sources [yeast extract (YE) and monosodium glutamate (MSG)]. DHA content in the fatty acids produced by the native thraustochytrids was dependent on the fermented carbon source; the fatty acids from biomass grown on carbon sources that permitted a lower growth rate contained more DHA. The highest DHA productivity [55.1 mg/(day L)] was obtained in the fermentation of RB-YE-MSG by M12-X1 strain. In this medium, M12-X1 strain grew at a specific growth rate of 0.014 h^?1 and total fatty acid content in the biomass was 41.3%. Production of DHA by M12-X1 strain followed a non-growth rate associated pattern and DHA content in the biomass decreased significantly after growth ceased.     

Incorporation and accumulation of docosahexaenoic acid from the medium by Pichia methanolica HA-32

Yeast species were screened for the incorporation and accumulation of docosahexaenoic acid (DHA) with a yeast-malt medium containing 0.5% free fatty acid prepared from fish oil (DHA, 28% of total fatty acids in fish oil). The most suitable strain was Pichia methanolica HA-32. The optimum cultivation conditions for the accumulation of lipids and incorporation of DHA were as follows: 5% glucose, 20% yeast extract, and 3% free fatty acid in the medium, at pH 6.0 and with incubated at 25 degrees C for 3 days. Under these conditions, about 200 mg of total lipids and 60 mg of DHA were recovered from 1 g of dry cells. The accumulation of DHA in cells increased in conjunction with the amount of yeast extract added to the medium. Vitamin B groups and minerals also had an effect on the accumulation of DHA. Choline and K2HPO4, which caused browning of the medium, promoted the accumulation of DHA in cells.     

Browning reduces the availability—but not the transfer—of essential fatty acids in temperate lakes

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Glycation of lens proteins by the oxidation products of ascorbic acid

Bovine lens water-soluble proteins were incubated with [I-14C]ascorbic acid (ASA) for 6 days, and the incorporation into protein was measured at daily intervals. Aliquots were also withdrawn to determine the distribution of label among the various ASA oxidation products. A linear incorporation into protein was observed in the presence of NaCNBH3, however, little or no incorporation was seen in its absence. TLC analysis showed a complete loss of ASA by day 3, whereas both dehydroascorbate (DHA) and diketogulonic acid (DKG) remained constant for 6 days, consistent with the linear incorporation into protein. The amino acid composition of the proteins glycated in the presence of NaCNBH3 was identical to controls except for a 70% reduction in lysine residues and a corresponding increase in an unknown product which eluted slightly earlier than methionine. In the absence of NaCNBH3 lysine decreased linearly to 20% with an additional decrease in arginine and histidine at later times concurrent with protein crosslinking. DHA and DKG were prepared and incubated directly with lens proteins for an 8 day period. Both compounds glycated lens protein as evidenced by an increased binding to a boronate affinity column. SDS-PAGE showed that both compounds were also capable of causing protein crosslinking. DHA is apparently capable of reacting directly with protein since glycation was observed with the ASA analog, reductic acid, which can be oxidized to dehydroreductic acid, but which cannot be hydrolyzed to an open chain structure. DHA also produced a lysine adduct which was not obtained with DKG, supporting the idea that both species have glycating ability.     

Stabilization of L-ascorbic acid by superoxide dismutase and catalase

The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.