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  • To investigate if WP increased CAT activity in cells C

    2024-08-03

    To investigate if WP increased CAT activity in cells, C2C12 muscle AZD0156 were treated with 0.1 to 0.4 mg/mL of WP (80.05% protein) for 24 h and then stressed with 0.75 mM H2O2 for 1 h (Xu et al., 2011). The CAT activity was significantly enhanced from 15.1 ± 0.7 to 23.7 ± 1.3 U/mg of total cellular protein (P < 0.05) by WPC. Similarly, CAT activity was increased 141% in HUVEC cells after a 48-h incubation with 1 mg/mL of 1-kDa permeate of WP hydrolyzed with Corolase PP compared with media alone (P < 0.01) (O'Keeffe and FitzGerald, 2014). In addition, CAT activity also increased in H2O2-stressed lung fibroblasts (MRC-5 cell line) after 24 h of treatment with 100 μg/mL of subtilisin WPH from 25 to 65 U/mg of protein (Kong et al., 2012). The SOD activity was also determined in stressed C2C12 cells (Xu et al., 2011). Once again, cells were preincubated with WP (0.1–0.4 mg/mL) for 24 h, and then stressed for 1 h with 0.75 mM H2O2. Pretreatment with WP significantly increased SOD levels (11.7 ± 0.5 U/mg of protein) in stressed cells compared with cells that did not receive WP treatment (5.27 ± 0.41 U/mg of protein). In addition, WP also increased SOD activity in nonstressed cells from 13.4 ± 0.82 to 19.4 ± 0.6 U/mg of protein. Similarly, 24 h of pretreatment with subtilisin hydrolysates of WPI (20 μg of WPH/mL) increased SOD activity in H2O2-stressed lung fibroblasts compared with non-whey–treated cells by 248% (Kong et al., 2012). The vast majority of experiments to date expose cells lines to whey test samples. The usual mode of delivery of whey products is via food consumption, so the physiological relevance of such experiments is questionable. Target cells will only be exposed to whey components arriving in the bloodstream from the gut. Bovine WP are easily and rapidly digested to individual AA in the gastrointestinal tract, showing maximum concentration of total AA in plasma at 69 min post-WPI consumption (Purpura et al., 2014). Indeed, WPI has a digestible indispensable AA score of 1.09 (Rutherfurd et al., 2015). Power-Grant et al. (2015) performed a simulated gastrointestinal digestion of intact WPC and then measured its antioxidant activity by ORAC. Gastric digestion of WPC increased its ORAC values by 2.5 fold compared with intact WPC. However, when the WPC was in a hydrolyzed form, gastric digestion resulted in a 22% decrease in ORAC values, which indicates that bioactivity of hydrolyzed whey samples was reduced during gut transit (Power-Grant et al., 2015). To assess the antioxidant benefit to intestinal cells exposed to gastric-digested whey products, the intestinal epithelial cell line, Caco-2, was stressed with 0.25 mM H2O2 for 1 h and then treated with gastric-digested WPI (0–2 mg/mL) for 23 h. The ROS activity in Caco-2 cells were reduced by 32.5% when cells were treated with 2 mg/mL of gastric-digested WPI compared with ROS values from stressed cells (Piccolomini et al., 2012). Picariello et al. (2013) recently reported whey peptides that are bioavailable across the Caco-2 intestinal barrier model, postgastrointestinal digestion. Interestingly, the iron-binding peptide TPEVDDEALEK (125–135 AA) from β-LG was found to be transported across the intestinal barrier (Picariello et al., 2013). Whether any of the other whey peptides are antioxidant has yet to be determined.
    DO WHEY PRODUCTS ACT AS ANTIOXIDANT PROTECTOR IN VIVO? Although physiological biomarkers are limiting, human or animal intervention trials with diets that include whey products are the best assessment of antioxidant benefit. Table 5 summarizes the most recent animal trials that tested the antioxidant effect of WP rich diets. Bounous et al. (1989) proposed that a diet rich in GSH AA precursors, such as Cys, would boost cellular GSH production. As WP are Cys-rich, Bounous et al. (1989) fed elderly (17–20 mo old) C7BL/6NIA male mice a diet rich in WPC (20 g/100 g of diet) for 3 mo. Animals were euthanized and GSH levels in liver and heart were measured. Mice on WPC diets had significantly higher GSH levels in liver (9 μmol GSH/g of liver) and heart (1.6 μmol of GSH/g of heart) than those animals fed a casein-rich diet (20 g/100 g of diet) or a control chow diet (8 μmol of GSH/g of liver and 1.3–1.5 μmol of GSH/g of heart; P < 0.05) for the same time period. In addition, the WP-rich diet appeared to extend the lifetime of the aged mice, with a 55% mortality rate reached at 125.0 ± 41.6 d compared with 92.2 ± 55.2 and 92.7 ± 31.7 d for mice fed casein-rich or chow diets, respectively (P < 0.05). Liver GSH was also increased in Fisher rats fed with WP (150 g/1,000 g of diet; 55 μmol of GSH/mL of tissue extract) during 8 wk compared with those on a casein-rich diet (44 μmol of GSH/mL; Haraguchi et al., 2011). Interestingly, a diet supplemented with 10% whey powder protected Wistar rats against induced CCl4 hepatotoxicity (Ashoush et al., 2013). Ashoush et al. (2013) proposed that this protection was as a result of an increase in total GSH plasma levels (CCl4 plus WP = 16.74 ± 1.2 mg/dL vs. CCl4 = 9.94 ± 0.84 mg/dL). As a model of oxidative stress, Sprague-Dawley rats were fed a diet high in iron (2,000 mg/kg) for 6 wk. Those animals that also received 10% WP had increased GSH in blood erythrocytes (11.43 ± 0.71 μM) compared with controls (GSH = 8.75 ± 0.71 μM; Kim et al., 2013). However, CAT levels were not significantly increased after WP supplementation (Kim et al., 2013). In agreement, a combination of exercise and WP intake over an 8-wk test period had little effect on liver CAT activity in Fisher rats fed a WP-rich diet (150 g/1,000 g) compared with those on a casein-rich diet (30 U/mg of protein; Haraguchi et al., 2011). In contrast, Athira et al. (2013) observed a significant increase in liver CAT levels in Swiss albino mice who received an intraperitoneal injection of WP hydrolyzed by subtilisin (4 mg/kg of BW; CAT = 193.66 ± 18.61 U/mg of protein) compared with mice without WP administration (149.67 ± 12.83 U/mg of protein). All of the Swiss albino mice in their study received paracetamol orally (300 mg/kg of BW) for 2 d to induce oxidative stress before WP administration (Athira et al., 2013).