J. Dairy Sci. 97:1–10 http://dx.doi.org/10.3168/jds.2014-7954 © american Dairy Science association®, 2014.
Antialcoholic liver activity of whey fermented by Lactobacillus casei isolated from koumiss Z. W. Zhao,* D. D. Pan,*1 Z. Wu,† Y. Y. Sun,* Y. X. Guo,† and X. Q. Zeng*
*Food Science and technology Department of the Marine Science School, Ningbo University, Ningbo 315211, P. R. China †Food Science and Nutrition Department, Nanjing Normal University, Nanjing 210097, P. R. China
ABSTRACT
past few decades. Researchers have reported that ALD is caused by oxidative stress and inflammation, and is closely linked to reactive oxygen species (ROS), chemokines, and interactions between alcohol metabolism and hepatic cells (Dey and Cederbaum, 2006; Vidali et al., 2008). Alanine aminotransferase (ALT) is mainly found in the cell cytoplasm, whereas aspartate aminotransferase (AST) is mainly in the mitochondria. The ROS produced by the metabolism of ethanol damages the mitochondrial membrane and increases the permeability of cell membranes. The transaminase in liver cells then permeates the blood, causing an increase in ALT and AST activity in the blood (Wang et al., 2002). Whey is a general term that typically denotes the translucent liquid part of milk that remains following the process (coagulation and curd removal) of cheese manufacturing. Whey also is 1 of 2 major protein groups of bovine milk, accounting for 20% of the milk, whereas casein accounts for the remainder. In the current study, we found that whey was rich in branched-chain amino acids such as leucine, isoleucine, and valerian ammonia acid (Ha and Zemel, 2003). These amino acids have positive effects, including suppressing appetite, which leads to weight loss. Leucine, for example, can stimulate the hypothalamus to produce mammalian target of rapamycin signals, which reduce food intake (Brody, 2000). In addition, the proteins in whey also exhibit a wide variety of biological activities, such as antioxidation and the regulation of blood lipids, and are used to treat nonalcoholic fatty liver disease (Frid et al., 2005; Gholam et al., 2007). Furthermore, whey protein contains bioactive amino acid sequences that may be crucial to its various effects and may be released by using a specific protease to hydrolyze whey protein. A variety of physiological functions related to active polypeptides have been found in the degradation products of whey protein (Korhonen and Pihlanto, 2006; Hartmann and Meisel, 2007). These findings indicate that it is important to discover more effective and comprehensive ways to use whey. Whey fermented liquid (WFL), prepared by inoculating whey with Lactobacillus and allowing it to be incubated, has been found to be rich in amino acids, polypeptides, and proteins. These properties
Whey fermented liquid (WFL) was studied for its hepatoprotective effects by using chronic alcoholinduced mice. Whey fermented liquid, prepared by inoculating whey with 4% (vol/vol) Lactobacillus casei and then incubating at 41°C for 8 h, was used to orally treat alcohol-induced mice at 3 dosages for 5 wk. Ethanol consumption significantly reduced the activity of superoxide dismutase and glutathione peroxidase, while lowering glutathione content and increasing levels of aspartate aminotransferase, alanine aminotransferase, total triglyceride, malondialdehyde, and cytochrome P450 2E1. Treatment with WFL significantly attenuated the increased levels of alanine aminotransferase, aspartate aminotransferase, triglyceride, and cytochrome P450 2E1, while decreasing superoxide dismutase, glutathione peroxidase, malondialdehyde, and glutathione levels. Pathological changes in the livers of mice who had ingested alcohol were improved by the administration of WFL. These results suggest that WFL may exert a protective effect against alcoholic liver disease by increasing antioxidant activity, which supports the use of WFL as an antialcoholic liver disease treatment. Key words: Lactobacillus casei, alcoholic liver, fermented whey, antioxidation INTRODUCTION
Alcohol consumption is increasing as the standard of living of many people around the world improves. However, long-term excessive intake of alcohol causes alcoholic hepatitis, fatty liver disease, and cirrhosis (Williams, 2006). As the World Health Organization reported in 2011 (WHO, 2011), the abuse of alcohol is a public health problem that causes disease, toxicity, injury, and premature death. Alcoholic liver disease (ALD), whose pathogenesis is not yet fully understood, has been of growing interest to researchers over the Received January 16, 2014. Accepted March 9, 2014. 1 Corresponding author: [email protected]
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mean that WFL exhibits many physiological functions. Although some of the biological activities of WFL have been discussed in the published literature, its role in relation to alcoholic liver disease has not been analyzed. In this study, 4 strains of Lactobacillus isolated from koumiss, a dairy product similar to kefir, were identified using 16S rRNA analysis. Whey fermented liquid was prepared using the screened Lactobacillus that exhibited the strongest antioxidant activity. The effect of the whey fermented using Lactobacillus casei (ATCC 15008) on alcohol-induced mice was then analyzed in order to provide some hope for treatment of alcoholinduced alcoholic hepatitis, fatty liver disease, and cirrhosis. MATERIALS AND METHODS Chemicals and Reagents
Cow milk was obtained from the Ningbo Dairy Group (Zhengjiang, China). Methanol, acetonitrile, and monoclonal anti-β-actin antibody (chromatographic grade) were purchased from Sigma Chemical Co. (St. Louis, MO). Trifluoroacetic acid was obtained from TCI Chemical Co. (Tokyo, Japan). Assay kits for AST, ALT, total triglyceride (TG), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, P.R. China). The anti-cytochrome P450 2E1 (CYP2E1) antibody (ab28146) was from Abcam (Britain). The horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) were obtained from Beyotime (Jiangsu, China). The other chemicals were all of analytical grade from commercial sources. Strains and Culture Media
Four strains of Lactobacillus isolated from koumiss were selected. The strains were obtained from the Key Laboratory of Marine Biotechnology Application, School of Marine Sciences, Ningbo University (Ningbo, P. R. China). The strains were cultured at 37°C in de Man, Rogosa, and Sharpe (MRS) medium for 24 h before being used in the next experiment. Identification of Lactobacillus
Identification of different Lactobacillus strains was conducted using 16S rRNA analysis (Scarpellini et al., 2002). Genomic DNA of Lactobacillus was isolated using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R. China) according to the manufacturer’s Journal of Dairy Science Vol. 97 No. 7, 2014
instructions. To detect the amplification of the 16S rRNA sequence, PCR was performed using the forward (5′-AACTGAAGAGTTTGATCCTGGCTC-3′) and the reverse (5′-TACGGTTACCTTGTTACGACTT-3′) primers. The 50-μL PCR reaction mixture consisted of 4 μL of deoxyribonucleotide triphosphate (dNTP; each 2.5 mM), 5 μL of 10× PCR buffer, 1 μL of forward primer (50 pM), 1 μL of reverse primer (50 pM), 4 μL pf MgCl2 (25 mM), 2 μL of DNA template (100 ng/ μL), 0.5 μL of Taq polymerase (5 U/μL), and 32.5 μL of double-distilled H2O. The cycling program was as follows: 94°C for 5 min, 94°C for 1 min, 56°C for 45 s, 72°C for 2 min for the next 30 cycles, and then 72°C for 10 min and 4°C for 30 min. Then, the purified PCR products were cloned into the T vector and the DNA sequence was analyzed by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, P. R. China). The sequencings were performed using DNAMAN software (Lynnon Corp., Pointe-Claire, QC, Canada). A homology search to reference strains registered in GenBank was performed using the NCBI BLAST algorithm (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Preparation of Supernatant and Intracellular Cell-Free Extracts
