The effect of vitamin E analogues and long hydrocarbon chain compounds on calcium-induced muscle damage. A novel role for alpha-tocopherol?

Previous studies have demonstrated that supplemental alpha-tocopherol inhibited calcium-induced cytosolic enzyme efflux from normal rat skeletal muscles incubated in vitro and suggested that the protective action was mediated by the phytyl chain of alpha-tocopherol [1]. In order to investigate this further a number of hydrocarbon chain analogues of tocopherol (7,8-dimethyl tocol, 5,7-dimethyl tocol, tocol, alpha-tocotrienol, alpha-tocopherol [10], vitamin K1, vitamin K1 [10], vitamin K1 diacetate, vitamin K2 [20], phytyl ubiquinone and retinol) were tested for any ability to inhibit calcium ionophore, A23187, induced creatine kinase (CK) enzyme efflux. Some compounds were found to be very effective inhibitors and comparison of their structures and ability to inhibit TBARS production in muscle homogenates revealed that the effects did not appear related to antioxidant capacity or chromanol methyl groups, but rather the length and structure of the hydrocarbon chain was the important mediator of the effects seen.     

The effect of vitamin A deficiency and dietary α-tocopherol on the stability of rat-liver lysosomes

1. Vitamin A-deficient rats and pair-fed controls were maintained on either normal or raised amounts of dietary alpha-tocopherol. 2. Their livers were fractionated and ;free' and ;total' lysosomal phosphatase were determined in the various fractions. The rate of release of this enzyme was determined in the mitochondria-lysosome-rich fraction during incubation at pH5 and 37 degrees . 3. The deficient livers showed increased enzymic activity. 4. Prolonged incubation caused more rapid enzyme release from the mitochondria-lysosome-rich fraction of the vitamin A-deficient rats receiving the normal amount of dietary alpha-tocopherol than from the equivalent fraction of their pair-fed controls receiving vitamin A. Raised dietary alpha-tocopherol reversed this phenomenon.     

The influence of α-tocopherol and pyritinol on oxidative DNA damage and lipid peroxidation in human lymphocytes

Unscheduled DNA synthesis (UDS) and lipid peroxidation (LPO) were measured in human peripheral lymphocytes from healthy volunteers. These processes were induced by the catalytic system Fe²⁺-sodium ascorbate. The degree of induced LPO was measured spectrophotometrically by the thiobarbituric acid assay. UDS was detected by scintillometric measurement of the incorporation of ³H-thymidine into DNA. The protective action by fat-soluble vitamin E (d,l-α-tocopherol) and the artificial antioxidant pyritinol on UDS and LPO was also investigated.The system Fe²⁺ (2 μmole/1)-sodium ascorbate (30 μmole/1) increased the LPO level in healthy volunteers approximately 2.5 times and the incorporation of ³H-thymidine by 60–70%. α-Tocopherol (0.2 mmole/1) very efficiently suppressed LPO processes (p < 0.01) and the oxidative damage of DNA measured as UDS was also significantly diminished (p < 0.05). Pyritinol had no effect on LPO and UDS under our experimental conditions.     

The effect of α-tocopherol, α- and γ-tocotrienols on amyloid-β aggregation and disaggregation in vitro

One of the neuropathological hallmarks of Alzheimer's disease (AD)—causing neurodegeneration and consequent memory deterioration, and eventually, cognitive decline—is amyloid-β (Aβ) aggregation forming amyloid plaques. Our previous study showed the potential of a tocotrienol-rich fraction—a mixture of naturally occurring of vitamin E analogs—to inhibit Aβ aggregation and restore cognitive function in an AD mouse model. The current study examined the effect of three vitamin E analogs—α-tocopherol (α-TOC), α-tocotrienol (α-T3), and γ-tocotrienol (γ-T3)—on Aβ aggregation, disaggregation, and oligomerization in vitro. Thioflavin T (ThT) assay showed α-T3 reduced Aβ aggregation at 10 μM concentration. Furthermore, both α-T3 and γ-T3 demonstrated Aβ disaggregation, as shown by the reduction of ThT fluorescence. However, α-TOC showed no significant effect. We confirmed the results for ThT assays with scanning electron microscopy imaging. Further investigation in photo-induced cross-linking of unmodified protein assay indicated a reduction in Aβ oligomerization by γ-T3. The present study thus revealed the individual effect of each tocotrienol analog in reducing Aβ aggregation and oligomerization as well as disaggregating preformed fibrils.     

