4-HNE Immunohistochemistry and Image Analysis for Detection of Lipid Peroxidation in Human Liver Samples Using Vitamin E Treatment in NAFLD as a Proof of Concept

Maren C Podszun, Joon-Yong Chung, Kris Ylaya, David E Kleiner, Stephen M Hewitt, Yaron Rotman

J Histochem Cytochem . 2020 Sep;68(9):635-643. doi: 10.1369/0022155420946402.

Abstract

Lipid peroxidation is a common feature of liver diseases, especially non-alcoholic fatty liver disease (NAFLD). There are limited validated tools to study intra-hepatic lipid peroxidation, especially for small specimen. We developed a semi-quantitative, fully automated immunohistochemistry assay for the detection of 4-hydroxynoneal (4-HNE) protein adducts, a marker of lipid peroxidation, for adaptation to clinical diagnostics and research. We used Hep G2 cells treated with 4-HNE to validate specificity, sensitivity, and dynamic range of the antibody. Staining and semi-quantitative automated readout were confirmed in human needle-biopsy liver samples from subjects with NAFLD and normal liver histology. The ability to detect changes in lipid peroxidation was tested in paired liver biopsies from NAFLD subjects, obtained before and after 4 weeks of treatment with the antioxidant vitamin E (ClinicalTrials.gov NCT01792115, n=21). The cellular calibrator was linear and NAFLD patients had significantly higher levels of 4-HNE adducts compared to controls (p=0.02). Vitamin E treatment significantly decreased 4-HNE (p=0.0002). Our findings demonstrate that 4-HNE quantification by immunohistochemistry and automated image analysis is feasible and able to detect changes in hepatic lipid peroxidation in clinical trials. This method can be applied to archival and fresh samples and should be considered for use in assessing NAFLD histology.

Moamen M Elmassry, Eunhee Chung, Jay J Cao, Abdul N Hamood, Chwan-Li Shen

J Nutr Biochem . 2020 Sep 10;108492. doi: 10.1016/j.jnutbio.2020.108492. Online ahead of print.

Abstract

The role of the gut microbiome in bone health has received significant attention in the past decade. We investigated the effects of green tea polyphenols (GTP) and annatto-extracted tocotrienols (AT) on bone properties and gut microbiome in obese mice. Male mice were assigned to a two (no AT vs. 400 mg/kg diet AT)×two (no GTP vs. 0.5% w/v GTP) factorial design, namely control, G, T, and G+T group respectively, for 14 weeks. The 4th lumbar vertebra (LV-4) and femur were harvested for bone microstructural analysis using μ-CT. Microbiome analysis using 16S rRNA gene sequencing of cecal feces was performed. AT increased bone volume at distal femur. GTP increased serum procollagen type 1 N-terminal propeptide concentration, bone volume at the distal femur and the LV-4, and trabecular number at distal femur; whereas GTP decreased trabecular separation at distal femur. Interactions between GTP and AT were observed in serum C-terminal telopeptide of type I collagen level (control>G=T=G+T) as well as the cortical bone area (control<G=T=G+T) and thickness (T≥G+T≥G≥control) at femur mid-diaphysis. Redundancy analysis showed a significant difference in the gut microbiome profile among different groups and the relative abundance of Akkermansia muciniphila, Clostridum saccharogumia, and Subdoligranulum variabile was increased in the GTP- and AT-supplemented groups. Functional profiling of the gut microbiome showed the combination of GTP and AT induced biosynthetic pathways for vitamin K₂. Our results suggest that GTP and AT supplementation benefits bone properties in obese mice through modifying gut microbiome composition and function.

Sangita MaitiDutta, Guangping Chen, Smarajit Maiti

Cell Biochem Biophys . 2020 Sep 8. doi: 10.1007/s12013-020-00938-x. Online ahead of print.

