Polarographic Studies on Fat-soluble Vitamins in Nonaqueous Media

Vitamin D₂ and vitamin D_s gave a single reduction-wave, respectively, in polarography in acetonitrile containing 0.05 M-tetrabutylammonium iodide. The eduction-wave of vitamin D_s was indistinguishable from that of vitamin D₂, whose half-wave potentials were evaluated identically at -2.1₀ V vs. Hg. Proportionalities between wave-height and concentration were held in the concentration range fronl 4× 10^-4 M (ca. 6000 IU·ml^-1) to 1.2×10 M (ca. 18000 IUD·ml^-1)

In both cases, the conjugated three double-bond system may be reduced to two double-bond system, which corresponds to the third reduction-step of vitamin A.

Ergosterol which has a conjugated two double-bond system and an isolated double-bond gave no reduction-wave under the present conditions.     

Biocatalytic synthesis of vitamin A palmitate using immobilized lipase produced by recombinant Pichia pastoris

In this work, the Candida antarctica lipase B (CALB), produced by recombinant Pichia pastoris , was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent. The reaction conditions including the type of solvent, temperature, rotation speed, particle size, and molar ratio between the two substrates were investigated. It turned out that the macroporous resin HPD826 serving as a carrier showed the highest activity (ca. 9200 U g^?1) among all the screened carriers. It was found that the transesterification kinetic of the immobilized CALB followed the ping pong Bi‐Bi mechanism and the reaction product acetic acid inhibited the enzymatic reaction with an inhibition factor of 2.823 mmol L^?1. The conversion ability of the immobilized CALB was 54.3% after 15 cycles. In conclusion, the present work provides a green route for vitamin A palmitate production using immobilized CALB to catalyze the transesterification of vitamin A acetate and palmitic acid.     

Direct electrochemistry of thioredoxins and glutathione at a lipid bilayer-modified electrode.

By using direct electrochemical analysis we have established that the reduction of Escherichia coli thioredoxin (EcT), T4 thioredoxin (T4T), and glutathione (GSSG) occurs at a self-assembled lipid bilayer-modified gold electrode via two separate one-electron processes. The first electron transfer has half-wave potentials of -0.05 +/- 0.01, -0.07 +/- 0.01, and -0.06 +/- 0.01 V, whereas the second one has values of -0.48 +/- 0.01, -0.39 +/- 0.01, and -0.45 +/- 0.01 V, for EcT, T4T, and GSSG, respectively. The scan-rate dependence of the cyclic voltammetry indicates, for both waves, that the process of electron transfer is dominated by a bulk diffusion of free species to and from the electrode, and that strongly adsorbed species do not significantly contribute at the scan rates used. The voltage separation of the peak currents indicates a quasi-reversible electron transfer process with an electrochemical rate constant which is larger for the second (lower potential) electron than for the first one. Using the above half-wave potentials of the one-electron steps, one can calculate a thermodynamic half-wave potential for the two-electron reduction processes. The values of these potentials are -0.265, -0.23, and -0.25 V for EcT, T4T, and GSSG, respectively. These are in excellent agreement with literature values obtained from equilibrium measurements of enzyme-catalyzed reactions involving these species. It is quite clear from these results that lipid bilayer-modified electrodes provide a biocompatible and direct means of efficiently carrying out electrochemical reactions with sulfur-based redox systems, as we have previously shown to be the case with metalloproteins.     

Micellar oleic and eicosapentaenoic acid but not linoleic acid influences the β-carotene uptake and its cleavage into retinol in rats