Specificity of the α-tocopherol (Vitamin E) effect on lifespan and fecundity of bdelloid rotifers

Experiments were undertaken to determine molecular specificity of Vitamin E-effects on lifespan and fecundity in four bdelloid rotifers (Habrotrocha sp., Philodina sp., Pleuretra sp., and Rotaria sp.). Results indicate that lifespan and fecundity could be significantly increased by addition of any one of three tocopherol compounds (d--, -, and -tocopherol) to the rotifer medium. Life table functions were increased the most by the d--tocopherol form. Improvement of these life table functions was not achieved by substitution of tocopherol analogs or other antioxidants in the rotifer medium.     

The role of αB-crystallin in skeletal and cardiac muscle tissues

All organisms and cells respond to various stress conditions such as environmental, metabolic, or pathophysiological stress by generally upregulating, among others, the expression and/or activation of a group of proteins called heat shock proteins (HSPs). Among the HSPs, special attention has been devoted to the mutations affecting the function of the αB-crystallin (HSPB5), a small heat shock protein (sHsp) playing a critical role in the modulation of several cellular processes related to survival and stress recovery, such as protein degradation, cytoskeletal stabilization, and apoptosis. Because of the emerging role in general health and disease conditions, the main objective of this mini-review is to provide a brief account on the role of HSPB5 in mammalian muscle physiopathology. Here, we report the current known state of the regulation and localization of HSPB5 in skeletal and cardiac tissue, making also a critical summary of all human HSPB5 mutations known to be strictly associated to specific skeletal and cardiac diseases, such as desmin-related myopathies (DRM), dilated (DCM) and restrictive (RCM) cardiomyopathy. Finally, pointing to putative strategies for HSPB5-based therapy to prevent or counteract these forms of human muscular disorders.     

The vitamin E isoforms α-tocopherol and γ-tocopherol have opposite associations with spirometric parameters: the CARDIA study

Background

Clinical studies of the associations of vitamin E with lung function have reported conflicting results. However, these reports primarily examine the α-tocopherol isoform of vitamin E and have not included the isoform γ-tocopherol which we recently demonstrated in vitro opposes the function of α-tocopherol. We previously demonstrated, in vitro and in animal studies, that the vitamin E isoform α-tocopherol protects, but the isoform γ-tocopherol promotes lung inflammation and airway hyperresponsiveness.

Methods

To translate these findings to humans, we conducted analysis of 4526 adults in the Coronary Artery Risk Development in Young Adults (CARDIA) multi-center cohort with available spirometry and tocopherol data in blacks and whites. Spirometry was obtained at years 0, 5, 10, and 20 and serum tocopherol was from years 0, 7 and 15 of CARDIA.

Results

In cross-sectional regression analysis at year 0, higher γ-tocopherol associated with lower FEV₁ (p = 0.03 in blacks and p = 0.01 in all participants) and FVC (p = 0.01 in blacks, p = 0.05 in whites, and p = 0.005 in all participants), whereas higher α-tocopherol associated with higher FVC (p = 0.04 in blacks and whites and p = 0.01 in all participants). In the lowest quartile of α-tocopherol, higher γ-tocopherol associated with a lower FEV₁ (p = 0.05 in blacks and p = 0.02 in all participants). In contrast, in the lowest quartile of γ-tocopherol, higher α-tocopherol associated with a higher FEV₁ (p = 0.03) in blacks. Serum γ-tocopherol >10 μM was associated with a 175–545 ml lower FEV₁ and FVC at ages 21–55 years.

