Article pubs.acs.org/JAFC
Potato Chip Intake Increases Ascorbic Acid Levels and Decreases Reactive Oxygen Species in SMP30/GNL Knockout Mouse Tissues Yoshitaka Kondo,*,† Rui Sakuma,‡ Megumi Ichisawa,‡ Katsuyuki Ishihara,‡ Misako Kubo,‡ Setsuko Handa,† Hiroyuki Mugita,‡ Naoki Maruyama,† Hidenori Koga,‡ and Akihito Ishigami† †
Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan Research and Development Department, CALBEE, Inc., 23-6 Kiyohara Kougyoudanchi, Utsunomiya, Tochigi 321-3231, Japan
‡
S Supporting Information *
ABSTRACT: Potato chips (PC) contain abundant amounts of the free radical scavenger ascorbic acid (AA) due to the rapid dehydration of potato tubers (Solanum tuberosum) that occurs during frying. To evaluate the antioxidant activity of PC, this study examined reactive oxygen species (ROS) levels in tissues from SMP30/GNL knockout (KO) mice that cannot synthesize AA and determined AA and ROS levels after the animals were fed 20 and 10% PC diets for 7 weeks. Compared with AA-sufficient mice, AA-depleted SMP30/GNL KO mice showed high ROS levels in tissues. SMP30/GNL KO mice fed a PC diet showed high AA and low ROS levels in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, eyeball, and epididymal fat compared with AA-depleted mice. The data suggest that PC intake increases AA levels and enhances ROS scavenging activity in tissues of SMP30/GNL KO mice, which are a promising model for evaluating the antioxidant activity of foods. KEYWORDS: antioxidant, bioavailability, mouse model, potato, vitamin C
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INTRODUCTION Reactive oxygen species (ROS) such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical are generated continuously by intracellular oxidative events and the mitochondrial electron transport chain. ROS can modify the biological activity of enzymes, modulate intracellular signaling events, and damage biological macromolecules.1 Oxidative stress caused by ROS is a risk factor for many diseases,2 whereas antioxidants, especially those present in various foods, are expected to prevent disease and maintain health. L-Ascorbic acid (AA or vitamin C) is a water-soluble, hexonic sugar acid that exists as an ascorbate monovalent anion at physiological pH. AA is an electron donor and therefore can act as a free radical scavenger and cofactor in reactions catalyzed by Cu+-dependent monooxygenases such as dopamine-βhydroxylase and Fe2+/α-ketoglutarate-dependent dioxygenases such as collagen prolyl and lysyl hydroxylases. Due to the existence of many mutations in the gene encoding L-gulono-γlactone oxidase (Gulo), the capacity to synthesize AA has been lost in guinea pigs and primates, including humans. However, many vertebrates have retained the ability to synthesize AA.3,4 The senescence marker protein-30 (SMP30) is an ageassociated protein that was originally identified in rat liver, and its levels were found to decrease with age.5 Recently, we identified SMP30 as a gluconolactonase (GNL) that catalyzes the lactonization of L-gulono-γ-lactone in the penultimate step of mammalian AA biosynthesis.6,7 We have also established SMP30/GNL knockout (KO) mice.8 Because SMP30/GNL KO mice cannot synthesize AA in vivo, we could determine the influence of AA deficiency on ROS in these animals. AA depletion in SMP30/GNL KO mice increases superoxide anion radical generation in the brain9 and in the lungs causes © 2014 American Chemical Society
pulmonary emphysema due to oxidative stress and a decrease in collagen synthesis.10 Thus, these SMP30/GNL KO mice could be utilized for evaluating the antioxidant activity of foods. However, whether AA depletion affects ROS levels in various tissues in SMP30/GNL KO mice remains unclear. Potato tubers (Solanum tuberosum) are a staple food in many countries and provide an excellent variety of nutrients such as carbohydrates, protein, lipids, dietary fiber, minerals (Zn, Fe, Mg, Ca, K, and Na), and vitamins (vitamin C, vitamin B1, vitamin B2, vitamin B6, niacin, pantothenic acid, and folic acid).11 Besides AA, potatoes contain efficient polyphenolic antioxidants, such as isomers of chlorogenic acid and caffeic acid, whereas fleshcolored potatoes also carry anthocyanin pigments.12,13 Potato chips (PC) contain high amounts of AA per gram wet weight when the fast-dry technology for tuber slices is used in PC production,14 but not with other methods. Recently, we reported that human consumption of mashed potatoes and PC provides AA that is effectively absorbed in the intestine and transferred to the blood.15 Thus, we consider that PC would be a superior dietary source of AA and a good antioxidant food. However, in human clinical studies, whether AA from PC is transported into tissues and exhibits possible antioxidant activity remains to be determined. To establish a novel animal model for evaluating the antioxidant activity of foods, we examined ROS levels in various tissues of SMP30/GNL KO mice using a fluorescent probe to detect ROS in tissue homogenates. Furthermore, to evaluate the Received: Revised: Accepted: Published: 9286