Improving the bioavailability of β-carotene is vital to manage vitamin A deficiency. The influence of micellar oleic (OA), linoleic (LA) and eicosapentaenoic (EPA) acids on plasma β-carotene response and its conversion to retinol has been studied in rats employing single (9 h time course) and repeated (10 days) dose administrations. After a single dose, the levels (area under the curve) of plasma β-carotene and retinyl palmitate in OA and EPA groups were higher (p < 0.05) by 13, 7 and 11, 6 folds than LA group. The liver β-carotene level in OA and EPA groups were higher (p < 0.05) by 3 and 1.2 folds than LA group. After repeated dose, the plasma β-carotene and retinyl palmitate levels in OA (6.2%, 51.7%) and EPA (25.4%, 17.23%) groups were higher (p < 0.05) than LA group. The liver β-carotene level in OA (21.2%) and EPA (17.6%) groups were higher (p < 0.05) than LA group. In both the experiments, the activity of β-carotene 15,15′-dioxygenase in the intestinal mucosa and plasma triglyceride levels were also higher in OA and EPA groups than LA group. β-Carotene excreted through urine and feces of OA and EPA groups was lower than the LA group. These results demonstrate an improved absorption and metabolism of β-carotene when fed mixed micelles with OA or EPA compared with LA. Although the mechanism involved in selective absorption of fatty acids needs further studies, intestinal β-carotene uptake and its conversion to vitamin A can be modulated using specific fatty acids.     

Carotenogenic and abscisic acid biosynthesizing activity in a cell-free system

Abscisic acid is considered an apocarotenoid formed by cleavage of a C-40 precursor and subsequent oxidation of xanthoxin and abscisic aldehyde. Confirmation of this reaction sequence is still awaited, and might best be achieved using a cell-free system capable of both carotenoid and abscisic acid biosynthesis. An abscisic acid biosynthesizing cell-free system, prepared from flavedo of mature orange fruits, was used to demonstrate conversion of farnesyl pyrophosphate, geranylgeranyl pyrophosphate and all-trans-β-carotene into a range of β,β-xanthophylls, xanthoxin, xanthoxin acid, 1′,4′-trans-abscisic acid diol and abscisic acid. Identification of product carotenoids was achieved by high-performance liquid chromatography and on-line spectral analysis of individual components together with co-chromatography. Putative C-15 intermediates and product abscisic acid were identified by combined capillary gas chroma-tography-mass spectrometry. Kinetic studies revealed that β-carotene, formed from either famesyl pyrophosphate or geranylgeranyl pyrophosphate, reached a maximum within 30 min of initiation of the reaction. Thereafter, β-carotene levels declined exponentially. Catabolism of substrate β-carotene into xanthophylls, putative abscisic acid precursors and product abscisic acid was restricted to the all-trans-isomer. However, when a combination of all-trans- and 9-cis-β-carotene in the ratio 1:1 was used as substrate, formation of abscisic acid and related metabolites was enhanced. Biosyn-thetically prepared [¹⁴C]-all-trans-violaxanthin, [¹⁴C]-all-trans-neoxanthin and [¹⁴C]-9′-cis-neoxanthin were used as substrates to confirm the metabolic interrelationship between carotenoids and abscisic acid. The results are consistent with 9′-cis-neoxan-thin being the immediate carotenoid precursor to ABA, which is oxidatively cleaved to produce xanthoxin. Formation of abscisic aldehyde was not observed. Rather, xanthoxin appeared to be converted to abscisic acid via xanthoxin acid and 1′,4′-trans-abscisic acid diol. An alternative pathway for abscisic acid biosynthesis is therefore proposed.     

Blood antioxidant status and urinary levels of catecholamine metabolites in β-thalassemia