Conclusion

Increasing serum concentrations of γ-tocopherol were associated with lower FEV1 or FVC, whereas increasing serum concentrations of α-tocopherol was associated with higher FEV1 or FVC. Based on the prevalence of serum γ-tocopherol >10 μM in adults in CARDIA and the adult U.S. population in the 2011 census, we expect that the lower FEV₁ and FVC at these concentrations of serum γ-tocopherol occur in up to 4.5 million adults in the population.     

Simple GC-FID method development and validation for determination of α-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of α-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of α-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. α-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min^− 1. Calibration curves were linear over the concentration range 1–30 μg ml^− 1 (for standard solutions and solutions without endogenous α-tocopherol in plasma) and 5–34 μg ml^− 1 (for solutions with endogenous α-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of α-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 μg.ml^− 1 and 0.30 μg.ml^− 1, respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of α-tocopherol. The endogenous α-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

The mechanism of apoptosis,cell membrane lipid peroxidation and a novel in vivo function for antioxidant vitamin E (α-tocopherol)

A critical novel function for the antioxidant vitamin E (α-tocopherol) may be its involvement in the control of apoptosis in which it acts as a ‘gate keeper’ in the regulation of membrane lipid peroxidation. The biochemical and biophysical antioxidant properties of the molecule are discussed together with recent evidence for its involvement in a possible model of the apoptotic mechanism. The original observation that led to the discovery of vitamin E (foetal resorption in pregnant rats fed on tocopherol-deficient diets) is considered as an unrecognised example of apoptosis.     

Fluidity,permeability and antioxidant behaviour of model membranes incorporated with α-tocopherol and vitamin E acetate

Magnetic resonance studies reveal a marked difference between the binding of α-tocopherol and that of the corresponding acetate (vitamin E acetate) with dipalmitoylphosphatidylcholine (DPPC) vesicles. This is reflected in differences in the phase-transition curves of the DPPC vesicles incorporated with the two compounds, as well as in the ¹³C relaxation times and line widths. A model for the incorporation of these molecules in lipid bilayers has been suggested. α-Tocopherol binds strongly with the lipids, possibly through a hydrogen bond formation between the hydroxyl group of the former and one of the oxygen atoms of the latter. The possibility of such a hydrogen bond formation is excluded in vitamin E acetate, which binds loosely through the normal hydrophobic interaction. The model for lipid-vitamin interaction explains the in vitro decomposition of H₂O₂ by α-tocopherol. α-Tocopherol in conjuction with H₂O₂ can also act as a free-radical scavenger in the lipid phase. The incorporation of α-tocopherol and vitamin E acetate in DPPC vesicles enhances the permeability of lipid bilayers for small molecules such as sodium ascorbate.     

A novel role for α-tocopherol transfer protein (α-TTP) in protecting against chloroquine toxicity

Chloroquine (CQ) is a widely prescribed anti-malarial agent and is also prescribed to treat autoimmune diseases. Clinical treatment with CQ is often accompanied by serious side effects such as hepatitis and retinopathy. As a weak base, CQ accumulates in intracellular acidic organelles, raises the pH, and induces osmotic swelling and permeabilization of acidic organelles, which account for CQ-induced cytotoxicity. We reported previously that CQ treatment caused α-tocopherol transfer protein (α-TTP), a gene product of familial vitamin E deficiency, to change its location from the cytosol to the surface of acidic organelles. Here we show that α-TTP plays a novel role in protecting against CQ toxicity both in vitro and in vivo. In the presence of CQ, rat hepatoma McARH7777 cells, which do not express α-TTP endogenously, showed more severe cytotoxicity, such as larger vacuolation of acidic organelles and caspase activation, than α-TTP transfectant cells. Similarly, α-TTP knockout mice showed more severe CQ toxicity, such as hepatotoxicity and retinopathy, than wild-type mice. These effects were not ameliorated by vitamin E supplementation. In contrast to bafilomycin A1 treatment, which prevents CQ accumulation in cells by raising the pH of acidic organelles, α-TTP expression prevented CQ accumulation without affecting the pH of acidic organelles. Taken together, our data suggest that α-TTP protects against CQ toxicity by preventing CQ accumulation in acidic organelles through a mechanism distinct from vitamin E transport.     