After the strains were cultured at 37°C in MRS broth for 24 h, the supernatant was harvested by centrifugation at 3,000 × g for 20 min at 4°C. The antioxidant activity was then analyzed. For the preparation of intracellular cell-free extracts, cell pellets were washed twice with PBS (0.85% NaCl, 2.68 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4; pH 7.0) and resuspended in deionized water to adjust the total cell number to 109 cfu/mL before sonication. Ultrasonic disruption was performed for five 1-min intervals (60 cycles at 400 W) in an ice bath. The resulting supernatant was collected to determine the level of antioxidant activity after the cell debris was removed by centrifugation at 6,500 × g for 20 min at 4°C. Scavenging of α,α-Diphenyl-β-Picrylhydrazyl Radical
The α,α-diphenyl-β-picrylhydrazyl (DPPH) clearance rate of the sample was analyzed by using the DPPH method (Shimada et al., 1992; Lin and Chang, 2000). Two milliliters of sample and 2.0 mL of freshly prepared DPPH-ethanol solution (0.2 mmol/L) were mixed and allowed to react in the dark for 30 min. The scavenged DPPH· was monitored by measuring the decrease in absorbance at 517 nm (Ai). Meanwhile, 2.0 mL of ethanol was used to take the place of DPPH· in the blank group (Aj). Control samples contained deionized water (A0). The scavenging ability was defined as follows:
ANTIALCOHOLIC LIVER ACTIVITY OF WHEY FERMENTED BY LACTOBACILLUS CASEI
DPPH scavenging ability = [1 − (Ai − Aj)/A0] × 100%. Superoxide Anion Scavenging Assay
The superoxide anion scavenging assay was analyzed using a modification of the method used by Marklund and Marklund (1974). First, 4.5 mL of Tris-HCl buffer (0.1 mol/L; pH 8.2) was mixed with 1 mL of EDTA (1.0 mL/L), 1.0 mL of sample, and 2.4 mL of distilled water. The mixture was left to react in a water bath (25°C) for 10 min. Then, the incubated mixture was mixed with 2.0 mL of pyrogallol (9.0 mmol/L) and allowed to react for 60 min. Finally, the mixture was mixed with 100 µL of HCl (12.0 mol/L) to determine the absorbance at 325 nm (As). Meanwhile, 1.0 mL of distilled water was used to take the place of the sample in the control group (Ac). The scavenging ability was defined as (Ac − As)/Ac × 100%. Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activity of the sample was determined by using the method of Smirnoff and Cumbes (1989). The mixture, which consisted of 6 mM FeSO4, 1 mL of sample, and 6 mM H2O2, was incubated at 25°C and allowed to react for 10 min (Aj). The reaction mixture was then mixed with 9 mM salicylic acid and 4 mL of distilled water and left to react for 30 min. The absorbance was determined at 510 nm (Ai). Meanwhile, 1.0 mL of distilled water was used to take the place of the sample in the control group (A0). The scavenging activity was defined as [1 − (Ai − Aj)/ A0] × 100%. Preparation of WFL
Cow milk was centrifuged for 20 min at 2,000 × g at 20°C to remove fat. The centrifuged milk was acidified by hydrochloric acid to pH 4.6 and filtrated using a ceramic membrane and stored at 4°C in a refrigerator. Then, the Lactobacillus casei (H4), which was cultivated in MRS liquid medium for 2 to 3 generations at 37°C, was used as starter culture to produce WFL. The preparation method involved adding 4% (vol/vol) Lactobacillus casei (H4) inoculum to whey liquid and culturing for 16 h at 41°C. The fermentation broth was collected to determine the antioxidant activity and stored at 4°C before the next experiment. Biological Experiment
Fifty mice (20 ± 2 g; male) were purchased from the Animal Division of Zhejiang University (Zhejiang,
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China), and acclimatized for 2 d in the animal experimental research laboratory of Ningbo University. The mice had free access to rodent chow and mineral water, and maintained at 22 ± 2°C with 55 ± 5% relative humidity and a 12 h:12 h light-dark cycle (Lian et al., 2010). The mice were randomly divided into 5 groups, with 10 mice in each group: (A) control group: 6.25 mL of distilled water/kg per day, (B) model alcohol-fed mice: 6.25 mL of ethanol/kg per day, (C) model mice treated with a low-WFL (LWFL) dose: 6.25 mL of ethanol/kg and 10 mL of WFL (20 times diluted)/kg per day, (D) model mice treated with a medium WFL (MWFL) dose: 6.25 mL of ethanol/kg and 10 mL of WFL (10 times diluted)/kg per day, and (E) model mice treated with a high-WFL (HWFL) dose: 6.25 mL of ethanol/kg and 10 mL of WFL/kg per day. In the model groups, mice were intragastrically administered with ethanol (20% in distilled water) at 0900 h. The various WFL doses were administered orally via a stomach tube every day around 1400 h (Tipoe et al., 2008; Hou et al., 2010; Cheng and Pan, 2011). All experiments lasted 5 wk. Changes in BW were monitored weekly. Analysis of Serum Enzymes
At the end of the animal experiment, blood samples were collected from the eyeballs of the mice. The serum was separated from blood samples for analysis after centrifugation at 1,000 × g for 20 min at 4°C (Kolankaya et al., 2002). Then serum enzymes, such as ALT and AST, were measured using the ALT and AST activity assay kits. Liver Function and Oxidative Stress Parameter
Liver homogenate (10%, vol/vol) was prepared according to a method described previously (Lieber and DeCarli, 1970; Raza et al., 2004). The liver (1 g), which was cut into small pieces, was fully ground using a tissue blender with 9 volumes of precooling medium (pH 7.4). Supernatant was collected from the liver homogenate by centrifugation at 1,000 × g for 10 min at 4°C. The medium comprised 0.1 mol/L Tris-hydrochloric acid, 0.25 M sucrose, 0.1 mM EDTA, and 0.05 mmol of phenylmethylsulfonyl fluoride/L. Finally, malonaldehyde, liver TG, and the enzyme activity of the liver tissue were analyzed using the corresponding assay kits. Liver Histopathological Studies
Liver tissues, which were immediately taken from the dead mice, were put into freshly prepared formalin solution (10%; 4°C). Frozen sections of liver (5–6 μm) were Journal of Dairy Science Vol. 97 No. 7, 2014
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cut using a microtome, and then washed with ethanol and water. The frozen section was counterstained with hematoxylin for 45 s. An image was then captured under a light microscope at 200× magnification (Park et al., 2012). Levels of CYP2E1
Levels of CYP2E1 in 10% liver homogenate were determined by Western blotting (WB). Liver tissue was homogenized in 9 volumes of cold buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin at pH 7.4) at 4°C. Then, the homogenates were centrifuged at 1,000 × g for 15 min at 4°C, and the supernatant was stored for analysis. Meanwhile, the protein content of the supernatant was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime). The CYP2E1 protein in the supernatant had been isolated in the gel by electrophoresis and then transferred to the polyvinylidene difluoride membrane. Finally, the polyvinylidene difluoride membrane was blocked with 5% Tris-buffered saline with Tween 20 (TBST) for 30 min at 25°C before incubation with ab28146 diluted 1/1,000 for 12 h at 4°C. Horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) was used as the secondary antibody. The position of CYP2E1 in the hepatic cells was measured using immunohistochemistry (frozen sections) according to the manufacturer’s instructions. Cells were fixed in acetone and blocked with 1% BSA for 5 min at 25°C before incubation with ab28146 diluted 1/300 for 30 min at 25°C. The Alexa Fluor 488-labeled goat antirabbit IgG (H+L) was used as the secondary antibody. Statistical Analysis
Data were expressed as mean values ± standard deviation. All data were analyzed using SPSS software (version 16.0; SPSS Inc., Chicago, IL). The differences between groups were compared using one-way ANOVA, followed by Dunnett’s test and P < 0.05 was considered to be significant. RESULTS AND DISCUSSION 16S rRNA Identification of Lactobacillus
The taxonomic identification of the 4 strains is shown in Figure 1. The homology tree was constructed using DNAMAN software. The 16S rRNA gene sequence similarities of strains H1 and H4 (H2 and H3) were found to be 100% (Figure 1). The analysis of the 16S rRNA gene sequence showed that the 4 strains Journal of Dairy Science Vol. 97 No. 7, 2014
Figure 1. Homology tree of 16S rRNA sequences in 4 different Lactobacillus strains.