It has been reported that iron overload in β-thalassemia leads to an enhanced generation of reactive oxygen species and to oxidative stress. We have studied the oxidant/antioxidant imbalance in the blood of 48 transfusion-dependent β-thalassemic patients (TLP) (17 males, 31 females, 11–22 year), under chelation therapy, and in 40 sex and age matched healthy controls (CTR). Plasma and lymphocyte levels of vitamin E (Vit E), ubiquinol (CoQ₁₀H₂), ubiquinone (CoQ₁₀), plasma concentrations of vitamin A (Vit A), β-carotene, lycopene, vitamin C (Vit C), total thiols, fatty acid patterns of phospholipids (PL-FA), and plasma and urinary markers of lipoperoxidation (TBA-RM, conjugated dienes, and azelaic acid (AZA), as well as the urinary levels of catecholamine and serotonin metabolites, were evaluated by gas chromatography-mass spectrometry (GC-MS), HPLC and spectrophotometry. Routine laboratory blood analyses were performed on the same samples; 39/48 TLP were HCV positive. Blood samples were collected just before transfusion, the 24 h urine samples the day before. Our results clearly showed that a severe oxidative stress occurs in the plasma of TLP in comparison with CTR. In fact, the levels of lipophilic antioxidants and ascorbate were severely depleted: CoQ₁₀H₂ (-62.5%), total CoQ₁₀ (-35.1%), Vit E (-43.8%, β-carotene (-31.1%), lycopene (-63.7%), Vit A (-35.9%), Vit C (-23.1%). The impairment of the antioxidant status was associated with elevated plasma levels of by-products of lipoperoxidation and urinary concentrations of catecholamine metabolites and of AZA, indicating a high degree of both neurological stress and lipoperoxidation. A significant positive correlation was found between vitamin E and non-transferrin-bound iron (NTBI) (r = -0.81; p < 0.001), while no correlation was found between antioxidant depletion and ferritin serum levels, average blood consumption, or the presence of clinical complications. The administration of selective antioxidants along with an appropriate diet might represent a promising way of counteracting oxidative damage and its deleterious effects on the progression of the disease.     

Sensory evoked potentials in unanesthetized unrestrained cuttlefish: a new preparation for brain physiology in cephalopods

Summary Up to five microelectrodes inserted through short hypodermic needles in the cranial cartilage of Sepia officinalis recorded potentials while the cuttlefish moved freely in a small enclosure. Compound field potentials and unit spikes were seen during ongoing, spontaneous activity and after sensory stimulation.Ongoing activity resembles that reported for octopus, with maximum power usually below 20 Hz. Amplitude varies greatly but has not been seen to shut off or turn on abruptly and globally as in octopus.Evoked potentials, focally large after flashes of light consist of several waves; the first is largest, positive and peaks at ca. 35 ms (called P35), followed by ca. P75, P95, N110 and smaller waves or oscillations lasting more than 0.5 s. The Upper Following Frequency (highest flashing rate the potentials can follow 1:1), without averaging, is >15 flashes/s (20–22 °C); at 20/s the 11 following lasts for 1 or 2 s. The Lower Fusion Frequency of averaged responses is < 30/s. Gentle tapping of the tank wall evokes local, brief, fast potentials. No responses have been found to loud air-borne clicks and tone bursts with principal energy at 300 Hz or to electric fields in the bath at 50–100 V/cm.In a few loci relatively large slow Omitted Stimulus Potentials have been seen following the end of a train of flashes at more than 5/s; these are by definition event related potentials and a special, central form of OFF response.Abbreviations EP evoked potential - ISI interstimulus interval - OSP omitted stimulus potential - VEP visual evoked potential     

Characterization of human β,β-carotene-15,15′-monooxygenase (BCMO1) as a soluble monomeric enzyme

The formal first step in in vitamin A metabolism is the conversion of its natural precursor β,β-carotene (C40) to retinaldehyde (C20). This reaction is catalyzed by the enzyme β,β-carotene-15,15′-monooxygenase (BCMO1). BCMO1 has been cloned from several vertebrate species, including humans. However, knowledge about this protein’s enzymatic and structural properties is scant. Here we expressed human BCMO1 in Spodoptera frugiperda 9 insect cells. Recombinant BCMO1 is a soluble protein that displayed Michaelis–Menten kinetics with a K_M of 14 μM for β,β-carotene. Though addition of detergents failed to increase BCMO1 enzymatic activity, short chain aliphatic detergents such as C₈E₄ and C₈E₆ decreased enzymatic activity probably by interacting with the substrate binding site. Thus we purified BCMO1 in the absence of detergent. Purified BCMO1 was a monomeric enzymatically active soluble protein that did not require cofactors and displayed a turnover rate of about 8 molecules of β,β-carotene per second. The aqueous solubility of BCMO1 was confirmed in mouse liver and mammalian cells. Establishment of a protocol that yields highly active homogenous BCMO1 is an important step towards clarifying the lipophilic substrate interaction, reaction mechanism and structure of this vitamin A forming enzyme.     

