Dried Apple Peel Extract Prevents Oxidative Stress and Inflammation in Intestinal Cells

Nutrition & Health 114 Reduction of Tocopherolquinone Back To Vitamin E by Stannous Chloride (II) and Chromium (III) Isso Bayala1, Ryan K David1, and Eugene Rogers1 1 University of Massachusetts Lowell Vitamin E, also called tocopherol (TOH), belongs to a class of vitamins referred to as lipid-soluble vitamins and well known for its antioxidant effects in living cells. Vitamin E is not produced in the body; it can only be obtained by mean of food from plant sources or through natural and synthetic supplements. Vitamin E is commercially extracted primarily from vegetable oils such as palm, canola, corn, cotton or soybean oils. Studies have shown that during industrial production a significant loss of tocopherols occurs in the range of 25% to 35% due to either thermal breakdown or chemical reactions. the majority of the tocopherol loss during oil processing is due to oxidation which renders the molecule ineffective with respect to its vitamin’s function. Tocopherolquinone is one of the major oxidation products of vitamin E. The present study is the first to demonstrate that stannous chloride (SnCl2.2H2O) ion is capable of reducing αtocopherolquinone (TQ) back to α-tocopherol. Experiments conducted by reacting pure commercial standard α-TQ with stannous chloride (II) in organic solvent (methanol) yield promising results by reducing 95% of the α-TQ and producing vitamin E. These results led us to the isolation by HPLC of a fraction of TQ that was successfully reduced back to TOH. the TQ was obtained by oxidation reaction in vitro of a small quantity of commercial standard α-TOH and copper chloride (CuCl2). in this study, stannous chloride has also proven to provide an important antioxidation effect by preventing the oxidation of TOH when exposed to a powerful oxidation catalyst such as CuCl2. the present study has shown as well for the first time the reaction of chromium (III) hexahydrate CrCl3.6 (H2O) with αtocopherolquinone resulting in the formation of α-tocopherol. This discovery presents a double advantage with regard to its applications: on one hand it can help reduce significantly the loss of vitamin E during industrial processing and on the other hand it could explain the still unanswered question on the potential antioxidant properties of chromium (III) reported in several publications.

doi:10.1016/j.freeradbiomed.2011.10.140

composition, as well as significant differences in antioxidant content, especially with respect to vitamin E content. Trace metal analysis of oils are under way. Conclusion: Malaysian red palm oil supplementation reduces myocardial ischaemia/reperfusion injury and infarct size, through its natural antioxidant and fatty acid content. We hypothesize that African palm oil will have similar effects than the Malaysian counterpart with regards to cardiovascular protection. Further studies need to be done with the South-American palm oil to determine its effects on cardiovascular protection in the rat model.

doi : 10.1016/j.freeradbiomed.2011.10.141

116 Assessment of Mitochondrial Dysfunction and Cytotoxicity of Bisphenol a in Isolated Hepatocytes Angela M Betancourt1, and Shannon Bailey1 1 University of Alabama at Birmingham Bisphenol a (BPA), an important occupational and environmental chemical, causes toxicity to several organ systems including the liver. However, very little is known regarding the precise molecular mechanisms and cellular targets responsible for BPA toxicity. Studies in experimental animal models report that mitochondrial damage could be involved in BPA-induced liver injury. Given that BPA adversely affects the liver, and considering the central role of the liver in whole body energy metabolism and homeostasis, it is critical to understand the mechanisms behind BPA-induced hepatotoxicity. the aim of the present study was to evaluate the effects of BPA on metabolism and mitochondrial function by measuring the oxygen consumption rate (OCR) in primary rat hepatocytes using an XF24 analyzer (Seahorse Bioscience). Primary rat hepatocytes were plated in collagencoated Seahorse 24-well plates, allowed to attach for 5 h, and then exposed to various concentrations of BPA (0, 1,10, and 100 µM) for 12 h before measuring OCR. Basal and maximal respiration, as well as the proton leak, were significantly increased following exposure to 100 µM BPA compared to untreated control hepatocytes. Cell viability was unaffected in rat hepatocytes exposed to 1 µM BPA, and decreased by only 22% and 30% with 10 µM and 100 µM BPA concentrations, respectively. Importantly, these results are in agreement with previous studies showing BPA-mediated increases in the rate of state 4 oxygen consumption in isolated mitochondria. These findings suggest that BPA may cause bioenergetic stress in cells by uncoupling. in summary, our results show that BPA negatively impacts mitochondrial function leading to critical alterations in the metabolic profile of hepatocytes.