clearly belong to Lactobacillus casei (Figure 1, strains H1 and H4) and Lactobacillus fermentum (Figure 1, strains H2 and H3). Free Radical Scavenging Effect of Lactobacillus and WFL
According to the free radical metabolism theory proposed by Harman (1994), free radicals, which exhibit high activity levels, easily react with other molecules and free radicals. Such interactions can initiate a chain reaction that produces new free radicals. A large number of free radicals, produced by the body during such metabolic processes, can cause tissue injury by attacking biological macromolecules (Cutler, 1991; Takemura et al., 1992). The free radical rate of metabolism increases as alcohol intake levels increase. Therefore, the ability of Lactobacillus and WFL to reduce free radical toxicity was analyzed directly by monitoring DPPH and hydroxyl radical scavenging activity, as well as the superoxide anion scavenging assay. To screen out Lactobacillus antioxidant activity, 4 Lactobacillus strains were cultivated in MRS broth and then antioxidant activity was measured in the supernatants and intracellular cell-free extracts. The screening results revealed that the antioxidant activity of Lactobacillus strains H1 and H4 were higher than that of the other strains, and both H1 and H4 were Lactobacillus casei (Figure 1). The scavenging effects of the 4 strains on DPPH, superoxide anions, and hydroxyl radicals are shown in Table 1. Because Lactobacillus strain H4 exhibited greater antioxidant activity, H4 was chosen from the 4 strains to use to ferment the whey. As shown in Table 1, WFL inoculated with Lactobacillus casei H4 showed the strong antioxidant capability.
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Table 1. Free radical scavenging effect of Lactobacillus and whey fermented liquid (WFL; mean ± SD; n = 5)1 Scavenging of DPPH (%) Item2 H1 H2 H3 H4 WFL
A 50.84 45.97 45.30 54.48 62.35
± ± ± ± ±
B 0.04 0.05 0.03 0.05 0.06
31.21 26.52 27.47 33.16 34.02
± ± ± ± ±
0.05 0.04 0.05 0.02 0.03
50.49 45.59 33.87 50.75 65.41
Scavenging of O2−· (%)
Scavenging of OH· (%)
A
A
± ± ± ± ±
B 0.07 0.06 0.02 0.06 0.04
24.85 13.57 23.56 28.08 30.75
± ± ± ± ±
0.03 0.03 0.03 0.01 0.03
51.37 42.35 43.90 52.44 61.73
± ± ± ± ±
B 0.04 0.04 0.05 0.03 0.07
27.63 13.53 11.58 29.36 30.43
± ± ± ± ±
0.06 0.04 0.08 0.05 0.03
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DPPH = α,α-diphenyl-β-picrylhydrazyl; A = supernatant; B = intracellular cell-free extracts. H1, H2, H3, and H4 = Lactobacillus strains.
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Effect of WFL on Body and Organ Weight
The BW of the mice was monitored every week. The results indicated that, after 5 wk, the BW of the alcohol-diet group was lighter than that of the control mice (Figure 2A). Also, the weights of the alcohol-diet group mice fed different doses of WFL were significantly different from those not fed WFL. The difference between the liver weight-to-BW ratio in the ALD and control groups was statistically significant (Figure 2B). The liver weight-to-BW ratio in the control group was lower than that found in the ALD group. Mice with ALD fed the HWFL dose gained in terms of their liver weight-to-BW ratio at a normal rate. Results of Serum Biochemical Analysis
The protective effect of WFL on the serum levels of ALT and AST in ALD mice is illustrated in Figure 3A and 3B. In the ALD group, the serum levels of ALT and AST were 40.7 ± 3.3 and 98.3 ± 1.7 U/L, respectively. Compared with the ALT and AST levels in the control group, no significant change occurred in the serum levels of ALT, whereas after 5 wk, the serum levels of AST significantly increased. Chronic daily feeding of WFL significantly reduced AST levels in serum, and ALD mice fed the HWFL dose showed a significant reduction in the alcohol-induced increase in AST levels (from 98.3 ± 1.7 to 72.6 ± 2.5 U/L). In addition, a significant difference existed in ALT levels between the MWFL, HWFL, and ALD mice. It could be suggested that WFL had a protective effect on the liver by inhibiting the increase in aminotransferase activity in the serum, protecting the cell and mitochondrial membranes from the damage caused by alcohol. Effect of WFL on MDA and TG Content in Liver Tissue
The effect of chronic alcohol ingestion on MDA content and the total TG in ALD mice are presented in
panels A and B of Figure 4. The content of hepatic TG and MDA in the ALD mice was significantly increased and matched the levels in the control mice, which suggested that the lipid metabolism of ALD mice was disorganized. Long-term consumption of alcohol increases TG levels in liver cells by greatly increasing TG synthetase activity, but alcohol metabolism can also influence the synthesis of apolipoprotein and increase the difficulty of transporting TG into the liver. Moreover, the significant increase in MDA content leads to the formation of Mallory corpuscles via cytoskeleton protein crosslinking, which leads to fibrosis or cirrhosis by activating Kupffer cells, which in turn promote the synthesis of collagen fibers (Niemelä et al., 2000). However, the hepatic TG content in all of the treatment groups was significantly reduced compared with that of the ALD mice. A significant reduction in hepatic MDA content was detected in the MWFL and HWFL mice. These findings indicated that WFL can help ALD mice maintain a balance between fat synthesis and fat metabolism. Effect of WFL on the GSH and Enzyme Activity in Liver Tissue
Superoxide dismutase can protect organisms, prevent aging, and treat diseases by catalyzing superoxide anions into H2O2 and O2 (Dean et al., 1997). Glutathione plays a key role in the process of protecting the liver from the ravages of alcoholic liver disease. Glutathione not only scavenges free radicals, but also removes drugs, chemicals, and reactive molecules from cells. Furthermore, GPx catalyzes GSH to become oxidized GSH by consuming H2O2 and LOOH. Therefore, the generation of hydroxyl radicals and lipid peroxidation is prevented, and the structure and function of cell membranes is protected. Ethanol becomes acetaldehyde via the catalytic action of alcohol dehydrogenase. Acetaldehyde can combine with glutathione, causing a decrease in GSH levels in the cytoplasm of liver cells, thereby reducing the ability of liver cells to eliminate free radicals (Cheng et al., 2006). Moreover, ethanol also interferes with Journal of Dairy Science Vol. 97 No. 7, 2014
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Figure 2. Effects of whey fermented liquid (WFL) on the liver and body. (A) Body weight; (B) liver-to-BW ratio. Results are expressed as the mean ± SD (n = 10). *P < 0.05 compared with the control group; #P < 0.05 compared with the alcoholic liver disease (ALD) group. LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dosetreated mice; HWFL = high-WFL dose-treated mice.
