Ingrid Elisia

Tryptophan Released From Mother's Milk Has Antioxidant Properties

Bioactive factors in human milk (HM) are crucial to the health of newborns, especially preterm infants. These compounds assist in reducing the oxidative stress that may occur as a result of combined exposure to supplemental oxygen and immature physiologic defenses. To identify the components in HM that contribute to its greater resistance to oxidative stress compared with infant formulae, enzymatic hydrolysates of HM were prepared, ultrafiltered, separated, and analyzed for antioxidant potential. The antioxidant activity [μM Trolox equivalent (TE/g)] of nondigested milk, whole digested milk, and derived ultrafiltrates were 80.4 ± 13.3, 159.0 ± 5.6, and 127.4 ± 3.1, respectively. An HPLC fraction denoted as fraction 23 (5274 ± 630 μM TE/g) was obtained and its constituents identified as tryptophan (Trp), peptides HNPI, and PLAPQA. Scavenging activity was not observed for PLAPQA, whereas moderate activity was associated with HNPI (144 ± 10.7 μM TE/g) and very high activity to Trp (7986 ± 468 μM TE/g). Trp addition to HM and two infant formulas significantly increased formulae antioxidant properties. Trp appeared to be a powerful free radical scavenger naturally present in HM. Its antioxidant effects and potential application in the diets of infants, particularly preterm, must be examined further.

Quantification of hexanal as an index of lipid oxidation in human milk and association with antioxidant components

Hexanal, a secondary product of lipid oxidation, was identified as the major volatile aldehyde generated from lipid peroxidation in human milk. Hexanal was quantified in human milk using solid phase microextraction-gas chromatography/flame ionization detection that required correction for recovery based on the fat content of human milk. Alpha-tocopherol was the only tocopherol isomer in human milk found to be significantly correlated with hexanal (R = −0.374, p<0.05) and the total antioxidant capacity of human milk (ORACFl (R = 0.408, p<0.01)). Ascorbic acid content was negatively correlated (R = −0.403, p<0.05) with hexanal, but not to ORACFl in human milk. The effect of Holder pasteurization on oxidative status of human milk was determined using multiple parameters that included, hexanal level and malondialdehyde as markers of lipid oxidation, vitamins C and E content and antioxidant capacity (e.g. ORACFl). Pasteurization did not affect the oxidative status of milk as measured by hexanal level, ORACFl and malondialdehyde content. We conclude that hexanal is a sensitive and useful chemical indicator for assessing peroxidation reactions in human milk and that alpha tocopherol and ascorbic acid are two key antioxidant components in milk that contribute to protection against oxidation of milk lipids.

Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design

INTRODUCTION The co-culture of Caco-2 and HT29 cells for testing intestinal drug and nutrient transport and metabolism provides the presence of both absorptive and goblet cells, both of which have different culture requirements for optimal growth and function. The research on the co-culture of Caco-2 and HT29 cells is very limited in respect to refining specific conditions that reduce intra- and inter-laboratory variations. In the present study we reported conditions that enable reproducible results to be obtained for drug permeability using in vitro co-culture of Caco-2 and HT29-MTX based on Taguchi experimental design. METHODS The selection of four factors that specified cell culture conditions, namely culture medium, seeding time, seeding density, and Caco-2:HT29-MTX ratio on TEER value and individual permeability coefficients of propranolol, ketoprofen and furosemide was established. Based on the selected conditions for co-culture, we also confirmed the functionality of the final chosen culture condition using nitric oxide as an indicator of intestinal inflammation. RESULTS Choice of cell culture time and culture medium represented two of the most important factors that affected TEER values and the permeability coefficients of the model drugs. On the other hand, the seeding density and the Caco-2:HT29-MTX ratio exerted no significant influence on TEER values and the drug permeability coefficients. No absolute optimal cell culture condition could be obtained for all drugs; however subsequent confirmation experiments concluded that excellent precision for TEER values and drug permeability coefficients was obtained from the two operators using the following combination of conditions, namely an initial seeding density of 1 x 10(5) Caco-2 and HT29-MTX cells/cm(2) at a ratio of 9:1, followed by a 21day culture time in MEM medium. Finally, functionality of the co-culture model system using the above selected in vitro conditions resulted in comparable nitric oxide synthesis to that of a Caco-2 cell monolayer. DISCUSSION Taguchi experimental design enabled us to define a combination of in vitro culture conditions that resulted in excellent operator reproducibility for determining drug permeability coefficients in a Caco-2 and HT29-MTX co-culture system. Moreover, the selected conditions used in co-culture of absorptive and goblet intestinal cells did not compromise the synthesis of nitric oxide, an indicator of inflammation, measured in Caco-2 monolayers.

Evaluation of viability assays for anthocyanins in cultured cells

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely accepted cytotoxicity assay which can produce inaccurate results due to possible interference with the antioxidant property of anthocyanins. Alternative methods to the MTT assay, such as BrdU (DNA-based) and CellTiter-Glo (ATP-based) assays were evaluated to assess anthocyanin cytotoxicity, derived from blackberry in LNCaP, MCF-7 and MDA-MB-453 cell lines. The standard cell counting method was the reference assay. Greater correlation of cell viability values following anthocyanin exposure was obtained from multiple cell lines with the alternative assays when compared with cell counting. MTT and cell counting results were not always correlated, albeit this was a function of cell type. In particular, poor correlations between cell counting and MTT procedures used to assess cytotoxicity of anthocyanins were observed in the MDA-MB-453 cell lines. Comparison of cytotoxicity derived from alternative assays and the MTT assays with the cell counting method was dependent on the assay procedure and the cell type. The LC(50) of blackberry crude extract ranged from 0.4 to 9.4 mg/mL between assays and across all cell lines, whereas a semi-purified anthocyanin extract was not cytotoxic. Cytotoxicity evaluation of polyphenolic-rich extracts using BrdU and CellTiter-Glo assays as alternatives to the MTT method is recommended.