Biotransformation of testosterone and other androgens by suspension cultures of Nicotiana tabacum “Bright yellow”

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ⁴-androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ⁴-Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4-¹⁴C]testosterone is actively incorporated in these transformations.     

N-Benzylideneaniline and N-Benzylaniline are Potent Inhibitors of Lignostilbene-α,β-dioxygenase,a Key Enzyme in Oxidative Cleavage of the Central Double Bond of Lignostilbene

Lignostilbene-α,β-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and β-carotene 15,15′-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC₅₀ = 0.3 µM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC₅₀ = 10 µM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.     

Serum α- and γ-tocopherols, retinol, retinyl palmitate, and carotenoid concentrations in captive and free-ranging bottlenose dolphins (Tursiops truncatus)

Concentrations of retinol, retinyl palmitate, β-carotene, α-carotene, cryptoxanthin, lutein, lycopene, α-tocopherol, and γ-tocopherol were measured in blood samples collected from 15 captive and 55 free-ranging bottlenose dolphins (Tursiops truncatus). From June 1991 to June 1994, blood samples were collected from captive animals residing at two locations; at Seven Seas (Brookfield Zoo, Brookfield, IL) and Hawk’s Cay (Marathon Key, FL). Blood samples were collected from free-ranging animals from June 1991 to June 1996. Retinol levels were not significantly different between captive dolphin groups. However, Seven Seas animals had higher (P<0.01) serum retinol concentrations compared to free-ranging animals (0.061 vs 0.041 μg/ml). Retinyl palmitate was not detected in the serum of captive or free-ranging dolphins. Alpha-tocopherol levels were significantly (P<0.05) higher for Seven Seas dolphins (16.4 μg/ml) than for Hawk’s Cay (13.0 μg/ml) and free-ranging dolphins (12.5 μg/ml). Gamma-tocopherol concentrations were similar among captive and free-ranging dolphins. Free-ranging dolphins showed levels of circulating carotenoids (lutein and β-carotene) while the captive animals did not. Additional carotenoids (lycopene, α-carotene and cryptoxanthin) were analyzed but not detected in any samples. Serum vitamin differences between captive and free-ranging dolphins may reflect the natural diet or indicate some potential biological or nutritional status significance.     

Antioxidant actions of plant foods: Use of oxidative DNA damage as a tool for studying antioxidant efficacy

Plant-food-derived antioxidants and active principles such as flavonoids, hydroxycinnamates (ferulic acid, chlorogenic acids, vanillin etc.), β-carotene and other carotenoids, vitamin E, vitamin C, or rosemary, sage, tea and numerous extracts are increasingly proposed as important dietary antioxidant factors. In this endeavor, assays involving oxidative DNA damage for characterizing the potential antioxidant actions are suggested as in vitro screens of antioxidant efficacy. The critical question is the bioavailability of the plant-derived antioxidants.     

The degradation of (all-E)-β-carotene by cigarette smoke

The effects of cigarette smoke in promoting the degradation of (all-E)-β-carotene have been studied, but some conflicting results promoted a further study. β-Carotene was solubilized in hexane and challenged with filtered cigarette smoke both at room temperature and at ?20°C. The products arising from smoke-induced oxidation were assessed using a combination of HLPC-DAD, LC-MS and GC-MS. At room temperature the degradation of β-carotene was very rapid, with only a few products being detected using HPLC-DAD. A range of volatile products including β-ionone, β-cyclocitral and 5,6-epoxy-β-ionone were detected using GC-MS. In contrast, when the reaction was slowed (by reducing the reaction temperature), a much wider range of products could be detected by HPLC-DAD, including 4-nitro-β-carotene and several of its geometric isomers. These degradation products suggest that the C4 position on the β-carotene end-group plays a key role in initiating free radical attack.     