the function of carrier proteins on the mitochondrial membrane, reducing GSH levels in the mitochondria and causing the mitochondria to swell. In our study, the activity of SOD, GSH, and GPx were measured to analyze the antioxidant ability of the body. Superoxide dismutase, GSH, and GPx activity in the liver tissue is shown in panels A, B, and C, respectively, of Figure 5. Hepatic SOD, GSH, and GPx activity in Journal of Dairy Science Vol. 97 No. 7, 2014
Figure 3. Effects of whey fermented liquid (WFL) on the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity of normal and experimental mice. Data are expressed as means ± SD (n = 10). Values are significant compared with the following: control (*P < 0.05) and alcohol (#P < 0.05). ALD = alcoholic liver disease group; LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dose-treated mice.
the ALD mice were significantly reduced compared with those of the control mice. However, SOD, GSH, and GPx activity in all the treatment groups were significantly elevated compared with those of the ALD
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Figure 4. Effects of whey fermented liquid (WFL) on hepatic malondialdehyde (MDA) and triglyceride (TG) of normal and experimental mice. Data are expressed as means ± SD (n = 10). Values are significant compared with the following: control (*P < 0.05) and alcohol (#P < 0.05). ALD = alcoholic liver disease group; LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dose-treated mice.
mice. Notably, SOD, GSH, and GPx activities in the HWFL group were significant elevated (increased 28.9, 69.2, and 64.3%, respectively). The results show that WFL can enhance the antioxidant capability of the body and reduce lipid peroxidation. Effects of WFL on the Liver Histopathology of ALD Mice
Chronic daily feeding of alcohol caused liver injury to mice, as determined by serum markers for liver damage and changes in hepatic histopathology. As shown in
Figure 5. Effects of whey fermented liquid (WFL) on the hepatic superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) activity of normal and experimental mice. Data are expressed as means ± SD (n = 10). Values are significant compared with the following: control (*P < 0.05) and alcohol (#P < 0.05). ALD = alcoholic liver disease group; LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dosetreated mice. Journal of Dairy Science Vol. 97 No. 7, 2014
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Figure 6. Effects of whey fermented liquid (WFL) on liver histopathology of alcoholic liver disease (ALD) mice using hematoxylin and eosin staining: (A) liver tissue of control mice; (B) ALD group; (C) low-WFL (LWFL) dose-treated group; (D) medium-WFL (MWFL) dose-treated group; (E) high-WFL (HWFL) dose-treated group. Color version available in the online PDF.
Figure 6, alcoholic foamy degeneration was found in the liver tissue of mice in the ALD group. The hepatic cells were swollen and the cytoplasm was full of fat foam. These pathological changes decreased after treatment with WFL. In the HWFL group, the hepatic cells of the treated mice had returned to a normal structure. The hepatic cells of treated mice in the MWFL group also displayed a certain degree of recovery. However, compared with the ALD group, the hepatocytes of treated mice in the LWFL group had not changed. These findings show that WFL could protect liver tissue. This may be due to the fact that WFL can reduce the production of free radicals, thereby allowing the restoration of hepatic tissue after damage caused by free radicals related to alcohol ingestion (Figure 6). Effects of WFL on Levels of CYP2E1 of ALD Mice
Cytochrome P450 2E1 is a major type of cytochrome CYP450 oxidase whose expression level is affected by many factors, and it plays an important role in detoxification and metabolism. After consuming alcohol, the metabolic substrate of ethanol leads to increased levels of CYP2E1 expression. As the level of CYP2E1 expression and metabolic substrate increases, a large number of free radicals, such as ROS, are produced (Gonzalez, 2005; Cederbaum et al., 2009). This change causes oxygen stress, lipid peroxidation, and biological membrane injury. Furthermore, ethanol oxidation by CYP2E1 does not produce ATP. This leads to an increase in the oxygen consumption of liver cells, which Journal of Dairy Science Vol. 97 No. 7, 2014
affects the normal metabolism of liver cells. Moreover, a high level of CYP2E1 in Kupffer cells increases the sensitivity of the liver to endotoxin damage and promotes hepatocyte apoptosis during the liver immune response (Cao et al., 2005). Therefore, it is important to study CYP2E1 levels. The position of CYP2E1 in the hepatic cells is shown in Figure 7A, and the CYP2E1 levels are shown in Figure 7B. Compared with the control group, the level of CYP2E1 in the liver tissue of ALD mice was significantly elevated by 63.64%. This showed that the increase in CYP2E1 expression, which was induced by ethanol, may be one of the mechanisms by which alcoholic liver disease causes damage. Meanwhile, CYP2E1 levels in LWFL, MWFL, and HWFL mice were reduced by 16.67, 33.34, and 44.45%, respectively, compared with mice in the ALD group. These results show that the protection of WFL against alcoholic fatty liver may be related to its ability to inhibit CYP2E1 expression, which contributes to the strong antioxidant capability of WFL. CONCLUSIONS
Chronic alcohol consumption increases lipid peroxidation and decreases antioxidant status, which then damages liver cells. However, these negative effects were reversed by WFL. Whey fermented liquid can lower CYP2E1 expression levels in alcohol-fed mice. Whey fermented liquid also lowered ALT, AST, TG, and MDA levels, and increased SOD, GSH, and GPx
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Technology Department of Ningbo University (Ningbo, China; 2012B82017; 2011C11017), and the K. C. Wong Magna Fund at Ningbo University. REFERENCES
Figure 7. Effects of whey fermented liquid (WFL) on levels of cytochrome P450 2E1 (CYP2E1) of alcoholic liver disease (ALD) mice: (A) immunohistochemistry (frozen sections) of liver tissue in which CYP2E1 was labeled with fluorescein isothiocyanate [FITC; green (light-colored) spots]; (B) Western blotting of CYP2E1 in liver tissue of 5 groups of mice. *P < 0.05 compared with the control group; #P < 0.05 compared with the ALD group. LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dose-treated mice. Color version available in the online PDF.