115 Analysis of Various Antioxidant Rich Palm Oils in Order To Predict Their Efficacy As Dietary Supplements

doi:10.1016/j.freeradbiomed.2011.10.142

Dirk J Bester1, Adriaan J Esterhuyse1, Jacques Van Rooyen1, Dirho Gihwala1, and Ernest Truter1 1 Cape Peninsula University of Technology Background: It has been shown that dietary supplementation with Malaysian red palm oil improves functional recovery and reduces infarct size, following ischaemia/reperfusion (IR) in rat hearts. We subsequently investigated the composition of several other palm oils. These palm oils are consumed as a regular part of the diet in many parts of Africa and South-America. Materials and Methods: Oils were analyzed for fatty acid composition, antioxidant and trace metal content. Results: Fatty acid and antioxidant composition of palm oils from Africa yielded similar results to that of the Malaysian product. South-American palm oil however showed some differences in fatty acid

117 Dried Apple Peel Extract Prevents Oxidative Stress and Inflammation in Intestinal Cells Marie-Claude Denis1,2, Alexandra Furtos3, Yves Desjardins4,5, Carole Garofalo1, Edgard Delvin1,3, and Emile Levy1,2 1 2 CHU Sainte-Justine, Department of Nutrition, University of 3 Montreal, Department of Biochemistry, University of Montreal, 4 5 University of Laval, INAF Gastrointestinal mucosa is constantly exposed to luminal oxidants from ingested foods. Additionally, the intestinal mucosa is subject to prolonged oxidative stress from reactive oxygen species (ROS) generated during aerobic metabolism. Finally, the influx of neutrophils and monocytes associated with inflammation can

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generate further ROS via respiratory burst enzymes as well as those involved in prostaglandin and leukotriene metabolism. Since polyphenols can act as antioxidants and may help to maintain the body’s natural defenses against a variety of diseases, we have hypothesized that polyphenols extracted from apple peels (DAPP) may offset oxidative stress and inflammation known to occur in inflammatory bowel diseases. Objectives: To characterize polyphenols extracted from DAPP and determine their antioxidant and anti-inflammatory potential in the intestine. Methods: a reversed phase LC-MS method has been developed to separate and identify masses and chemical structures of polyphenolic compounds in DAPP extracted from crude and purified fractions. Differentiated intestinal Caco-2 cells were used to study the role of these polyphenols in the induction of both lipid peroxidation by iron-ascorbate (200µM/2mM) and inflammation by lipopolysaccharide (LPS, 200 µg/mL). Results: Polyphenols were mainly composed of flavonols in crude extract, while purified fractions were mostly characterized by flavanols along with their oligomers. the addition of iron-ascorbate caused a three-fold increase (p < 0,001) in lipid peroxidation compared with control cells. the presence of crude or purified fractions prevented ironascorbate-mediated lipid peroxidation with more favorable effects of crude fractions. Furthermore, investigation of DAPP extractsmediated anti-inflammatory activity, following treatment of Caco-2 cells with iron-ascorbate or lipopolysaccharides (LPS), displayed a marked decline of cytokines (IL-6, TNF-α) and eicosanoids (PEG2). Mechanisms of action include a down-regulation of the nuclear factor-κΒ and cyclooxygenase-2 (COX-2), responsible for the increasing levels of cytokines and eicosanoids, respectively. Conclusions: Our findings provide evidence of the capacity of polyphenols extracted from DAPP to reduce oxidative stress and inflammation, two processes involved in inflammatory bowel disease.