June 18, 2014 August 26, 2014 September 2, 2014 September 2, 2014 dx.doi.org/10.1021/jf502587j | J. Agric. Food Chem. 2014, 62, 9286−9295
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AA-free water containing 10 μM EDTA, and powder CL-2 and water containing 1.5 g/L AA and 10 μM EDTA, that is, 20% PC, 10% PC, AA(−), and AA(+). Five mice were used in each group for these experiments. Mice were maintained on a 12 h light/dark cycle in a controlled environment. All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology (permit no. 12016). Collection of Blood and Tissue Samples. Mice were sacrificed, and each blood sample was drawn into an EDTA-containing syringe from the inferior vena cava. Plasma was obtained by centrifugation at 1700g for 15 min at 4 °C. Mice were then systemically perfused with ice-cold phosphate-buffered saline through the left ventricle to wash out any remaining blood cells. The tissues of interest were then collected. All samples were stored at −80 °C until use. Determination of AA and DHA. Amounts of AA and dehydroascorbic acid (DHA), an oxidized form of AA, were determined by high-performance liquid chromatography using an Atlantis dC18 5 μm column (4.6 × 150 mm; Nihon Waters K.K., Tokyo, Japan) as described previously.17 For determination of plasma AA, plasma was mixed with equal amounts of 10% metaphosphoric acid (MPA) and 1 mM EDTA and centrifuged at 21000g for 15 min at 4 °C. For analysis of AA content in PC, 20% PC powder diet, and 10% PC powder diet, 0.4 g samples were homogenized with 14 volumes of cold 5.4% MPA and 1 mM EDTA using a high-speed homogenizer (POLYTRON, Kinematica AG, Switzerland) and subsequently centrifuged at 21000g for 15 min at 4 °C. For AA determination in the brain, eyeball, heart, lung, testis, stomach, small intestine, large intestine, pancreas, kidney, liver, skin, and epididymal fat, tissues were homogenized with 14 volumes of cold 5.4% MPA and 1 mM EDTA using a Teflon homogenizer (model TH-M, Takashima, Tokyo, Japan). The soleus and plantaris muscles were homogenized with 14 volumes of cold 5.4% MPA and 1 mM EDTA using a Handy homogenizer (Moji-mojikun, Nippon Genetics Co., Ltd., Tokyo, Japan). Homogenates were then centrifuged at 21000g for 10 min at 4 °C. To reduce DHA to AA for determination of AA plus DHA, 90 μL of supernatant was incubated with 10 μL of 350 mM tris(2-carboxyethyl)phosphine hydrochloride for 2 h at 4 °C. The mobile phase was 50 mM phosphate buffer (pH 2.8), 0.2 g/L EDTA, and 2% methanol at a flow rate of 1.3 mL/min, and electrical signals were recorded using an electrochemical detector equipped with a glassy carbon electrode at +0.6 V. The value of DHA was determined by subtracting AA from AA plus DHA. Determination of ROS. ROS were measured using carboxyH2DCFDA as described previously.18,19 Carboxy-H2DCFDA was deesterified from diacetate by intracellular esterases and then oxidized to fluorescent 5-(and-6)-carboxy-2′,7′-dichlorofluorescein by various ROS such as hydroxyl radical, peroxynitrite anion, carbonate, nitrogen dioxide, peroxyl radical, and hydrogen peroxide according to the manufacturer’s handbook and Wardman et al.20 Briefly, tissues were homogenized with 14 volumes of cold homogenization buffer consisting of 50 mM sodium phosphate buffer (pH 7.4), 0.5 mM phenylmethanesulfonyl fluoride, 1 mM EDTA, and protease inhibitor cocktail using a Teflon homogenizer, and the homogenate was centrifuged at 900g for 15 min at 4 °C. One hundred microliters of 50 μM carboxy-H2DCFDA, 25 μL of supernatants containing 15 μg total protein, and 75 μL of homogenization buffer were mixed to yield a 200 μL total reaction volume. Changes in fluorescence intensity were measured every 5 min for 60 min at 37 °C using a SPECTRAmax Gemini (Life Technologies Corp.) with excitation and emission wavelengths set at 485 and 530 nm, respectively. ROS in tissue homogenates were calculated using the values from 30 to 60 min and expressed as “counts/min/mg protein”. The protein concentration was determined by BCA protein assay using bovine serum albumin as a standard. Statistical Analysis. The values are expressed as means ± standard error of the mean (SEM). Statistical analysis was performed using Turkey’s honestly significant difference test for multiple comparisons in body weight, AA plus DHA, and ROS among all groups, and in ORAC among all diets. All statistical analyses were performed using KaleidaGraph software (Synergy Software Inc., Reading, PA, USA). Statistically significant differences were defined as p < 0.05.