activity in alcohol-fed mice, thereby decreasing liver damage. In conclusion, WFL can protect the liver from alcohol-induced damage. This research provides some hope for treatment of alcohol-induced alcoholic hepatitis, fatty liver disease, and cirrhosis. ACKNOWLEDGMENTS
This work was supported by the Natural Science Funding of China (Beijing, China; 41276121) and Zhejiang Province (China; Z3110211), the Science and
Brody, E. P. 2000. Biological activities of bovine glycomacropeptide. Br. J. Nutr. 84:S39–S46. Cao, Q., K. M. Mak, and C. S. Lieber. 2005. Cytochrome P4502E1 primes macrophages to increase TNF-alpha production in response to lipopolysaccharide. Am. J. Physiol. Gastrointest. Liver Physiol. 289:G95–G107. Cederbaum, A. I., Y. Lu, and D. Wu. 2009. Role of oxidative stress in alcohol-induced liver injury. Arch. Toxicol. 83:519–548. Cheng, C., and T.-M. Pan. 2011. Protective effect of Monascus-fermented red mold rice against alcoholic liver disease by attenuating oxidative stress and inflammatory response. J. Agric. Food Chem. 59:9950–9957. Cheng, Z., M. Yan, D. Chai, B. He, H. Cheng, and Q. Liu. 2006. Experimental study of Radix notoginseng in resisting alcoholic fatty liver. China J. Traditional Chinese Med. Pharmacy 21:614–616. Cutler, R. G. 1991. Antioxidant and aging. Am. J. Clin. Nutr. 53:373S–379S. Dean, R. T., S. Fu, R. Stocker, and M. J. Davies. 1997. Biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324:1–18. Dey, A., and A. I. Cederbaum. 2006. Alcohol and oxidative liver injury. Hepatology 43:S63–S74. Frid, A. H., M. Nilsson, J. J. Holst, and I. M. E. Björck. 2005. Effect of whey on blood glucose and insulin response to composite breakfast and lunch meals in type 2 diabetic subjects. Am. J. Clin. Nutr. 82:69–75. Gholam, P. M., L. Flancbaum, J. T. Machan, D. A. Charney, and D. P. Kotler. 2007. Nonalcoholic fatty liver disease in severely obese subjects. Am. J. Gastroenterol. 102:399–408. Gonzalez, F. J. 2005. Role of cytochromes P450 in chemical toxicity and oxidative stress: Studies with CYP2E1. Mutat. Res. 569:101– 110. Ha, E., and M. B. Zemel. 2003. Functional properties of whey, whey components, and essential amino acids: mechanisms underlying health benefits for active people (review) . J. Nutr. Biochem. 14:251–258. Harman, D. 1994. Free-radical theory of aging. Increasing the functional life span. Ann. N.Y. Acad. Sci. 717:1–15. Hartmann, R., and H. Meisel. 2007. Food-derived peptides with biological activity: From research to food applications. Curr. Opin. Biotechnol. 18:163–169. Hou, Z., P. Qin, and G. Ren. 2010. Effect of anthocyanin-rich extract from black rice (Oryza sativa L. japonica) on chronically alcoholinduced liver damage in rats. J. Agric. Food Chem. 58:3191–3196. Kolankaya, D., G. Selmanoğlu, K. Sorkun, and B. Salih. 2002. Protective effects of Turkish propolis on alcohol-induced serum lipid changes and liver injury in male rats. Food Chem. 78:213–217. Korhonen, H., and A. Pihlanto. 2006. Bioactive peptides: Production and functionality. Int. Dairy J. 16:945–960. Lian, L.-H., Y.-L. Wu, S.-Z. Song, Y. Wan, W.-X. Xie, X. Li, T. Bai, B.-Q. Ouyang, and J.-X. Nan. 2010. Gentiana manshurica Kitagawa reverses acute alcohol-induced liver steatosis through blocking sterol regulatory element-binding protein-1 maturation. J. Agric. Food Chem. 58:13013–13019. Lieber, C. S., and L. M. DeCarli. 1970. Hepatic microsomal ethanoloxidizing system: In vitro characteristics and adaptive properties in vivo. J. Biol. Chem. 245:2505–2512. Lin, M.-Y., and F.-J. Chang. 2000. Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708 and lactobacillus acidophilus ATCC 4356. Dig. Dis. Sci. 45:1617–1622. Marklund, S., and G. Marklund. 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47:469–474. Journal of Dairy Science Vol. 97 No. 7, 2014
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Niemelä, O., S. Parkkila, R. O. Juvonen, K. Viitala, H. V. Gelboin, and M. Pasanen. 2000. Cytochromes P450 2A6, 2E1, and 3A and production of protein-aldehyde adducts in the liver of patients with alcoholic and non-alcoholic liver diseases. J. Hepatol. 33:893–901. Park, H.-Y., Y. Park, Y. Lee, S. K. Noh, E.-G. Sung, and I. Choi. 2012. Effect of oral administration of water-soluble extract from citrus peel (Citrus unshiu) on suppressing alcohol-induced fatty liver in rats. Food Chem. 130:598–604. Raza, H., S. K. Prabu, M.-A. Robin, and N. G. Avadhani. 2004. Elevated mitochondrial cytochrome P450 2E1 and glutathione Stransferase A4-4 in streptozotocin-induced diabetic rats tissue-specific variations and roles in oxidative stress. Diabetes 53:185–194. Scarpellini, M., D. Mora, S. Colombo, and L. Franzetti. 2002. Development of genus/species-specific PCR analysis for identification of Carnobacterium strains. Curr. Microbiol. 45:24–29. Shimada, K., K. Fujikawa, K. Yahara, and T. Nakamura. 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40:945–948. Smirnoff, N., and Q. J. Cumbes. 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28:1057–1060.