Abstract

Oxidative stress is generated in biological system by several endogenous/exogenous factors like environmental-pollution/toxicity/diseases and by daily-life-stress. We previously showed that oxidative-stress impaired the activities/expressions of phase-II drug-metabolizing enzyme, sulfotransferases (SULTs). The SULT catalyzes sulfation of endogenous/exogenous compounds. Vitamin E is globally consumed by a large number of individuals for the cellular protection from oxidative stress and aging. Here, vitamin E (tocopherol; α/γ and tocotrienol; α/γ; 0, 1, 10, or 100 μM) was tested in human carcinoma cell line, HepG2 for their influences on SULTs expression/(western blotting). The effects of oxidant (glutathione-oxidized/GSSG) or reductant (glutathione-reduced/GSH, Dithiothreitol/DTT) on SULT activities were studied in rat-liver/human intestinal tissues. Results suggest, tocopherol is more inductive to monoamine-SULT (MPST) and Dehydroepiandrosterone-SULT (DHEAST) compared to that of tocotrienol (inconsistent change in PPST, phenol sulfotransferase/MPST/EST, estrogen sulfotransferase). The nuclear-factor constitutive androstane receptor (CAR) was found to be induced moderately. This study overall describes that vitamin E moderately influences SULTs expression. The induction ability of tocopherol should be judged taking into account its long-term consummation. Oxidative stress activates rat and human SULTs activities and expressions. Further studies are necessary in this regard.

Kiandokht Ghamari, Ladan Kashani, Morteza Jafarinia, Borna Tadayon Najafabadi, Kamyar Shokraee, Sophia Esalatmanesh, Shahin Akhondzadeh

Women Health . 2020 Sep 6;1-10. doi: 10.1080/03630242.2020.1803465. Online ahead of print.

Abstract

Female sexual disorders (FSD) are a spectrum of disorders common among women, especially in their middle age, which can reduce the female quality of life substantially. We aimed to evaluate the effects of a combined vitamin E and ginseng supplement on amelioration of female sexual dysfunction. In a 6-week, double-blind, randomized, placebo-controlled clinical trial, participants, suffering from sexual dysfunction based on the female sexual function index (FSFI) questionnaire, were randomly allocated to receive the supplement (100 IU vitamin E, 67 mg Korean ginseng, and 40 mg Siberian ginseng) or placebo daily. The primary outcome in our trial was the change in the FSFI total score. Sixty-nine participants were enrolled, but only 31 in each group completed the trial. Changes in the FSFI total score and its domain scores were significant during the trial course within each group. However, the supplement only ameliorated desire and satisfaction domains superior to the placebo. In case of the total score and other domains, the changes were insignificantly different between the treatment groups. Although our study could not find additional benefits for the vitamin E and ginseng supplement over placebo in enhancing sexual function overall, the supplement worked better in enhancing sexual desire and satisfaction.

Peter O Okebukola, Sonal Kansra, Joanne Barrett

Cochrane Database Syst Rev . 2020 Sep 6;9:CD009422. doi: 10.1002/14651858.CD009422.pub4.

Abstract

Background: People with cystic fibrosis are at an increased risk of fat-soluble vitamin deficiency, including vitamin E. Vitamin E deficiency can cause a host of conditions such as haemolytic anaemia, cerebellar ataxia and cognitive difficulties. Vitamin E supplementation is widely recommended for people with cystic fibrosis and aims to ameliorate this deficiency. This is an updated version of the review.

Objectives: To determine the effects of any level of vitamin E supplementation on the frequency of vitamin E deficiency disorders in people with cystic fibrosis.

Search methods: We searched the Cochrane Group’s Cystic Fibrosis Trials Register and also searched international online trial registries for any ongoing clinical trials that were not identified during our register search. Date of last search of the Register: 11 August 2020. Date of last search of international online trial registries: 20 July 2020.

Selection criteria: Randomised controlled trials and quasi-randomised controlled trials comparing any preparation of vitamin E supplementation to placebo or no supplement, regardless of dosage or duration.

Data collection and analysis: Two authors extracted outcome data from each study (published information) and assessed the risk of bias of each included study. They assessed the quality of the evidence using GRADE.

Main results: Four studies with a total of 141 participants were included in the review, two of these were in children (aged six months to 14.5 years), and two did not specify participants’ age. All studies used different formulations and doses of vitamin E for various durations of treatment (10 days to six months). Two studies compared the supplementation of fat-soluble as well as water-soluble formulations to no supplementation in different arms of the same study. A third study compared a water-soluble formulation to a placebo; and in the fourth study a fat-soluble formulation of vitamin E was assessed against placebo. There was limited detail about randomisation and blinding in the included studies which compromises the quality of the evidence base for the review. The heterogeneous mix of the formulations with differing biovailabilities among these studies also limits the generalisability of the data to the wider cystic fibrosis population. None of the studies in either comparison report the review’s primary outcomes of vitamin E total lipid ratio or the incidence of vitamin E-specific deficiency disorders, or the secondary outcomes lung function or quality of life. Water-soluble vitamin E Water-soluble vitamin E may improve serum vitamin E levels compared with control at six months, one study (45 participants), mean difference (MD) 19.74 umol/L (95% confidence interval (CI) 13.48 to 26.00) (low-quality evidence). Similar results were also seen at one month, two studies (32 participants), MD 17.66 umol/L (95% CI 10.59 to 24.74) and at three months, one study (45 participants), MD 11.61 umol/L (95% CI 4.77 to 18.45). Only one study (45 participants) reported weight (secondary outcome of growth and nutritional status) at one and six months, but showed no difference between treatment and control at either time point. Fat-soluble vitamin E Two studies (36 participants) reported higher levels of serum vitamin E at one month with fat-soluble vitamin E compared with control, MD 13.59 umol/L (95% CI 9.52 to 17.66); however, at three months one study (36 participants) showed no difference between treatment and control. No studies in this comparison reported on growth or nutritional status.