Effect of finishing period length with α-tocopherol supplementation on the expression of vitamin E-related genes in the muscle and subcutaneous fat of light lambs

The aim of this study was to investigate how different finishing period lengths with α-tocopherol supplementation or alfalfa grazing affect mRNA expression levels of genes related to vitamin E metabolism in L. thoracis (LT) muscle and subcutaneous fat (SF) from lambs of the Rasa Aragonesa breed. Indoors, concentrate-fed light lambs (n = 48) were supplemented with 500 dl-α-tocopheryl acetate/kg concentrate for an average finishing period length of 0 (C), 10.7 (VE10d), 21.2 (VE20d) and, 32.3 (VE30d) days before slaughtering. Simultaneously, 8 lambs with their dams were alfalfa-grazed. The α-tocopherol affected in a short-term the expression of genes in LT muscle (ABCA1, LPL, APOE, and SREBP1) and SF (ABCA1, SCARB1, LPL, and PPARG). On the contrary, PPARA gene expression showed a long-term α-tocopherol effect because the highest levels of PPARA mRNA were found in the VE30d.     

Effects of oral micellized natural vitamin E (d-α-tocopherol) v. synthetic vitamin E (dl-α-tocopherol) in feed on α-tocopherol levels,stereoisomer distribution,oxidative stress and the immune response in piglets

This study evaluated the strategy of supplementing oral micellized natural vitamin E (d-α-tocopherol) to either piglets and/or sows on α-tocopherol concentrations in piglets serum and tissues after weaning. One first experiment tested the influence of the vitamin E supplementation source (natural form in water v. the synthetic form in feed) and dose administered to piglets and/or sows on serum α-tocopherol concentration, α-tocopherol stereoisomer accumulation, antioxidant capacity and immune response of weaned piglets. A second experiment studied the effect of sow source and dose vitamin E supplementation on some of these parameters in piglets. Oral supplementation to sows with natural vitamin E as a micellized form (d-α-tocopherol) at the lowest dose produced a similar concentration of α-tocopherol in serum at days 2, 14 and 28 postpartum to those supplemented with threefold higher dose of the synthetic form in feed. At day 39 of age, neither piglet supplementation source nor dose significantly affected α-tocopherol accumulation in the serum, muscle, subcutaneous fat or liver. Those piglets from sows supplemented with the micellized alcohol form had higher RRR-α-tocopherol stereoisomers (P<0.001) and lower (P<0.001) RRS- RSS- and RSR-α-tocopherol, at day 39 of age than those from sows supplemented with the synthetic form. A predominant importance of sow over piglet vitamin E supplementation was observed on stereoisomer distribution in piglets. Low doses of oral natural vitamin E supplementation to sows or piglets did not increase the oxidative stress of piglets when compared with the use of the synthetic form in feed. Immunoglobulin levels in piglet serum at day 39 were not affected by natural vitamin E supplementation at low doses in drinking water of piglets or sows when compared with the synthetic form in feed. IgA tended to be higher (P=0.145) at day 39 in piglets supplemented with natural vitamin E when compared with those supplemented with the synthetic form. Low doses of oral micellized natural vitamin E supplementation to sows is an interesting feeding strategy, when compared with the use of high doses of the synthetic form in feed, because it results in similar α-tocopherol concentrations, allows a predominant –R stereoisomer distribution in piglets and also maintains their oxidative status in vivo.     

Perlecan mediates the antiproliferative effect of apolipoprotein E on smooth muscle cells. An underlying mechanism for the modulation of smooth muscle cell growth?

