Reduced infiltration of T-regulatory cells in tumours from mice fed daily with gamma-tocotrienol supplementation

Shonia Subramaniam, Jeya Seela Anandha Rao, Premdass Ramdas, Mei Han Ng, Methil Kannan Kutty, Kanga Rani Selvaduray, Ammu Kutty Radhakrishnan

Clin Exp Immunol . 2021 Jul 31. doi: 10.1111/cei.13650. Online ahead of print.


Gamma-tocotrienol (γT3) is an analogue of vitamin E with beneficial effects on the immune system, including immune-modulatory properties. This study reports the immune-modulatory effects of daily supplementation of γT3 on host T-helper (Th) and T-regulatory (Treg) populations in a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with either γT3 or vehicle (soy oil) for 2-weeks via oral gavage before they were inoculated with syngeneic 4T1 mouse mammary cancer cells (4T1 cells). Supplementation continued until the mice were sacrificed. Mice (n=6) were sacrificed at specified time-points for various analysis (blood leucocyte, cytokine production, and immunohistochemistry). Tumour volume was measured once every seven days. Gene expression studies were carried out on tumour-specific T-lymphocytes isolated from splenic cultures. Supplementation with γT3 increased CD4+ (p<0.05), CD8+ (p<0.05) T-cells and natural killer cells (p<0.05) but suppressed Treg cells (p<0.05) in peripheral blood when compared to animals fed with the vehicle. Higher interferon-gamma (IFNγ) and lower transforming growth factor-beta (TGF-ꞵ) levels were noted in the γT3 fed mice. Immunohistochemistry findings revealed higher infiltration of CD4+ cells, increased expression of interleukin-12 receptor-beta-2 (IL-12ꞵ2R), interleukin-24 (IL-24) and reduced expression of cells that express the forkhead box P3 (FoxP3) in tumours from the γT3 fed animals. Gene expression studies showed the downregulation of seven prominent genes in splenic CD4+ T-cells isolated from γT3-fed mice. Supplementation with γT3 from palm oil-induced T-cell dependent cell-mediated immune responses and suppressed Treg cells in the tumour microenvironment in a syngeneic mouse model of BC.

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Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy

Erin N Hales, Hadi Habib, Gianna Favro, Scott Katzman, R Russell Sakai, Sabin Marquardt, Matthew H Bordbari, Brittni Ming-Whitfield, Janel Peterson, Anna R Dahlgren, Victor Rivas, Carolina Alanis Ramirez, Sichong Peng, Callum G Donnelly, Bobbi-Sue Dizmang, Angelica Kallenberg, Robert Grahn, Andrew D Miller, Kevin Woolard, Benjamin Moeller, Birgit Puschner, Carrie J Finno

J Vet Intern Med . 2021 Jul 31. doi: 10.1111/jvim.16233. Online ahead of print.


Background: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM.

Hypothesis/objectives: Based on the association of α-tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α-tocopherol, but not γ-tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM-affected horses.

Animals: Vitamin E metabolism: Proof of concept (POC) study; eNAD/EDM-affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM-affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM-affected (n = 12) and age- and sex-matched control (n = 11-12) horses.

Methods: The rates of α-tocopherol/tocotrienol and γ-tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative reverse-transcriptase PCR (qRT-PCR) and droplet digital (dd)-PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog.

Results: Metabolic rate of α-tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ-tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number.

Conclusions and clinical importance: Increased α-tocopherol metabolism in eNAD/EDM-affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high-dose supplementation to prevent the clinical phenotype in genetically susceptible horses.

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A Fast and Efficient Ultrasound-Assisted Extraction of Tocopherols in Cow Milk Followed by HPLC Determination

Archimede Rotondo, Giovanna Loredana La Torre, Teresa Gervasi, Giacomo di Matteo, Mattia Spano, Cinzia Ingallina, Andrea Salvo

Molecules . 2021 Jul 30;26(15):4645. doi: 10.3390/molecules26154645.


A fast HPLC method with fluorescence detector (FD) was developed for the determination of three tocopherols (TOCs) in milk samples from Modicana cattle breed. The ultrasound-assisted procedure was optimized for the extraction of TOCs prior to HPLC/FD analysis, reducing sample preparation time and allowing a fast quantification of α-tocopherol, δ-tocopherol and γ tocopherol. The optimized ultrasonic extraction combines an efficient and simple saponification at room temperature and a rapid HPLC quantification of TOCs in milk. The precision of the full analytical procedure was satisfactory and the recoveries at three spiked levels were between 95.3% and 87.8%. The linear correlations were evaluated (R2 > 0.99) and the relative standard deviation (RSD) values for intra-day and inter-day tests at three spiked levels were below 1% for the retention time and below 5.20% for the area at low level spiking. The proposed procedure, reducing the experimental complexity, allowed accurate extraction and detection of three TOCs in milk samples from Modicana cattle breed.