Modulation of NF-κB and Nrf2 control of inflammatory responses in FHs 74 Int cell line is tocopherol isoform-specific

The present study investigates the relative ability of α-, γ-, and δ-tocopherol (Toc) to modulate cell signaling events that are associated with inflammatory responses in fetal-derived intestinal (FHs 74 Int) cells. Secretion of the proinflammatory cytokine IL-8 in FHs 74 Int cells was stimulated in the following order: α-Toc<γ-Toc<δ-Toc. A similar proinflammatory response was observed when inflammation was induced in FHs 74 Int cells. Modulation of IL-8 expression by Toc corresponded to an isoform-specific modulation of NF-κB and nuclear factor-erythroid 2-related factor 2 (Nrf2) cell signaling pathways involved in expression of proinflammatory cytokines and antioxidant enzymes, respectively. δ-Toc and, to a lesser extent, γ-Toc activated NF-κB and Nrf2 signaling, as indicated by the greater nuclear translocation of transcription factors. Activation of NF-κB signaling by γ- and δ-Toc was accompanied by upregulation of NF-κB target genes, such as IL-8 and prostaglandin-endoperoxide synthase 2, with and without a prior IFNγ-PMA challenge. Nevertheless, γ- and δ-Toc, particularly δ-Toc, concurrently downregulated glutamate-cysteine ligase, a Nrf2 target gene that encodes for glutathione biosynthesis. This observation was substantiated by confirmation that γ- and δ-Toc were effective at decreasing glutamate-cysteine ligase protein expression and cellular glutathione content. Downregulation of glutathione content in fetal intestinal cells corresponded to induction of apoptosis-mediated cytotoxicity. In conclusion, γ- and δ-Toc are biologically active isoforms of vitamin E and show superior bioactivity to α-Toc in modulating cell signaling events that contribute to a proinflammatory response in fetal-derived intestinal cells.

Different tocopherol isoforms vary in capacity to scavenge free radicals, prevent inflammatory response, and induce apoptosis in both adult‐ and fetal‐derived intestinal epithelial cells

Gamma-tocopherol (γ-Toc) and δ-Toc are two vitamin E isoforms for which biological activities are not well established, yet these isoforms are present in many different sources of vegetable oils and, therefore, contribute significantly to the total dietary intake of vitamin E. Infant formula also contains relatively high amounts of γ-Toc and δ-Toc, compared with that found in human milk. The efficacy of γ-Toc and δ-Toc to modulate cellular events that include oxidative stress, inflammatory response, and apoptosis-mediated cytotoxicity, relative to α-Toc, was determined using differentiated Caco-2 and primary FHs 74 Int cells intestinal epithelial cell lines. Antioxidant capacity of Toc-isoforms followed the order of δ-Toc > γ-Toc > α-Toc against peroxyl radical-induced membrane oxidation in both Caco-2 and FHs 74 Int cells, respectively. The different Toc-isoforms suppressed inflammatory response in interferon (IFN) γ/phorbol myristate acetate (PMA)-induced Caco-2 adult-derived intestinal epithelial cells, but exacerbated both IL8 and PGE2 secretion in fetal-derived FHs 74 Int intestinal epithelial cells. Lastly, Toc exhibited an isoform-dependent apoptosis-mediated cytotoxicity, whereby δ-Toc elicited the greatest apoptosis followed by γ-Toc, whereas α-Toc was not cytotoxic. Cellular uptake of non-α-Toc isoforms were greater (P < 0.05) than that observed for α-Toc in both intestinal epithelial cell lines which in part explains the superior bioactive function observed for both γ-Toc and δ-Toc, compared with α-Toc. We conclude that the non-α-Toc isoforms of vitamin E have distinct roles that influence oxidative stress and inflammatory responses in both adult and fetal-derived intestinal epithelial cell lines.

Differences in Vitamin E and C Profile Between Infant Formula and Human Milk and Relative Susceptibility to Lipid Oxidation

The vitamin E isoforms and vitamin (vit) C content of infant formulas were compared to human milk and related to relative susceptibilities to lipid peroxidation. We report that a highly distinct vitamin E and C profile exists between formula and human milk. Whileα-tocopherol (α-Toc) is the dominant vit E isoform in human milk, formula contains a substantial amount of α-Toc and δ-Toc that was greater than the level found in human milk (12- and 32-fold, respectively). Vitamin C was also two- fold higher in infant formula compared to human milk. Despite the higher vitamin E and C content, we also observed higher rates of lipid oxidation in the formula when compared to human milk. Storing human milk for one day at refrigeration temperatures did not produce hexanal in human milk, but this storage resulted in an increase in hexanal in formulas. We conclude that the higher concentrations of γ-Toc and δ-Toc in infant formulas did not provide similar protection from lipid oxidation as human milk. We also observed that vit C content was reduced during storage in both infant formula and human milk, which did not occur with the Toc isoforms.