antioxidant activity of PC, we determined ROS and AA levels in SMP30/GNL KO mouse tissues after long-term oral ingestion of PC.
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MATERIALS AND METHODS
Chemicals. Ethylenediaminetetraacetic acid (EDTA) was purchased from Dojindo Laboratories (Kumamoto, Japan). Tris(2-carboxyethyl)phosphine hydrochloride was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 5-(and 6)-carboxy-2′,7′dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) was purchased from Life Technologies Corp. (Carlsbad, CA, USA). Protease inhibitor cocktail (cOmplete, EDTA-free) was purchased from Roche Diagnostics GmbH (Mannheim, Germany). BCA protein assay reagent was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). L-Ascorbic acid used in the HPLC analysis and other reagents and chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Preparation of PC and PC Diet. White potato (S. tuberosum cv. Toyoshiro) tubers of similar size and appearance were obtained for an experimental field in Hokkaido, Japan, in September 2010 and stored for several days. The unpeeled potatoes were washed with tap water and air-dried on filter paper. PC were prepared as the commercial product “Olivee” and were manufactured by CALBEE, Inc. (Tokyo, Japan). Briefly, peeled potatoes were sliced and immersed in tap water and then fried in olive oil for several minutes. After frying, the PC were finely ground and passed through a 1.5 mm sieve. To avoid AA degradation, the crushed PC were stored at −80 °C in a nitrogen gas-filled pouch with an aluminum vapor-deposited film. The stored-crushed PC were thawed before use and mixed with the AA-free powder CL-2 (CLEA Japan, Inc., Tokyo, Japan) to produce a final 20% (w/w) and 10% PC content. Diet compositions (moisture, crude protein, crude fat, crude fiber, crude ash, and nitrogen-free extract) of 20% PC CL-2, 10% PC CL-2, and CL-2 were analyzed at the Laboratories for Food and Environmental Science (Tokyo, Japan) and are shown in Table 1.
Table 1. Nutritional Composition of PC Diets (in 100 Grams)
a
nutritional component
20% PC CL-2
10% PC CL-2
CL-2
moisture (g) crude protein (g) crude fat (g) crude fiber (g) crude ash (g) nitrogen-free extract (g) calorie (kcal) ascorbic acid (mg) dehydroascorbic acid (mg)
6.6 20.7 9.0 3.3 7.9 52.5 387 13.6 0.5
7.1 22.6 7.5 3.4 8.4 51.0 376 6.0 0.6
7.6 24.4 6.0 3.6 8.9 49.5 364 nda nd
nd, not detectable.