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Controlled Release of the α-Tocopherol-Derived Metabolite α-13′-Carboxychromanol from Bacterial Nanocellulose Wound Cover Improves Wound Healing

Jessica Hoff, Berit Karl, Jana Gerstmeier, Uwe Beekmann, Lisa Schmölz, Friedemann Börner, Dana Kralisch, Michael Bauer, Oliver Werz, Dagmar Fischer, Stefan Lorkowski, Adrian T Press

Nanomaterials (Basel) . 2021 Jul 28;11(8):1939. doi: 10.3390/nano11081939.


Inflammation is a hallmark of tissue remodeling during wound healing. The inflammatory response to wounds is tightly controlled and well-coordinated; dysregulation compromises wound healing and causes persistent inflammation. Topical application of natural anti-inflammatory products may improve wound healing, in particular under chronic pathological conditions. The long-chain metabolites of vitamin E (LCM) are bioactive molecules that mediate cellular effects via oxidative stress signaling as well as anti-inflammatory pathways. However, the effect of LCM on wound healing has not been investigated. We administered the α-tocopherol-derived LCMs α-13′-hydroxychromanol (α-13′-OH) and α-13′-carboxychromanol (α-13′-COOH) as well as the natural product garcinoic acid, a δ-tocotrienol derivative, in different pharmaceutical formulations directly to wounds using a splinted wound mouse model to investigate their effects on the wounds’ proinflammatory microenvironment and wound healing. Garcinoic acid and, in particular, α-13′-COOH accelerated wound healing and quality of the newly formed tissue. We next loaded bacterial nanocellulose (BNC), a valuable nanomaterial used as a wound dressing with high potential for drug delivery, with α-13′-COOH. The controlled release of α-13′-COOH using BNC promoted wound healing and wound closure, mainly when a diabetic condition was induced before the injury. This study highlights the potential of α-13′-COOH combined with BNC as a potential active wound dressing for the advanced therapy of skin injuries.

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Vitamin E Can Ameliorate Oxidative Damage of Ovine Hepatocytes In Vitro by Regulating Genes Expression Associated with Apoptosis and Pyroptosis, but Not Ferroptosis

Luyang Jian, Ying Xue, Yuefeng Gao, Bo Wang, Yanghua Qu, Shuanghong Li, Heqiong Li, Zhen Li, Bing Wang, Hailing Luo

Molecules . 2021 Jul 27;26(15):4520. doi: 10.3390/molecules26154520.


(1) Background: the current research was conducted to investigate the potential non-antioxidant roles of vitamin E in the protection of hepatocysts from oxidative damage. (2) Methods: primary sheep hepatocytes were cultured and exposed to 200, 400, 600, or 800 μmol/L hydrogen peroxide, while their viability was assessed using a CCK-8 kit. Then, cells were treated with 400 μmol/L hydrogen peroxide following a pretreatment with 50, 100, 200, 400, and 800 μmol/L vitamin E and their intracellular ROS levels were determined by means of the DCF-DA assay. RNA-seq, verified by qRT-PCR, was conducted thereafter: non-treated control (C1); cells treated with 400 μmol/L hydrogen peroxide (C2); and C2 plus a pretreatment with 100 μmol/L vitamin E (T1). (3) Results: the 200-800 μmol/L hydrogen peroxide caused significant cell death, while 50, 100, and 200 μmol/L vitamin E pretreatment significantly improved the survival rate of hepatocytes. ROS content in the cells pretreated with vitamin E was significantly lower than that in the control group and hydrogen-peroxide-treated group, especially in those pretreated with 100 μmol/L vitamin E. The differentially expressed genes (DEGs) concerning cell death involved in apoptosis (RIPK1TLR7CASP8, and CASP8AP2), pyroptosis (NLRP3IL-1β, and IRAK2), and ferroptosis (TFRC and PTGS2). The abundances of IL-1βIRAK2NLRP3CASP8CASP8AP2RIPK1, and TLR7 were significantly increased in the C1 group and decreased in T1 group, while TFRC and PTGS2 were increased in T1 group. (4) Conclusions: oxidative stress induced by hydrogen peroxide caused cellular damage and death in sheep hepatocytes. Pretreatment with vitamin E effectively reduced intracellular ROS levels and protected the hepatocytes from cell death by regulating gene expression associated with apoptosis (RIPK1TLR7CASP8, and CASP8AP2) and pyroptosis (NLRP3IL-1β, and IRAK2), but not ferroptosis (TFRC and PTGS2).