Apolipoprotein E (apoE) is known to inhibit cell proliferation; however, the mechanism of this inhibition is not clear. We recently showed that apoE stimulates endothelial production of heparan sulfate (HS) enriched in heparin-like sequences. Because heparin and HS are potent inhibitors of smooth muscle cell (SMC) proliferation, in this study we determined apoE effects on SMC HS production and cell growth. In confluent SMCs, apoE (10 microg/ml) increased (35)SO(4) incorporation into PG in media by 25-30%. The increase in the medium was exclusively due to an increase in HSPGs (2.2-fold), and apoE did not alter chondroitin and dermatan sulfate proteoglycans. In proliferating SMCs, apoE inhibited [(3)H]thymidine incorporation into DNA by 50%; however, despite decreasing cell number, apoE increased the ratio of (35)SO(4) to [(3)H]thymidine from 2 to 3.6, suggesting increased HS per cell. Purified HSPGs from apoE-stimulated cells inhibited cell proliferation in the absence of apoE. ApoE did not inhibit proliferation of endothelial cells, which are resistant to heparin inhibition. Analysis of the conditioned medium from apoE-stimulated cells revealed that the HSPG increase was in perlecan and that apoE also stimulated perlecan mRNA expression by >2-fold. The ability of apoE isoforms to inhibit cell proliferation correlated with their ability to stimulate perlecan expression. An anti-perlecan antibody completely abrogated the antiproliferative effect of apoE. Thus, these data show that perlecan is a potent inhibitor of SMC proliferation and is required to mediate the antiproliferative effect of apoE. Because other growth modulators also regulate perlecan expression, this may be a key pathway in the regulation of SMC growth.     

Oxidative stress and potential free radical damage associated with photocopying. A role for ozone?

OBJECTIVE: To study the relationship between oxidative stress and potential free radical damage associated with photocopying and to explore a role for ozone emitted during the photocopying process. METHODS: 80 photocopying operators (PO) and 80 healthy volunteers (HV) were enrolled in a random control study design, in which the level of lipoperoxide (LPO, thiobarbituric acid reactive substances, TBARS) in erythrocytes and the levels of vitamin C (VC), vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as the activities of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes were determined by spectrophotometric methods. RESULTS: Compared with the average values of the above biochemical parameters in the HV group, the average value of LPO (TBARS) in erythrocytes in the PO group was significantly increased (P < 0.0001), while the average values of VC, VE and beta-CAR in plasma as well as those of SOD and CAT in erythrocytes in the PO group were significantly decreased (P < 0.0001). Pearson product-moment correlation analysis showed that with the increase of the ozone level in photocopying sites and the PO duration of exposure to ozone, the level of LPO in erythrocytes in the operators was increased (P < 0.001), while the levels of VC, VE and beta-CAR in plasma as well as the activities of SOD and CAT in erythrocytes in the operators were decreased (P < 0.01-0.0001). CONCLUSION: The findings in this study suggest that ozone causes oxidative damage in copier operatives.     

The long term effect of α-amanitin on RNA synthesis in adult female Aedes aegypti

The effect of the eukaryotic RNA polymerase inhibitor α-amanitin on RNA synthesis in adult female Aedes aegypti was studied. Administration of the toxin 24 hr, prior to a 2 hr pulse of radioactive precursors inhibited incorporation of the label into total RNA by approximately 40%. Nuclear RNA and microsomal RNA showed 25% and 15% less radioactivity, respectively, than the saline injected controls. Total soluble 4S RNA was unaffected by α-amanitin but heterodisperse RNA incorporated 50% less label than controls. Using both nitrocellulose membrane filters and poly(U)-sepharose chromatography it was found that the synthesis of poly(A)-containing microsomal RNA was severely inhibited by the toxin. It was concluded that the major long term effect of α-amanitin in these adults is to inhibit messenger RNA synthesis and to have little if any effect on ribosomal or 4S RNA.     