Assays of Oxygen Radical Absorbance Capacity (ORAC). ORAC for hydrophilic and lipophilic antioxidants was measured using a previously described method.16 Animals. SMP30/GNL KO mice were established and maintained as described previously.8 SMP30/GNL KO and C57BL/6N (wild type, WT) male mice were fed a commercial autoclaved chow diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and had free access to water containing 1.5 g/L AA (DSM Nutrition Japan, Tokyo, Japan) and 10 μM EDTA until weaning. After weaning at the age of 4 weeks, all mice were used for the following two experiments. (1) All mice were fed CL-2 and divided into four groups: SMP30/GNL KO and WT mice given water with or without AA, that is, AA(−) KO, AA(+) KO, AA(−) WT, and AA(+) WT, whereas AA(+) mice had free access to water containing 1.5 g/L AA and 10 μM EDTA. AA(−) mice had free access to AA-free water containing 10 μM EDTA. (2) SMP30/GNL KO mice were divided into four groups that were given either 20% PC powder in CL-2 and AA-free water containing 10 μM EDTA, 10% PC powder in CL-2 and AA-free water containing 10 μM EDTA, powder CL-2 and 9287
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RESULTS Body Weight of SMP30/GNL KO Mice during AA Depletion. Compared to AA(+) KO mice, the body weights of AA(−) KO mice were reduced by 12 and 48% at 7 and 14 weeks, respectively, after weaning (p < 0.05 and p < 0.001, respectively) (Table S1 in the Supporting Information). Meanwhile, there were no significant differences in body weight among AA(+) KO, AA(−) WT, and AA(+) WT mice at 7 and 14 weeks. AA(−) KO mice at 7 weeks showed marginal initial manifestations of AA deficiency (e.g., bleeding from the knee junctions), and they had developed appreciable symptoms of scurvy (rachitic rosary and an abnormal gait) by 14 weeks. Depletion of AA and DHA Levels in SMP30/GNL KO Mice Fed an AA-free Diet and Water. We first determined the AA and DHA levels in tissues and plasma from AA(−) KO mice during AA depletion. Seven weeks after weaning that occurred at 4 weeks, AA plus DHA concentrations in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, pancreas, kidney, liver, and plasma of AA(−) KO mice were 2.6, 3.4, 4.1, 8.5, 2.0, 3.6, 4.1, 3.1, 3.9, 3.5, 1.0, 2.0, and 0% that of AA(+) KO mice, respectively (Figure 1). There were no significant differences in AA plus DHA concentrations in tissues and plasma from AA(+) KO and AA(−) WT mice. The AA plus DHA concentrations in the lung, stomach, and liver were significantly (17, 20, and 29%, respectively) higher in AA(+) WT mice than those of AA(−) WT mice (Figure 1C,G,L). DHA per AA plus DHA levels in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, pancreas, kidney, liver, and plasma of AA(+) KO, AA(−) WT, and AA(+) WT mice were 1.6−4.9, 19.1−24.3, 6.4−20.2, 0−0.5, 25.2−29.6, 11.7−12.0, 4.3−7.8, 4.6−5.0, 1.0−2.2, 4.8−5.2, 5.6−9.0, 0−0.5, and 15.0−18.4%, respectively. As shown in Figure S1 (Supporting Information), the AA plus DHA levels were lower in AA(−) KO mice at 14 weeks compared to 7 weeks. AA plus DHA concentrations in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, pancreas, kidney, liver, and plasma of AA(−) KO mice were 1.5, 2.2, 3.0, 5.6, 3.9, 2.0, 1.9, 1.9, 2.5, 2.1, 1.4, 0.9, and 0% that of AA(+) KO mice, respectively. AA plus DHA concentrations in these tissues and plasma did not differ significantly between AA(+) KO and AA(−) WT mice. The values in the testis and stomach of AA(+) WT mice were significantly (9 and 25%, respectively) higher than those of AA(−) WT mice. The DHA per AA plus DHA in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, pancreas, kidney, liver, and plasma of AA(+) KO, AA(−) WT, and AA(+) WT mice were 1.0−1.8, 19.6−27.5, 17.8−22.3, 0−0.1, 23.4−30.9, 20.7−22.0, 4.9−6.7, 3.4−8.0, 0−2.3, 4.5−7.2, 2.8−16.4, 0−0.7, and 15.6−38.9%, respectively. Increased ROS Levels in SMP30/GNL KO Mice Fed an AA-free Diet and Water. To establish an evaluation method of the antioxidant activity of various foods using SMP30/GNL KO mice, we next investigated ROS levels in tissues from AA(−) KO mice using a simple method for ROS detection.18 ROS levels in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, and large intestine of AA(−) KO mice at 7 weeks were significantly (27, 39, 29, 32, 81, 28, 43, 42, and 64%, respectively) higher than those of AA(+) KO mice (Figure 2A−I). In the pancreas and kidney, ROS levels in AA(−) KO mice were 17 and 31% higher, respectively, than those of AA(+) KO mice, although the difference did not reach significant