doi:10.1016/j.freeradbiomed.2011.10.143

118 Can a Soy Bean Diet Pave the Way To Calcium Oxalate Stones? Asokan Devarajan1, Shukkur Muhammed Farooq2, Sowmini Kumuran3, and Periandavan Kalaiselvi3 1 2 3 University of California, Wayne State University, University of Madras Membrane injury facilitated the fixation of calcium oxalate crystals and subsequent growth into urolithiasis (kidney stone). Oxalate induced free radical damage is the major detrimental and prerequisite factor for calcium oxalate retention in urothelium. Soy, a proteinaceous diet, which is consumed widely, contains oxalate and antioxidants. a spotlight has been given to find out an answer for the problem of whether a soy diet influences the calcium oxalate lithogenesis due to richness of oxalate or prevent of calcium oxalate crystal retention in urothelium due to its antioxidant property. We have demonstrated that urinary risk factors such as oxalate, calcium, urea and uric acid were significantly increased in urolithic rats, whereas in soy fed rats, the above parameters were found to be within the normal limits except oxalate. Renal antioxidant enzyme activities such as superoxide dismutase, catalase, glutathione-S transferase, glucose 6 phosphate dehydrogenase and glutathione reductase were lowered in urolithic rats with increased lipid peroxidation. It was found to be within normal limits in soy fed rats. There was no tissue injury in control and soy fed rats, while in urolithic rats, tissue injury was associated with calcium oxalate crystal deposition. Furthermore, the urinary excretion of 45 KDa calcium oxalate binding protein, a marker for kidney stone disease was found to be increased in urolithic rats whereas in animals fed with soy, the excretion was found similar to that of the control rats. for the first time, we here report that rats fed with soy diet showed an increased excretion of oxalate as harmless crystalluria or in its

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soluble form without any retention. the calcium oxalate crystal retention process in the kidney is presumed to be inhibited by antioxidants present in soy. Hence, dietary intake of soy by healthy rats may not induce stone formation.

doi:10.1016/j.freeradbiomed.2011.10.144

119 Low Methionine Adenosyltransferase Activity in Newborn Animals Infused With Peroxide Or Parenteral Nutrition Wesam Elremaly1, Thérèse Rouleau1, and Jean-Claude Lavoie1 1 Université de Montréal, Department of Pediatrics & Nutrition, Canada Introduction: the low glutathione levels in premature newborns can partly explain the high incidence of complications associated with oxidative stress in this population. Since the synthetic activity of glutathione is mature, a lack of substrate, especially cysteine, is suspected. Methionine provided by their parenteral nutrition (PN) is the in vivo precursor of cysteine. Since, methionine adenosyltransferase (MAT), the first enzyme in methionine transformation, has redox sensitive cysteines, we hypothesize that peroxides contaminating PN inhibit the activity of MAT, leading to a lower availability of cysteine for glutathione synthesis. Methods: At 3 days of life, guinea pigs with a catheter fixed in jugular vein were separated in 3 groups: 1) Control: animals fed with regular chow, a node closed their catheter; 2) PN: animals fed exclusively with parenteral nutrition (dextrose, amino acids, fat, vitamins) containing about 400 µM peroxides; 3) H2O2: animals fed with regular chow and received continuously 350 µM H2O2 through the catheter. Four days later, liver was sampling for determination of GSH & GSSG, redox potential and activity of MAT. Means ± sem (n=4-7 per group) were compared by ANOVA, P<0.05. Results: MAT activity was lower in groups receiving PN or H2O2 (PN: 3.1±0.6; H2O2: 2.4± 0.2; C: 5.6±0.7 nmol/min/mg prot). the redox potential was more oxidized in PN (PN: -203±3; H2O2: 229± 4; C: -227±2 mV). GSH was not affected (29.4±1.9 nmol/mg prot) whereas GSSG was higher in PN (PN: 0.8±0.1; H2O2: 0.2±0.1; C: 0.4±0.1 nmol/mg prot). Conclusion: Differences between PN and H2O2 in glutathione system suggest that the inhibition of MAT and the oxidation of redox by PN are two independent phenomena. in our model, the oxidant property of PN on glutathione seems stronger than that of H2O2 suggesting the presence of others oxidant molecules in the parenteral nutrition received by newborns.

doi:10.1016/j.freeradbiomed.2011.10.145

120 Optimal Storage of Human Milk James Friel1, and Milana Abramovich1 1 University of Manitoba, Canada Current official protocols for frozen storage of human milk (HM) aim at avoiding bacterial growth rather than preserving nutrients. Based on little data, most authorities recommend HM storage for o o 5-8 days at 4 C or for 6 or more months at -20 C. Therefore in HM samples, we assessed losses of fatty acids (by GC-FID) linoleic, α-linolenic, arachidonic and docosahexaenoic, vitamin B2 o and C (by HPLC) during storage of pasteurized (62.5 C for 30 min) and unpasteurized HM. Analysis of treatments: every other o day for 8 days at 4 C; weekly for one month and monthly for 6

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