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Administration of vitamin E attenuates airway inflammation through restoration of Nrf2 in a mouse model of asthma

Quang Luu Quoc, Tra Cao Thi Bich, Seo-Hee Kim, Hae-Sim Park, Yoo Seob Shin

J Cell Mol Med . 2021 Jul;25(14):6721-6732. doi: 10.1111/jcmm.16675. Epub 2021 Jun 4.


Accumulating evidence reveals that ROS is one of the key mediators that contribute to the development of asthma. Studies on antioxidants have shown to have beneficial effects on asthma management. However, we still do not know the precise mechanism, and the effects depend on age. This study was conducted to assess the levels of ROS and the effect of antioxidants in younger and older mice using an eosinophilic asthma model. We analyzed airway hyperresponsiveness (AHR), cytokines in bronchoalveolar lavage fluid (BALF), inflammatory cell counts, and the expression levels of NFκB, Nrf2, EPx, and EDN in the lung tissue, as well as the level of ROS in the lung tissue and BALF. The degree of eosinophilia and the levels of IL-5, ROS, and NFκB were significantly increased, whereas the endogenous levels of vitamin E and Nrf2 were decreased in the lung and BALF in the older mice compared to younger mice. The administration of vitamin E attenuated AHR, airway inflammation, and the level of IL-13 and ROS and enhanced the Nrf2 level in the older mice compared to the younger mice. Taken together, vitamin E treatment may have the therapeutic potential through restoration of the Nrf2 level, especially in elderly asthma.

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Effects of tocotrienols supplementation on markers of inflammation and oxidative stress: A systematic review and meta-analysis of randomized controlled trials

Ban-Hock Khor, Hui-Ci Tiong, Shing Cheng Tan, Sok Kuan Wong, Kok-Yong Chin, Tilakavati Karupaiah, Soelaiman Ima-Nirwana, Abdul Halim Abdul Gafor

PLoS One . 2021 Jul 23;16(7):e0255205. doi: 10.1371/journal.pone.0255205. eCollection 2021.


Studies investigating the effects of tocotrienols on inflammation and oxidative stress have yielded inconsistent results. This systematic review and meta-analysis aimed to evaluate the effects of tocotrienols supplementation on inflammatory and oxidative stress biomarkers. We searched PubMed, Scopus, and Cochrane Central Register of Controlled Trials from inception until 13 July 2020 to identify randomized controlled trials supplementing tocotrienols and reporting circulating inflammatory or oxidative stress outcomes. Weighted mean difference (WMD) and corresponding 95% confidence interval (CI) were determined by pooling eligible studies. Nineteen studies were included for qualitative analysis, and 13 studies were included for the meta-analyses. A significant reduction in C-reactive protein levels (WMD: -0.52 mg/L, 95% CI: -0.73, -0.32, p < 0.001) following tocotrienols supplementation was observed, but this finding was attributed to a single study using δ-tocotrienols, not mixed tocotrienols. There were no effects on interleukin-6 (WMD: 0.03 pg/mL, 95% CI: -1.51, 1.58, p = 0.966), tumor necrosis factor-alpha (WMD: -0.28 pg/mL, 95% CI: -1.24, 0.68, p = 0.571), and malondialdehyde (WMD: -0.42 μmol/L, 95% CI: -1.05, 0.21, p = 0.189). A subgroup analysis suggested that tocotrienols at 400 mg/day might reduce malondialdehyde levels (WMD: -0.90 μmol/L, 95% CI: -1.20, -0.59, p < 0.001). Future well-designed studies are warranted to confirm the effects of tocotrienols on inflammatory and oxidative stress biomarkers, particularly on different types and dosages of supplementation. PROSPERO registration number: CRD42020198241.

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Association of measures of body fat with serum alpha-tocopherol and its metabolites in middle-aged individuals

Fleur L Meulmeester, Jiao Luo, Leon G Martens, Nadia Ashrafi, Renée de Mutsert, Dennis O Mook-Kanamori, Hildo J Lamb, Frits R Rosendaal, Ko Willems van Dijk, Kevin Mills, Diana van Heemst, Raymond Noordam

Nutr Metab Cardiovasc Dis . 2021 Jul 22;31(8):2407-2415. doi: 10.1016/j.numecd.2021.05.001. Epub 2021 May 18.