levels (p = 0.09 and 0.08, respectively) (Figure 2J,K). There were no significant differences in ROS levels in these tissues among AA(+) KO, AA(−) WT, and AA(+) WT mice. Interestingly, ROS levels in the liver of AA(+) KO mice were significantly (19%) higher than that of AA(−) WT mice. The ROS levels for AA(−) KO mice were 13% higher in the liver compared to AA(+) KO mice, although there were no significant differences (p = 0.07) (Figure 2L). Likewise, at 14 weeks AA(−) KO mice showed increased ROS levels in tissues. ROS levels in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, pancreas, and kidney of AA(−) KO mice were significantly (74, 108, 117, 72, 95, 48, 29, 45, 35, 31, and 26%, respectively) higher compared with those of AA(+) KO mice (Figure S2A−K in the Supporting Information). No significant differences were observed in tissue ROS levels among AA(+) KO, AA(−) WT, and AA(+) WT mice. In livers, the ROS levels of AA(−) KO and AA(+) KO mice were 22 and 19%, respectively, higher than that of AA(−) WT mice, although the difference did not reach significant levels (p = 0.14 and 0.24, respectively) (Figure S2L in the Supporting Information). Nutritional Composition and ORAC of PC Diets. For feeding PC to SMP30/GNL KO mice, we prepared PC diets by mixing the AA-free powder diet CL-2 and crushed PC to yield a final PC content of 10 or 20%. The AA content of the PC used in this study was 78.0 ± 1.6 mg/100 g, whereas DHA was under the detection limit. The nutritional composition of the PC diets was determined as listed in Table 1. Whereas moisture, crude protein, crude fiber, and crude ash were lower in the 20 and 10% PC diets compared with CL-2, crude fat, nitrogen-free extract, AA, and DHA, and calorie amounts of the 20 and 10% PC diets were higher than those of CL-2. Furthermore, we determined the radical scavenging ability of the PC diet and PC using the wellestablished ORAC method. The ORAC values of the 20 and 10% PC diets were significantly higher compared with PC, whereas the values were significantly (25 and 17%, respectively) lower than that of CL-2 (Figure S3 in the Supporting Information). Body Weight Change, Food Intake, and Water Intake of SMP30/GNL KO Mice Fed PC Diets. To investigate the effect of the PC diet on growth, we compared SMP30/GNL KO mice fed a 20% PC diet and AA-free water, a 10% PC diet and AA-free water, an AA-free diet and water, or an AA-free diet and 1.5 g/L AA water after weaning at 4 weeks of age. Each group gained equal amounts of weight compared to the initial body weights (Figure 3A). Food and water intakes throughout the experimental period were 3.5 ± 0.1 g/day/mouse and 3.7 ± 0.1 mL/day/mouse in the 20% PC group, although there were no significant differences among all four groups (Figure 3B,C). Increase in AA and DHA Levels in SMP30/GNL KO Mice Fed PC Diets. Because whether AA and DHA in PC diets were absorbed in intestines and transported into tissues was unclear, we determined the AA and DHA levels in tissues and plasma from SMP30/GNL KO mice fed PC diets (Figure 4). The AA plus DHA levels of the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, eyeball, epididymal fat, skin, pancreas, kidney, liver, and plasma of the 10% PC group were 28, 34, 35, 36, 36, 32, 29, 24, 29, 51, 31, 25, 27, 18, 19, and 14%, respectively, those of the AA(+) group, which were significantly higher than those of the AA(−) group, although the differences in epididymal fat, liver, and plasma did not reach significant levels (p = 0.830, 0.222, and 0.179, respectively). The DHA per AA plus DHA levels in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small 9288
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Figure 1. Ascorbic acid (AA) and dehydroascorbic acid (DHA) levels in SMP30/GNL KO mice fed an AA-free diet and water for 7 weeks: AA (black bars) and DHA (gray bars) concentrations in (A) brain, (B) heart, (C) lung, (D) testis, (E) soleus muscle, (F) plantaris muscle, (G) stomach, (H) small intestine, (I) large intestine, (J) pancreas, (K) kidney, (L) liver, and (M) plasma of AA(−) KO, AA(+) KO, AA(−) WT, and AA(+) WT mice. SMP30/ GNL KO and WT mice were given water with or without AA for 7 weeks after weaning at 4 weeks of age. Values are given as means ± SEM (AA plus DHA) of five animals.