Authors’ conclusions: Vitamin E supplementation may lead to an improvement in vitamin E levels in people with cystic fibrosis, although evidence we assessed was low quality. No data on other outcomes of interest were available to allow conclusions about any other benefits of this therapy. In future, larger studies are needed, especially in people already being treated with enteric-coated pancreatic enzymes and supplemented with vitamin E, to look at more specific outcome measures such as vitamin E status, lung function and nutritional status. Future studies could also look at the optimal dose of vitamin E required to achieve maximal clinical effectiveness.

Ahmed A Morsy, Nagwa A Sabri, Abdelrehim M Mourad, Eman M Mojahed, May A Shawki

J Obstet Gynaecol Res . 2020 Sep 3. doi: 10.1111/jog.14467. Online ahead of print.

Abstract

Aim: To evaluate the effect of vitamin E on ovulation and pregnancy in women with clomiphene citrate (CC)-resistant polycystic ovary syndrome (PCOS).

Methods: A prospective, randomized, controlled, open label study was conducted on women with CC-resistant PCOS. Patients were randomized, to either control group (n = 30), who received metformin 500 mg thrice daily, in addition to 150 mg/day CC for 5 days starting from day 3 of menstruation for three menstruation cycles, or vitamin E group (n = 30) who received vitamin E 1500 IU/day for the whole study period in addition to metformin and CC with the same previous regimen. The primary outcome was cumulative ovulation rate, while secondary outcomes were pregnancy rate, serum midluteal progesterone, mean follicular diameter, number of dominant follicles and endometrial thickness.

Results: Ovulation was reported in 57 (64.8%) of 88 cycles in the control group and 63 (73.3%) of 86 cycles in the vitamin E group (P = 0.227), while pregnancy was reported in 4 (4.5%) of 88 cycles in the control group and 6 (7%) of 86 cycles in the vitamin E group (P = 0.491).There were nonsignificant differences between groups regarding serum midluteal progesterone, number of dominant follicles and mean follicular diameter. Endometrial thickness was significantly higher in the vitamin E group compared to the control group.

Conclusion: The findings of this trial do not support the hypothesis that vitamin E may increase the ovulation and pregnancy rates in women with clomiphene citrate-resistant PCOS.

Milad Narimani, Gabriel da Silva

PLoS One . 2020 Sep 3;15(9):e0238140. doi: 10.1371/journal.pone.0238140. eCollection 2020.

Abstract

Vitamin E acetate (VEA) is strongly linked to the outbreak of electronic-cigarette or vaping product use-associated lung injury (EVALI). It has been proposed that VEA decomposition to ketene-a respiratory poison that damages lungs at low ppm levels-may play a role in EVALI. However, there is no information available on the temperature at which VEA decomposes and how this correlates with the vaping process. We have studied the temperature-dependent kinetics of VEA decomposition using quantum chemical and statistical mechanical modelling techniques, developing a chemical kinetic model of the vaping process. This model predicts that, under typical vaping conditions, the use of VEA contaminated e-cigarette products is unlikely to produce ketene at harmful levels. However, at the high temperatures encountered at low e-cigarette product levels, which produce ‘dry hits’, ketene concentrations are predicted to reach acutely toxic levels in the lungs (as high as 30 ppm). We therefore hypothesize that dry hit vaping of e-cigarette products containing VEA contributes to EVALI.

Kamaljit Kaur, Jasdeep Singh, Vipandeep Singh

J Food Sci Technol . 2020 Sep;57(9):3509-3517. doi: 10.1007/s13197-020-04386-6. Epub 2020 Apr 8.