Background and aims: The accumulation of fat increases the formation of lipid peroxides, which are partly scavenged by alpha-tocopherol (α-TOH). Here, we aimed to investigate the associations between different measures of (abdominal) fat and levels of urinary α-TOH metabolites in middle-aged individuals.

Methods and results: In this cross-sectional analysis in the Netherlands Epidemiology of Obesity study (N = 511, 53% women; mean [SD] age of 55 [6.1] years), serum α-TOH and α-TOH metabolites from 24-h urine were measured as alpha-tocopheronolactone hydroquinone (α-TLHQ, oxidized) and alpha-carboxymethyl-hydroxychroman (α-CEHC, enzymatically converted) using liquid-chromatography-tandem mass spectrometry. Body mass index and total body fat were measured, and abdominal subcutaneous and visceral adipose tissue (aSAT and VAT) were assessed using magnetic resonance imaging. Using multivariable-adjusted linear regression analyses, we analysed the associations of BMI, TBF, aSAT and VAT with levels of urinary α-TOH metabolites, adjusted for confounders. We observed no evidence for associations between body fat measures and serum α-TOH. Higher BMI and TBF were associated with lower urinary levels of TLHQ (0.95 [95%CI: 0.90, 1.00] and 0.94 [0.88, 1.01] times per SD, respectively) and with lower TLHQ relative to CEHC (0.93 [0.90, 0.98] and 0.93 [0.87, 0.98] times per SD, respectively). We observed similar associations for VAT (TLHQ: 0.94 [0.89, 0.99] times per SD), but not for aSAT.

Conclusions: Opposite to our research hypothesis, higher abdominal adiposity was moderately associated with lower levels of oxidized α-TOH metabolites, which might reflect lower vitamin E antioxidative activity in individuals with higher abdominal fat instead.

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Role of Vitamin E in Selected Malignant Neoplasms in Women

Anna Markowska, Michał Antoszczak, Janina Markowska, Adam Huczyński

Nutr Cancer . 2021 Jul 19;1-8. doi: 10.1080/01635581.2021.1952626. Online ahead of print.


Vitamin E, which is actually a mixture of eight isoforms (four tocopherols and four tocotrienols), is a powerful antioxidant that protects polyunsaturated fatty acids against oxidation and has the ability to break the chain lipid peroxidation, which is used in the treatment of heart disease, atherosclerosis, muscle disorders or infertility among men. Studies in-vitro show that one of the effects of tocopherol is the reduction of cancer stem cell activity which is connected to poor clinical course. In the scientific literature, reports on the participation of vitamin E not only in protection against the mutagenic effects of reactive oxygen species, but also in its anti-angiogenic activity and the ability to inhibit the invasion and metastasis of neoplastic cells are increasingly common. In this context, the role of vitamin E in preventing the neoplastic process and selected malignant neoplasms among women seems to be of particular interest. In this article, we present the results of research on the potential anticancer effects of vitamin E in the fight against breast, cervical, endometrial and ovarian cancer.

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Quercetin and vitamin E attenuate diabetes-induced testicular anomaly in Wistar rats via the mitochondrial-mediated apoptotic pathway

Oluwatoyin Osinimega Ojo, Olufunso Olabode Olorunsogo

Andrologia . 2021 Jul 18;e14185. doi: 10.1111/and.14185. Online ahead of print.


The role of quercetin and vitamin E treatment against streptozotocin (STZ)-induced testicular abnormalities in diabetic rats and the possible mechanism of action they use for protection were investigated. Diabetes was induced by STZ (45 mg/kg i.p. once) and blood glucose was determined. Plasmatic insulin, testosterone, luteinising hormone and follicle-stimulating hormone (FSH) were determined by ELISA. Levels of cytochrome c, caspase 3 and caspase 9 were evaluated by immunohistochemistry, while lesions were viewed by histology. Insulin played a role in testicular protection against male infertility through modulation of luteinising hormone (LH). This consequently increased Leydig and Sertoli cells and maturation of germ cells with the attached epididymis having abundant spermatozoa. The study showed a positive correlation in the levels of LH, FSH and testosterone; it was further established that all treatments normalised diabetes-induced alterations. Treatment with quercetin and vitamin E resulted in 34% decrease of apoptogenic cytochrome c release. This protected the testes against excessive apoptosis by decreasing caspase 3 and caspase 9 activation by up to 30 and 28% respectively (p < .05). Histology also showed that treatment prevented testicular cell death. The findings show that quercetin/vitamin E possess free radical scavenging properties that protected against testicular damage in diabetes. This suggests the possibility of pharmaco-therapeutic intervention.

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