intestine, large intestine, eyeball, epididymal fat, skin, pancreas, kidney, liver, and plasma of the 10% PC group were 0, 25.6, 25.4,
3.1, 38.0, 15.6, 4.4, 0.2, 0, 17.6, 18.2, 16.9, 5.5, 17.7, 0.3, and 26.2%, respectively. The AA plus DHA levels in the brain, heart, 9289
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Figure 2. Reactive oxygen species (ROS) levels in SMP30/GNL KO mice fed an ascorbic acid (AA)-free diet and water for 7 weeks in (A) brain, (B) heart, (C) lung, (D) testis, (E) soleus muscle, (F) plantaris muscle, (G) stomach, (H) small intestine, (I) large intestine, (J) pancreas, (K) kidney, and (L) liver of AA(−) KO, AA(+) KO, AA(−) WT, and AA(+) WT mice. SMP30/GNL KO and WT mice were given water with or without AA for 7 weeks after weaning at 4 weeks of age. Values are given as means ± SEM of five animals.
muscle, stomach, small intestine, large intestine, eyeball, epididymal fat, skin, pancreas, kidney, liver, and plasma of the 20% PC group were 0, 14.9, 7.2, 2.2, 20.2, 12.1, 1.9, 2.2, 0.1, 12.9, 3.9, 12.0, 4.8, 8.0, 1.7, and 25.2%, respectively. Decrease in ROS Levels of SMP30/GNL KO Mice Fed PC Diets. To evaluate the antioxidant activity of PC in mice, we next determined ROS levels of tissues in SMP30/GNL KO mice fed a PC diet (Figure 5). Compared with the AA(−) group, ROS levels in the brain, heart, testis, soleus muscle, plantaris muscle,
lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, eyeball, epididymal fat, skin, pancreas, kidney, liver, and plasma of the 20% PC group were 97, 99, 83, 81, 99, 99, 86, 86, 87, 105, 100, 85, 91, 80, 89, and 89% of those of the AA(+) group, which were significantly higher than those of the AA(−) group. There were no significant differences in the AA plus DHA levels between the 20% PC and AA(+) groups except in the testis, small intestine, and kidney. The DHA per AA plus DHA in the brain, heart, lung, testis, soleus muscle, plantaris 9290
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Figure 3. Body weight change, food intake, and water intake of SMP30/GNL KO mice fed a potato chip (PC) diet for 7 weeks: (A) body weight change of 20% PC (solid circles), 10% PC (open circles), AA(−) (solid squares), and AA(+) (open squares) groups from SMP30/GNL KO mice, that is, mice fed a 20% PC diet and AA-free water, a 10% PC diet and AA-free water, an AA-free diet and water, and an AA-free diet and 1.5 g/L AA water, respectively, for 7 weeks after weaning at 4 weeks of age (values are given as means ± SEM of five animals); (B) food intake and (C) water intake of each group (values are given as means ± SEM through the experimental period).
superoxide anion radical are reduced by the activity of superoxide dismutase (SOD), which catalyzes superoxide anion radical dismutation to hydrogen peroxide.22 Hydrogen peroxide is relatively stable and thus can act as a signal molecule. Although glutathione peroxidase (GPX) and catalase convert hydrogen peroxide to water and oxygen, the Fenton reaction with excess hydrogen peroxide and Fe2+ promote the production of the hydroxyl radical, which is extremely reactive and toxic. Peroxynitrite is generated from the reaction of the superoxide anion radical and nitric oxide produced by nitric oxide synthase and then converts into the hydroxyl radical and nitrogen dioxide radical. Accurate measurement of ROS in living tissues and animals is difficult because ROS are highly reactive and therefore have an extremely short life span. In our previous study, we used a real-time imaging system with lucigenin as a chemiluminescent probe to detect the superoxide anion radical,9 although this approach is applicable only in the brain. Thus, for simple detection of ROS in a variety of tissues, we used tissue homogenates and fluorescein carboxy-H2DCFDA to react with various ROS. Although the type of ROS generated during incubation of tissue homogenates was unclear, it is likely that superoxide anion radicals are generated first, and subsequently a series of ROS is produced. Therefore, this assay would be suitable for evaluating the antioxidant activity of foods that contain a wide variety of antioxidants. AA can scavenge a broad spectrum of free radicals such as superoxide anion radical, hydroxyl radical, nitrogen dioxide radical, and carbonate.22 A noteworthy finding of the present study is that compared to AA(+) KO mice, AA(−) KO mice exhibited much higher ROS levels in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, pancreas, and kidney (although not in the liver) with