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Takemura, G., T. Onodera, and M. Ashraf. 1992. Quantification of hydroxyl radical and its lack of relevance to myocardial injury during early reperfusion after graded ischemia in rat hearts. Circ. Res. 71:96–105. Tipoe, G. L., E. C. Liong, C. A. Casey, T. M. Donohue, P. K. Eagon, H. So, T. M. Leung, F. Fogt, and A. A. Nanji. 2008. A voluntary oral ethanol-feeding rat model associated with necroinflammatory liver injury. Alcohol. Clin. Exp. Res. 32:669–682. Vidali, M., M.-F. Tripodi, A. Ivaldi, R. Zampino, G. Occhino, L. Restivo, S. Sutti, A. Marrone, G. Ruggiero, E. Albano, and L. E. Adinolfi. 2008. Interplay between oxidative stress and hepatic steatosis in the progression of chronic hepatitis C. J. Hepatol. 48:399–406. Wang, G., M. Liu, X. Jiang, L. Ming, and Q. Xie. 2002. The value of gamma-glutamyltransferase in the diagnosis of chronic hepatitis B. Zhonghua Gan Zang Bing Za Zhi 10:120–122. WHO (World Health Organization). 2011. Global status report on alcohol and health. WHO, Geneva, Switzerland. Williams, R. 2006. Global challenges in liver disease. Hepatology 44:521–526.
Antialcoholic liver activity of whey fermented by Lactobacillus casei isolated from koumiss Z. W. Zhao,* D. D. Pan,*1 Z. Wu,† Y. Y. Sun,* Y. X. Guo,† and X. Q. Zeng*
*Food Science and technology Department of the Marine Science School, Ningbo University, Ningbo 315211, P. R. China †Food Science and Nutrition Department, Nanjing Normal University, Nanjing 210097, P. R. China
ABSTRACT
past few decades. Researchers have reported that ALD is caused by oxidative stress and inflammation, and is closely linked to reactive oxygen species (ROS), chemokines, and interactions between alcohol metabolism and hepatic cells (Dey and Cederbaum, 2006; Vidali et al., 2008). Alanine aminotransferase (ALT) is mainly found in the cell cytoplasm, whereas aspartate aminotransferase (AST) is mainly in the mitochondria. The ROS produced by the metabolism of ethanol damages the mitochondrial membrane and increases the permeability of cell membranes. The transaminase in liver cells then permeates the blood, causing an increase in ALT and AST activity in the blood (Wang et al., 2002). Whey is a general term that typically denotes the translucent liquid part of milk that remains following the process (coagulation and curd removal) of cheese manufacturing. Whey also is 1 of 2 major protein groups of bovine milk, accounting for 20% of the milk, whereas casein accounts for the remainder. In the current study, we found that whey was rich in branched-chain amino acids such as leucine, isoleucine, and valerian ammonia acid (Ha and Zemel, 2003). These amino acids have positive effects, including suppressing appetite, which leads to weight loss. Leucine, for example, can stimulate the hypothalamus to produce mammalian target of rapamycin signals, which reduce food intake (Brody, 2000). In addition, the proteins in whey also exhibit a wide variety of biological activities, such as antioxidation and the regulation of blood lipids, and are used to treat nonalcoholic fatty liver disease (Frid et al., 2005; Gholam et al., 2007). Furthermore, whey protein contains bioactive amino acid sequences that may be crucial to its various effects and may be released by using a specific protease to hydrolyze whey protein. A variety of physiological functions related to active polypeptides have been found in the degradation products of whey protein (Korhonen and Pihlanto, 2006; Hartmann and Meisel, 2007). These findings indicate that it is important to discover more effective and comprehensive ways to use whey. Whey fermented liquid (WFL), prepared by inoculating whey with Lactobacillus and allowing it to be incubated, has been found to be rich in amino acids, polypeptides, and proteins. These properties
Whey fermented liquid (WFL) was studied for its hepatoprotective effects by using chronic alcoholinduced mice. Whey fermented liquid, prepared by inoculating whey with 4% (vol/vol) Lactobacillus casei and then incubating at 41°C for 8 h, was used to orally treat alcohol-induced mice at 3 dosages for 5 wk. Ethanol consumption significantly reduced the activity of superoxide dismutase and glutathione peroxidase, while lowering glutathione content and increasing levels of aspartate aminotransferase, alanine aminotransferase, total triglyceride, malondialdehyde, and cytochrome P450 2E1. Treatment with WFL significantly attenuated the increased levels of alanine aminotransferase, aspartate aminotransferase, triglyceride, and cytochrome P450 2E1, while decreasing superoxide dismutase, glutathione peroxidase, malondialdehyde, and glutathione levels. Pathological changes in the livers of mice who had ingested alcohol were improved by the administration of WFL. These results suggest that WFL may exert a protective effect against alcoholic liver disease by increasing antioxidant activity, which supports the use of WFL as an antialcoholic liver disease treatment. Key words: Lactobacillus casei, alcoholic liver, fermented whey, antioxidation INTRODUCTION
Alcohol consumption is increasing as the standard of living of many people around the world improves. However, long-term excessive intake of alcohol causes alcoholic hepatitis, fatty liver disease, and cirrhosis (Williams, 2006). As the World Health Organization reported in 2011 (WHO, 2011), the abuse of alcohol is a public health problem that causes disease, toxicity, injury, and premature death. Alcoholic liver disease (ALD), whose pathogenesis is not yet fully understood, has been of growing interest to researchers over the Received January 16, 2014. Accepted March 9, 2014. 1 Corresponding author: [email protected]
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mean that WFL exhibits many physiological functions. Although some of the biological activities of WFL have been discussed in the published literature, its role in relation to alcoholic liver disease has not been analyzed. In this study, 4 strains of Lactobacillus isolated from koumiss, a dairy product similar to kefir, were identified using 16S rRNA analysis. Whey fermented liquid was prepared using the screened Lactobacillus that exhibited the strongest antioxidant activity. The effect of the whey fermented using Lactobacillus casei (ATCC 15008) on alcohol-induced mice was then analyzed in order to provide some hope for treatment of alcoholinduced alcoholic hepatitis, fatty liver disease, and cirrhosis. MATERIALS AND METHODS Chemicals and Reagents
Cow milk was obtained from the Ningbo Dairy Group (Zhengjiang, China). Methanol, acetonitrile, and monoclonal anti-β-actin antibody (chromatographic grade) were purchased from Sigma Chemical Co. (St. Louis, MO). Trifluoroacetic acid was obtained from TCI Chemical Co. (Tokyo, Japan). Assay kits for AST, ALT, total triglyceride (TG), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, P.R. China). The anti-cytochrome P450 2E1 (CYP2E1) antibody (ab28146) was from Abcam (Britain). The horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) and Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) were obtained from Beyotime (Jiangsu, China). The other chemicals were all of analytical grade from commercial sources. Strains and Culture Media