Comparative effect of N-substituted dehydroamino acids and α-tocopherol on rat liver lipid peroxidation activities

Free radical damage has been associated with a growing number of diseases and conditions, such as autoimmune diseases, neurodegenerative disorders and multiple types of cancer. Some dehydroamino acids and corresponding peptides can function as radical scavengers. In this study the in vitro effects on rat liver lipid peroxidation levels of fourteen N-substituted dehydroamino acid derivatives and α-tocopherol were investigated. α-Tocopherol is a powerful antioxidant that is beneficial in the treatment of many free radical related diseases. The results indicated that all the compounds showed very good inhibitory effect on the lipid peroxidation compound with α-tocopherol at 1 mM concentrations and the inhibition rate was in the range of 70–79 % with the exception of compound 5. At 0.1 mM concentrations compounds 1, 2 and 9 were found more active than α-tocopherol. The results confirmed that molecules such as dehydroamino acids which have reactive double bonds can act as a guard in vitro against oxidants.     

The effect of some glycolytic intermediates and long-chain acyl-CoA esters on rat skeletal muscle mitochondrial α-glycerophosphate dehydrogenase

Summary -Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: acceptor oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from rat skeletal muscle has been studied. The pH optimum of the enzyme activity was about 7.4 and the apparent K_m value for DL--glycerophosphate was approxinately 1.6mm. The activity of this enzyme was found to be inhibited by DL-glyceraldehyde-3-phosphate, phosphoenolpyruvate and 3-phosphoglycerate in a competitive manner: the apparent K_i values at pH 7.4 being 0.3mm, 1.5mm and 4.0mm respectively. The enzyme was found to be more sensitive to phosphoenolpyruvate at pH 7.0 than 7.6.The activity of -glycerophosphate dehydrogenase in rat skeletal muscle was also inhibited by palmitoyl-CoA and stearoyl-CoA in a competitive manner. The K_i values being about 9.0 m for both metabolites. This inhibition was partly reversed by Ca²⁺ and Mg²⁺ ions. Palmitoylcarnitine also exerted inhibitory effect on -glycerophosphate dehydrogenase activity but palmitate, carnitine and CoA added alone was without effect. It is proposed that the activity of -glycerophosphate dehydrogenase in rat skeletal muscle mitochondria may be controlled by changes of the cytosolic levels of some glycolytic intermediates and long-chain acyl-CoA esters. These results are discussed with respect to the regulation of -glycerophosphate shuttle activity in skeletal muscle.     

Regulation of sheep α-TTP by dietary vitamin E and preparation of monoclonal antibody for sheep α-TTP

α-Tocopherol transfer protein (α-TTP) is a cytosolic protein that plays an important role in regulating concentrations of plasma α-tocopherol (the most bio-active form of vitamin E). Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, we have only recently proved the existence of the α-TTP gene in sheep and, for the first time, cloned its full-length cDNA. However, the study of sheep α-TTP is still in its infancy. In the present study, thirty-five local male lambs of Tan sheep with similar initial body weight were randomly divided into five groups and fed with diets supplemented with 0 (control group), 20, 100, 200, 2000 IU·sheep^− 1·d^− 1 vitamin E for 120 days. At the end of the experiment, the plasma and liver vitamin E contents were analyzed first and then α-TTP mRNA and protein expression levels were determined by quantitative real-time PCR (qRT-PCR) and Western-blot analysis, respectively. In addition, as no sheep α-TTP antibody was available, a specific monoclonal antibody (McAb) against the ovine α-TTP protein was prepared. The effect of vitamin E supplementation was confirmed by the significant changes in the concentrations of vitamin E in the plasma and liver. As shown by qRT-PCR and Western-blot analysis, dietary vitamin E does not affect sheep α-TTP gene expression, except for high levels of vitamin E supplementation, which significantly increased expression at the protein level. Importantly, the specific sheep anti-α-TTP McAb we generated could provide optimal recognition in ELISA, Western-blot and immunohistochemistry assays, which will be a powerful tool in future studies of the biological functions of sheep α-TTP.