various levels for each tissue (Figure 2 and Figure S2 in the Supporting Information). Many previous studies have evaluated animal models that lack the capacity to synthesize AA. Harrison et al. reported that F4-neuroprostanes, a brain-specific marker of lipid peroxidation, and malondialdehyde were elevated in the cortex and cerebellum of mice lacking Gulo that received low levels of AA.23,24 AA-deficient Osteogenic Disorder Shionogi (ODS) rats were reported to exhibit increased lipid hydroperoxide levels in the brain, elevated thiobarbituric acid reactive substances (TBARS), and GPX activity in the heart and decreased amounts of glutathione in the plasma and liver.25 TBARS levels were also increased in the plasma and liver of ODS rats, and lipid peroxide concentrations in plasma LDL and liver
small intestine, and large intestine were significantly (40, 34, 18, 41, 29, 17, and 23%, respectively) lower in the 10% PC group, but the difference in tissues from the large intestine did not reach a significant level (p = 0.08). In the lung, stomach, eyeball, and epididymal fat, no significant differences were observed in ROS levels between the 10% PC and AA(−) groups. For the 20% PC group, ROS levels in the brain, heart, lung, testis, soleus muscle, plantaris muscle, stomach, small intestine, large intestine, eyeball, and epididymal fat were significantly (63, 46, 40, 27, 53, 54, 20, 33, 27, 33, and 42%, respectively) lower than those of the AA(−) group, and there were no significant differences between the 20% PC and AA(+) groups. Skin tissue showed a similar pattern, although it did not show significant differences. There were no differences among all four groups in tissue from the pancreas, kidney, and liver.
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DISCUSSION In this study, we showed that AA depletion increases ROS levels in various tissues of SMP30/GNL-KO mice. Furthermore, we demonstrated that the dietary intake of PC increases total AA (AA plus DHA) levels and reduces ROS levels in tissues from SMP30/GNL KO mice. Our data suggest that SMP30/GNL KO mice are a promising model for evaluating the antioxidant activity of foods. Here the ratio of DHA to total AA differed among tissues from AA(−) WT mice and was comparatively lower in the brain, testis, large intestine, and liver but higher in tissues from the heart, lung, soleus muscle, and plantaris muscle as well as in plasma (Figure 1 and Figure S1 in the Supporting Information). These results are consistent with our previous study.17 DHA is transported into cells or generated from AA by a two-electron oxidation, but is rapidly and continuously reduced by dehydroascorbate reductase (DHAR) via a glutathione-dependent mechanism catalyzed by glutaredoxin, protein disulfide isomerase, and omega class glutathione transferase or by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent mechanisms including reduction catalyzed by 3-α-hydroxysteroid dehydrogenase.3 DHAR activity was previously reported to be relatively higher in liver, adrenals, jejunum, ileum, colon, and testis, but lower in kidney, lung, thyroid, heart, and skeletal muscle.21 Thus, our results suggest that the ratio of DHA to total AA might depend on DHAR activity in addition to ROS oxidation in tissues. The superoxide anion radical is generated by the mitochondrial electron transport chain and in the enzymatic reaction that is catalyzed by NADPH oxidase. The reactivity and toxicity of the 9291
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Figure 4. Ascorbic acid (AA) and dehydroascorbic acid (DHA) levels in SMP30/GNL KO mice fed potato chip (PC) diets for 7 weeks: AA (black bars) and DHA (gray bars) concentrations in (A) brain, (B) heart, (C) lung, (D) testis, (E) soleus muscle, (F) plantaris muscle, (G) stomach, (H) small intestine, (I) large intestine, (J) eyeball, (K) epididymal fat, (L) skin, (M) pancreas, (N) kidney, (O) liver, and (P) plasma of 20% PC, 10% PC, AA(−), and AA(+) groups from SMP30/GNL KO mice, that is, mice fed a 20% PC diet and AA-free water, a 10% PC diet and AA-free water, an AA-free diet and water, and an AA-free diet and 1.5 g/L AA water, respectively, for 7 weeks after weaning at 4 weeks of age. Values are given as means ± SEM (AA plus DHA) of five animals.
were elevated in animals fed an AA-free diet.26 Lykkesfeldt et al. reported that guinea pigs 3 weeks after weaning showed decreases in tocopherols and glutathione as well as decreased SOD activity and an increase in protein oxidation in the liver.