Four strains of Lactobacillus isolated from koumiss were selected. The strains were obtained from the Key Laboratory of Marine Biotechnology Application, School of Marine Sciences, Ningbo University (Ningbo, P. R. China). The strains were cultured at 37°C in de Man, Rogosa, and Sharpe (MRS) medium for 24 h before being used in the next experiment. Identification of Lactobacillus
Identification of different Lactobacillus strains was conducted using 16S rRNA analysis (Scarpellini et al., 2002). Genomic DNA of Lactobacillus was isolated using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R. China) according to the manufacturer’s Journal of Dairy Science Vol. 97 No. 7, 2014
instructions. To detect the amplification of the 16S rRNA sequence, PCR was performed using the forward (5′-AACTGAAGAGTTTGATCCTGGCTC-3′) and the reverse (5′-TACGGTTACCTTGTTACGACTT-3′) primers. The 50-μL PCR reaction mixture consisted of 4 μL of deoxyribonucleotide triphosphate (dNTP; each 2.5 mM), 5 μL of 10× PCR buffer, 1 μL of forward primer (50 pM), 1 μL of reverse primer (50 pM), 4 μL pf MgCl2 (25 mM), 2 μL of DNA template (100 ng/ μL), 0.5 μL of Taq polymerase (5 U/μL), and 32.5 μL of double-distilled H2O. The cycling program was as follows: 94°C for 5 min, 94°C for 1 min, 56°C for 45 s, 72°C for 2 min for the next 30 cycles, and then 72°C for 10 min and 4°C for 30 min. Then, the purified PCR products were cloned into the T vector and the DNA sequence was analyzed by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, P. R. China). The sequencings were performed using DNAMAN software (Lynnon Corp., Pointe-Claire, QC, Canada). A homology search to reference strains registered in GenBank was performed using the NCBI BLAST algorithm (http:// blast.ncbi.nlm.nih.gov/Blast.cgi). Preparation of Supernatant and Intracellular Cell-Free Extracts
After the strains were cultured at 37°C in MRS broth for 24 h, the supernatant was harvested by centrifugation at 3,000 × g for 20 min at 4°C. The antioxidant activity was then analyzed. For the preparation of intracellular cell-free extracts, cell pellets were washed twice with PBS (0.85% NaCl, 2.68 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4; pH 7.0) and resuspended in deionized water to adjust the total cell number to 109 cfu/mL before sonication. Ultrasonic disruption was performed for five 1-min intervals (60 cycles at 400 W) in an ice bath. The resulting supernatant was collected to determine the level of antioxidant activity after the cell debris was removed by centrifugation at 6,500 × g for 20 min at 4°C. Scavenging of α,α-Diphenyl-β-Picrylhydrazyl Radical
The α,α-diphenyl-β-picrylhydrazyl (DPPH) clearance rate of the sample was analyzed by using the DPPH method (Shimada et al., 1992; Lin and Chang, 2000). Two milliliters of sample and 2.0 mL of freshly prepared DPPH-ethanol solution (0.2 mmol/L) were mixed and allowed to react in the dark for 30 min. The scavenged DPPH· was monitored by measuring the decrease in absorbance at 517 nm (Ai). Meanwhile, 2.0 mL of ethanol was used to take the place of DPPH· in the blank group (Aj). Control samples contained deionized water (A0). The scavenging ability was defined as follows:
ANTIALCOHOLIC LIVER ACTIVITY OF WHEY FERMENTED BY LACTOBACILLUS CASEI
DPPH scavenging ability = [1 − (Ai − Aj)/A0] × 100%. Superoxide Anion Scavenging Assay
The superoxide anion scavenging assay was analyzed using a modification of the method used by Marklund and Marklund (1974). First, 4.5 mL of Tris-HCl buffer (0.1 mol/L; pH 8.2) was mixed with 1 mL of EDTA (1.0 mL/L), 1.0 mL of sample, and 2.4 mL of distilled water. The mixture was left to react in a water bath (25°C) for 10 min. Then, the incubated mixture was mixed with 2.0 mL of pyrogallol (9.0 mmol/L) and allowed to react for 60 min. Finally, the mixture was mixed with 100 µL of HCl (12.0 mol/L) to determine the absorbance at 325 nm (As). Meanwhile, 1.0 mL of distilled water was used to take the place of the sample in the control group (Ac). The scavenging ability was defined as (Ac − As)/Ac × 100%. Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activity of the sample was determined by using the method of Smirnoff and Cumbes (1989). The mixture, which consisted of 6 mM FeSO4, 1 mL of sample, and 6 mM H2O2, was incubated at 25°C and allowed to react for 10 min (Aj). The reaction mixture was then mixed with 9 mM salicylic acid and 4 mL of distilled water and left to react for 30 min. The absorbance was determined at 510 nm (Ai). Meanwhile, 1.0 mL of distilled water was used to take the place of the sample in the control group (A0). The scavenging activity was defined as [1 − (Ai − Aj)/ A0] × 100%. Preparation of WFL
Cow milk was centrifuged for 20 min at 2,000 × g at 20°C to remove fat. The centrifuged milk was acidified by hydrochloric acid to pH 4.6 and filtrated using a ceramic membrane and stored at 4°C in a refrigerator. Then, the Lactobacillus casei (H4), which was cultivated in MRS liquid medium for 2 to 3 generations at 37°C, was used as starter culture to produce WFL. The preparation method involved adding 4% (vol/vol) Lactobacillus casei (H4) inoculum to whey liquid and culturing for 16 h at 41°C. The fermentation broth was collected to determine the antioxidant activity and stored at 4°C before the next experiment. Biological Experiment
Fifty mice (20 ± 2 g; male) were purchased from the Animal Division of Zhejiang University (Zhejiang,
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China), and acclimatized for 2 d in the animal experimental research laboratory of Ningbo University. The mice had free access to rodent chow and mineral water, and maintained at 22 ± 2°C with 55 ± 5% relative humidity and a 12 h:12 h light-dark cycle (Lian et al., 2010). The mice were randomly divided into 5 groups, with 10 mice in each group: (A) control group: 6.25 mL of distilled water/kg per day, (B) model alcohol-fed mice: 6.25 mL of ethanol/kg per day, (C) model mice treated with a low-WFL (LWFL) dose: 6.25 mL of ethanol/kg and 10 mL of WFL (20 times diluted)/kg per day, (D) model mice treated with a medium WFL (MWFL) dose: 6.25 mL of ethanol/kg and 10 mL of WFL (10 times diluted)/kg per day, and (E) model mice treated with a high-WFL (HWFL) dose: 6.25 mL of ethanol/kg and 10 mL of WFL/kg per day. In the model groups, mice were intragastrically administered with ethanol (20% in distilled water) at 0900 h. The various WFL doses were administered orally via a stomach tube every day around 1400 h (Tipoe et al., 2008; Hou et al., 2010; Cheng and Pan, 2011). All experiments lasted 5 wk. Changes in BW were monitored weekly. Analysis of Serum Enzymes
At the end of the animal experiment, blood samples were collected from the eyeballs of the mice. The serum was separated from blood samples for analysis after centrifugation at 1,000 × g for 20 min at 4°C (Kolankaya et al., 2002). Then serum enzymes, such as ALT and AST, were measured using the ALT and AST activity assay kits. Liver Function and Oxidative Stress Parameter