Carotenoid Components in Soybean Seeds Varying with Seed Color and Maturation Stage

The difference in carotenoid components among various color types of soybean seeds, and the changes in carotenoid composition during seed development were examined by reverse-phase high-performance liquid chromatogrphy (HPLC). Lutein was the major carotenoid component in seed extracts from the common yellow soybean and from a variety having a black seed coat. Green soybean seeds contained several xanthophylls in addition to lutein. None of the mature soybean seeds contained β-carotene, a part from a trace amount being detected in a local variety of green soybean. The total carotenoid and lutein contents were higher in green soybeans than in the yellow types, and the estimated total amount of carotenoids correlates with that of chlorophylls. The thylakoid membrane residue in the plastids of green soybean had lost its functional lamella structure. Immature soybean seeds contained a green-vegetable type of carotenoids including α- and β-carotene. The amount of β-carotene decreased more rapidly than that of lutein and chlorophylls during seeds maturation. These results suggest that β-carotene, which acts as a photo-protective agent in developing seeds, is susceptible to degradation in the course of seed maturation.     

Colchicine Modulates Oxidative Stress in Serum and Leucocytes from Remission Patients with Family Mediterranean Fever Through Regulation of Ca<Superscript>2+</Superscript> Release and the Antioxidant System

We investigated the effects of colchicine on oxidative stress and Ca²⁺ release in serum and polymorphonuclear leucocytes (PMNs) of Familial Mediterranean Fever (FMF) patients with attack, remission and unremission periods. Eighteen FMF patients and six age-matched healthy subjects in four groups were used. The first group was a control. The second group included patients with active FMF. The third and fourth groups were patients with remission and unremission, respectively. Colchicine (1.5 mg/day) was given to the third and fourth groups for 1 month. PMN cells, serum lipid peroxidation and intracellular Ca²⁺-release levels in the attack and unremission groups were higher than in those in controls, although they were lower in the remission group than in the attack group. Serum vitamin E and β-carotene concentrations were higher in the remission group than in the control and attack groups. However, PMN, serum lipid peroxidation and Ca²⁺-release levels were further increased in the unremission group compared to the attack group. Glutathione peroxidase, reduced glutathione and vitamin A values in the four groups did not change by FMF and colchicine. In conclusion, we observed that colchicine induced protective effects on oxidative stress by modulating vitamin E, β-carotene and Ca²⁺-release levels in FMF patients with a remission period.     

β-carotene improves fecal dysbiosis and intestinal dysfunctions in a mouse model of vitamin A deficiency

Vitamin A deficiency (VAD) results in intestinal inflammation, increased redox stress and reactive oxygen species (ROS) levels, imbalanced inflammatory and immunomodulatory cytokines, compromised barrier function, and perturbations of the gut microbiome. To combat VAD dietary interventions with β-carotene, the most abundant precursor of vitamin A, are recommended. However, the impact of β-carotene on intestinal health during VAD has not been fully clarified, especially regarding the VAD-associated intestinal dysbiosis. Here we addressed this question by using Lrat^?/-Rbp^?/? (vitamin A deficient) mice deprived of dietary preformed vitamin A and supplemented with β-carotene as the sole source of the vitamin, alongside with WT (vitamin A sufficient) mice. We found that dietary β-carotene impacted intestinal vitamin A status, barrier integrity and inflammation in both WT and Lrat^?/-Rbp^?/? (vitamin A deficient) mice on the vitamin A-free diet. However, it did so to a greater extent under overt VAD. Dietary β-carotene also modified the taxonomic profile of the fecal microbiome, but only under VAD. Given the similarity of the VAD-associated intestinal phenotypes with those of several other disorders of the gut, collectively known as Inflammatory Bowel Disease (IBD) Syndrome, these findings are broadly relevant to the effort of developing diet-based intervention strategies to ameliorate intestinal pathological conditions.     