They also observed an increase in lipid and DNA oxidation but a decrease in protein oxidation in the brain.27 On the other hand, the sodium-dependent vitamin C transporter (SVCT) 2 transgenic mouse showed that despite having increased AA levels 9292
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Figure 5. Reactive oxygen species (ROS) levels in SMP30/GNL KO mice fed potato chip (PC) diets for 7 weeks: ROS in (A) brain, (B) heart, (C) lung, (D) testis, (E) soleus muscle, (F) plantaris muscle, (G) stomach, (H) small intestine, (I) large intestine, (J) eyeball, (K) epididymal fat, (L) skin, (M) pancreas, (N) kidney, and (O) liver of 20% PC, 10% PC, AA(−), and AA(+) groups from SMP30/GNL KO mice, tht is, mice fed a 20% PC diet and AA-free water, a 10% PC diet and an AA-free water, an AA-free diet and water, and an AA-free diet and 1.5 g/L AA water, respectively, for 7 weeks after weaning at 4 weeks of age. Values are given as means ± SEM of five animals. AA, ascorbic acid.
activity was observed.10 Taken together, an AA deficiency is likely to have a different influence on ROS metabolism and oxidative stress in tissues. Because we do not presently have an explanation for the observed increase in ROS in AA-depleted SMP30/GNL KO mice, further work will be required to elucidate the molecular mechanisms involved in regulating oxidative stress markers such
in the cortex, spleen, kidney, heart, and lung, TBARS levels were not altered.28 We previously reported that AA-depleted SMP30/ GNL KO mice showed an increase in superoxide anion radical levels, although SOD activity was not altered in the brain.9 In lung tissue of AA-depleted SMP30/GNL KO mice there were increased ROS and TBARS levels, but no alteration of GPX 9293
dx.doi.org/10.1021/jf502587j | J. Agric. Food Chem. 2014, 62, 9286−9295
Journal of Agricultural and Food Chemistry
Article
Further studies will be required to define the exact molecular mechanism of the increase in ROS in AA-depleted SMP30/GNL KO mice and the antioxidant activity of PC in an animal model. However, our results suggest that PC intake increases total AA and enhances ROS scavenging activity in tissues of SMP30/GNL KO mice and that SMP30/GNL KO mice are a promising model for evaluating the antioxidant activity of various foods.
as lipid peroxidation, protein oxidation, and DNA oxidation, as well as the activity of antioxidants and antioxidative enzyme levels that could be responsible for this increase in ROS. To our knowledge, no paper describes the bioavailability of AA from PC in animal tissues. Our data showed that the total AA levels in tissues such as the brain, heart, lung, soleus muscle, plantaris muscle, stomach large intestine, eyeball, epididymal fat, skin, pancreas, liver, and plasma of the 20% PC diet group were similar to those of the AA(+) KO group, whereas levels differed in the testis, small intestine, and kidney (Figure 4). The AA contents of 20 and 10% PC diets used in this experiment were 13.6 and 6.0 mg/100 g, respectively. The average AA intake in the 20 and 10% PC groups of SMP30/GNL KO mice was calculated using the food intake values for each 3.5 g/day/mouse to be 0.47 and 0.21 mg/day/mouse, respectively. Because AA(+) KO mice consumed 3.7 mL/day/mouse of 1.5 g/L AA water and thus AA intake was calculated to be 5.5 mg/day/mouse, the AA intakes of the 20 and 10% PC groups were only 8.6 and 3.7%, respectively, of AA(+) KO. SVCTs have high affinity and specificity and are capable of generating a steep AA concentration gradient. Despite small amounts of oral AA intake, the total tissue AA can therefore be maintained at a subsaturating concentration in the 20% PC groups. In contrast, the total tissue AA concentration showed tissue specificity in the 10% PC groups. SVCT1 having a low affinity for AA is known to be abundantly expressed in the liver and kidney,29 which might be the reason why the ratio of total AA levels in the 10% PC group compared to AA(+) KO mice was lower in the kidney and liver (