Abstract

Microencapsulated α-tocopherol and wheat germ oil (WGO) were incorporated as WGO (5.0 ml) in liquid: WGO-L, encapsulated: WGO-E, encapsulated α-tocopherol as E1, E2 and E3 at 2.0, 3.0 and 4.0 g respectively in cookies and evaluated for physical, sensory and shelf life parameters. Spread ratio was decreased, whereas hardness was increased with encapsulated formulations and observed least in WGO-L (40.52 N) formulated cookies. During storage moisture content was observed increased (2.51-4.78%), vitamin E was retained in all formulations except WGO-L and was found maximum in E3 (4.45 mg/100 g) formulated cookies. Formulations brought the peroxide value to nil, free fatty acid development was very less, better antioxidant activity (41.1% maximum), total plate count was observed least in E3 (25 × 10² cfu/g) and good sensory acceptance of cookies up to 4 months of storage. The study concluded that encapsulated vitamin E elevated the antioxidant activity and consequently shelf life and nutritive value of cookies.

Julie Hviid Klaebel, Günaj Rakipovski, Birgitte Andersen, Jens Lykkesfeldt, Pernille Tveden-Nyborg

Antioxidants (Basel) . 2020 Sep 1;9(9):E808. doi: 10.3390/antiox9090808.

Abstract

Despite the lack of effective pharmacotherapy against nonalcoholic steatohepatitis (NASH) and liver fibrosis, vitamin E (vitE) supplementation and lifestyle modifications are recommended for the management of NASH due to promising clinical results. We recently reported a positive effect of supplementation with 800 IU vitE and atorvastatin on NASH resolution in guinea pigs. In the present study, we investigated the effect of high-dose vitE therapy combined with dietary intervention against progressive NASH and advanced fibrosis in the guinea pig model. Sixty-six guinea pigs received either high-fat (HF) or standard guinea pig chow diet (Control) for 25 weeks. Prior to eight weeks of intervention, HF animals were allocated into groups; dietary intervention (Chow) or dietary intervention with 2000 IU/d vitE supplementation (CvitE). Both Chow and CvitE reduced dyslipidemia, hepatic lipid accumulation and liver weight (p < 0.05), while CvitE further decreased hepatocellular ballooning (p < 0.05). Subanalyses of individual responses within intervention groups showed significant correlation between the hepatic hallmarks of NASH and lipid accumulation vs. inflammatory state (p < 0.05). Collectively, our results indicate that individual differences in sensitivity towards intervention and inflammatory status determine the potential beneficial effect of dietary intervention and high-dose vitE supplementation. Moreover, the study suggests that inflammation is a primary target in NASH treatment.

Maren C Podszun, Ahmad S Alawad, Shilpa Lingala, Nevitt Morris, Wen-Chun A Huang, Shanna Yang, Megan Schoenfeld, Adam Rolt, Ronald Ouwerkerk, Kristin Valdez, Regina Umarova, Yanling Ma, Syeda Zaheen Fatima, Dennis D Lin, Lakshmi S Mahajan, Niharika Samala, Pierre-Christian Violet, Mark Levine, Robert Shamburek, Ahmed M Gharib, David E Kleiner, H Martin Garraffo, Hongyi Cai, Peter J Walter, Yaron Rotman

Redox Biol . 2020 Sep 1;37:101710. doi: 10.1016/j.redox.2020.101710. Online ahead of print.

Abstract

Oxidative stress (OS) in non-alcoholic fatty liver disease (NAFLD) promotes liver injury and inflammation. Treatment with vitamin E (α-tocopherol, αT), a lipid-soluble antioxidant, improves liver injury but also decreases steatosis, thought to be upstream of OS, through an unknown mechanism. To elucidate the mechanism, we combined a mechanistic human trial interrogating pathways of intrahepatic triglyceride (IHTG) accumulation and in vitro experiments. 50% of NAFLD patients (n = 20) treated with αT (200-800 IU/d) for 24 weeks had a ≥ 25% relative decrease in IHTG by magnetic resonance spectroscopy. Paired liver biopsies at baseline and week 4 of treatment revealed a decrease in markers of hepatic de novo lipogenesis (DNL) that strongly predicted week 24 response. In vitro, using HepG2 cells and primary human hepatocytes, αT inhibited glucose-induced DNL by decreasing SREBP-1 processing and lipogenic gene expression. This mechanism is dependent on the antioxidant capacity of αT, as redox-silenced methoxy-αT is unable to inhibit DNL in vitro. OS by itself was sufficient to increase S2P expression in vitro, and S2P is upregulated in NAFLD livers. In summary, we utilized αT to demonstrate a vicious cycle in which NAFLD generates OS, which feeds back to augment DNL and increases steatosis.