β-Carotene generates thiobarbituric acid-reactive substances by interaction with nitrogen dioxide in air

Generation of thiobarbituric acid-reactive substances (TBARS) from methyl linoleate in the exposure of nitrogen dioxide/air was inhibited by β-carotene in a dose-dependent manner. However, introduction of nitrogen dioxide/air or oxygen into a solution of β-carotene generated a significant amount of TBARS accompanying loss of its characteristic yellow color. Storing β-carotene in a solid state at ambient temperatures in air generated a large amount of TBARS accompanying loss of its yellow color. TBARS from β-carotene may interfere the measurement of TBARS from polyunsaturted fatty acids, and may give undesirable effects on biomaterials.     

Acamprosate Modulates Alcohol-Induced Hippocampal NMDA Receptors and Brain Microsomal Ca2+-ATPase but Induces Oxidative Stress in Rat

We investigated the effects of acamprosate on alcohol-induced oxidative toxicity, microsomal membrane Ca²⁺-ATPase (MMCA) activity and N-methyl-d-aspartate receptor (NMDAR) subunits in rat brain. Forty male rats were equally divided into four groups. The first group was used as control, and the second group received ethanol. Acamprosate and acamprosate plus ethanol each day were administered to rats constituting the third and fourth groups for 21 days, respectively. Brain cortical and hippocampal samples were taken from the four groups after 21 days. Brain cortical lipid peroxidation (LP) levels and MMCA activity were higher in the alcohol group than in control, although glutathione peroxidase (GSH-Px), vitamin C, vitamin E and β-carotene values were lower in the alcohol group than in control. LP levels were further increased in the acamprosate and alcohol + acamprosate groups compared with the alcohol group. GSH-Px, vitamin A, vitamin C, vitamin E and β-carotene in the acamprosate and alcohol + acamprosate groups were further decreased compared with the alcohol group. Hippocampal NMDAR 2A and 2B subunit concentrations were lower in the alcohol group than in control, although they were increased by acamprosate and alcohol + acamprosate. Brain cortical MMCA activity was higher in the acamprosate group than in the alcohol-treated rats, although its activity was lower in the alcohol + acamprosate group than in the acamprosate group. Brain cortical reduced glutathione levels were not found to be statistically different in any of the groups. Oxidative stress has been proposed to explain the biological side effects of experimental alcohol intake. Acamprosate and alcohol-induced oxidative stress decreased brain antioxidant vitamins in the alcoholic rats.     

Lipids of Algae

The carotenoid pigments prepared from acetone extracts of chlorella were separated into epiphasic and hypophasic fractions by partition between petroleum ether and 90% methanol. Each fraction was subjected to column chromatography, using aluminium oxide, magnesium oxide, calcium hydroxide or calcium carbonate as adsorbent. The absorption maxima of the separated pigments in hexane and in carbon disulfide were compared with those of the known pigments. Some of the separated pigments were identified as those previously known which follow: α-carotene, β-carotene, rhodoxanthin, sarcinaxanthin, lutein and neoxanthin. Two unknown pigments with absorption maxima not yet reported were separated. The first showed absorption maxima at 478 m_μ in hexane and 518 m_μ in carbon disulfide, and the second at 383, 402 and 425 m_μ in hexane and 428 and 450 m_μ in carbon disulfide.     

Profiling of carotenoids in six microalgae (Eustigmatophyceae) and assessment of their β-carotene productions in bubble column photobioreactor

The profiles of carotenoids and production of β-carotene by six eustigmatophytes, Eustigmatos magnus, Eustigmatos polyphem, Eustigmatos vischeri, Vischeria helvetica, Vischeria punctata and Vischeria stellata, grown in a bubble column photobioreactor were measured. All eustigmatophytes contained β-carotene, violaxanthin and vaucheriaxanthin as their major carotenoids and accumulated large amount of β-carotene, which accounted for over 50?% of total carotenoids. Maximum intracellular β-carotene contents ranged 1.5–3.5?% of dry wt and in V. stellata it reached 5.9?% dry wt, accompanied by a biomass dry wt >7.3?g/l, with the highest up to 9.8?g/l. These eustigmatophytes are thus promising producers of β-carotene.