Liver homogenate (10%, vol/vol) was prepared according to a method described previously (Lieber and DeCarli, 1970; Raza et al., 2004). The liver (1 g), which was cut into small pieces, was fully ground using a tissue blender with 9 volumes of precooling medium (pH 7.4). Supernatant was collected from the liver homogenate by centrifugation at 1,000 × g for 10 min at 4°C. The medium comprised 0.1 mol/L Tris-hydrochloric acid, 0.25 M sucrose, 0.1 mM EDTA, and 0.05 mmol of phenylmethylsulfonyl fluoride/L. Finally, malonaldehyde, liver TG, and the enzyme activity of the liver tissue were analyzed using the corresponding assay kits. Liver Histopathological Studies
Liver tissues, which were immediately taken from the dead mice, were put into freshly prepared formalin solution (10%; 4°C). Frozen sections of liver (5–6 μm) were Journal of Dairy Science Vol. 97 No. 7, 2014
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cut using a microtome, and then washed with ethanol and water. The frozen section was counterstained with hematoxylin for 45 s. An image was then captured under a light microscope at 200× magnification (Park et al., 2012). Levels of CYP2E1
Levels of CYP2E1 in 10% liver homogenate were determined by Western blotting (WB). Liver tissue was homogenized in 9 volumes of cold buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, and leupeptin at pH 7.4) at 4°C. Then, the homogenates were centrifuged at 1,000 × g for 15 min at 4°C, and the supernatant was stored for analysis. Meanwhile, the protein content of the supernatant was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime). The CYP2E1 protein in the supernatant had been isolated in the gel by electrophoresis and then transferred to the polyvinylidene difluoride membrane. Finally, the polyvinylidene difluoride membrane was blocked with 5% Tris-buffered saline with Tween 20 (TBST) for 30 min at 25°C before incubation with ab28146 diluted 1/1,000 for 12 h at 4°C. Horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) was used as the secondary antibody. The position of CYP2E1 in the hepatic cells was measured using immunohistochemistry (frozen sections) according to the manufacturer’s instructions. Cells were fixed in acetone and blocked with 1% BSA for 5 min at 25°C before incubation with ab28146 diluted 1/300 for 30 min at 25°C. The Alexa Fluor 488-labeled goat antirabbit IgG (H+L) was used as the secondary antibody. Statistical Analysis
Data were expressed as mean values ± standard deviation. All data were analyzed using SPSS software (version 16.0; SPSS Inc., Chicago, IL). The differences between groups were compared using one-way ANOVA, followed by Dunnett’s test and P < 0.05 was considered to be significant. RESULTS AND DISCUSSION 16S rRNA Identification of Lactobacillus
The taxonomic identification of the 4 strains is shown in Figure 1. The homology tree was constructed using DNAMAN software. The 16S rRNA gene sequence similarities of strains H1 and H4 (H2 and H3) were found to be 100% (Figure 1). The analysis of the 16S rRNA gene sequence showed that the 4 strains Journal of Dairy Science Vol. 97 No. 7, 2014
Figure 1. Homology tree of 16S rRNA sequences in 4 different Lactobacillus strains.
clearly belong to Lactobacillus casei (Figure 1, strains H1 and H4) and Lactobacillus fermentum (Figure 1, strains H2 and H3). Free Radical Scavenging Effect of Lactobacillus and WFL
According to the free radical metabolism theory proposed by Harman (1994), free radicals, which exhibit high activity levels, easily react with other molecules and free radicals. Such interactions can initiate a chain reaction that produces new free radicals. A large number of free radicals, produced by the body during such metabolic processes, can cause tissue injury by attacking biological macromolecules (Cutler, 1991; Takemura et al., 1992). The free radical rate of metabolism increases as alcohol intake levels increase. Therefore, the ability of Lactobacillus and WFL to reduce free radical toxicity was analyzed directly by monitoring DPPH and hydroxyl radical scavenging activity, as well as the superoxide anion scavenging assay. To screen out Lactobacillus antioxidant activity, 4 Lactobacillus strains were cultivated in MRS broth and then antioxidant activity was measured in the supernatants and intracellular cell-free extracts. The screening results revealed that the antioxidant activity of Lactobacillus strains H1 and H4 were higher than that of the other strains, and both H1 and H4 were Lactobacillus casei (Figure 1). The scavenging effects of the 4 strains on DPPH, superoxide anions, and hydroxyl radicals are shown in Table 1. Because Lactobacillus strain H4 exhibited greater antioxidant activity, H4 was chosen from the 4 strains to use to ferment the whey. As shown in Table 1, WFL inoculated with Lactobacillus casei H4 showed the strong antioxidant capability.
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Table 1. Free radical scavenging effect of Lactobacillus and whey fermented liquid (WFL; mean ± SD; n = 5)1 Scavenging of DPPH (%) Item2 H1 H2 H3 H4 WFL
A 50.84 45.97 45.30 54.48 62.35
± ± ± ± ±
B 0.04 0.05 0.03 0.05 0.06
31.21 26.52 27.47 33.16 34.02
± ± ± ± ±
0.05 0.04 0.05 0.02 0.03
50.49 45.59 33.87 50.75 65.41
Scavenging of O2−· (%)
Scavenging of OH· (%)
A
A
± ± ± ± ±
B 0.07 0.06 0.02 0.06 0.04
24.85 13.57 23.56 28.08 30.75
± ± ± ± ±
0.03 0.03 0.03 0.01 0.03
51.37 42.35 43.90 52.44 61.73
± ± ± ± ±
B 0.04 0.04 0.05 0.03 0.07
27.63 13.53 11.58 29.36 30.43
± ± ± ± ±
0.06 0.04 0.08 0.05 0.03
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DPPH = α,α-diphenyl-β-picrylhydrazyl; A = supernatant; B = intracellular cell-free extracts. H1, H2, H3, and H4 = Lactobacillus strains.
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Effect of WFL on Body and Organ Weight
The BW of the mice was monitored every week. The results indicated that, after 5 wk, the BW of the alcohol-diet group was lighter than that of the control mice (Figure 2A). Also, the weights of the alcohol-diet group mice fed different doses of WFL were significantly different from those not fed WFL. The difference between the liver weight-to-BW ratio in the ALD and control groups was statistically significant (Figure 2B). The liver weight-to-BW ratio in the control group was lower than that found in the ALD group. Mice with ALD fed the HWFL dose gained in terms of their liver weight-to-BW ratio at a normal rate. Results of Serum Biochemical Analysis
The protective effect of WFL on the serum levels of ALT and AST in ALD mice is illustrated in Figure 3A and 3B. In the ALD group, the serum levels of ALT and AST were 40.7 ± 3.3 and 98.3 ± 1.7 U/L, respectively. Compared with the ALT and AST levels in the control group, no significant change occurred in the serum levels of ALT, whereas after 5 wk, the serum levels of AST significantly increased. Chronic daily feeding of WFL significantly reduced AST levels in serum, and ALD mice fed the HWFL dose showed a significant reduction in the alcohol-induced increase in AST levels (from 98.3 ± 1.7 to 72.6 ± 2.5 U/L). In addition, a significant difference existed in ALT levels between the MWFL, HWFL, and ALD mice. It could be suggested that WFL had a protective effect on the liver by inhibiting the increase in aminotransferase activity in the serum, protecting the cell and mitochondrial membranes from the damage caused by alcohol. Effect of WFL on MDA and TG Content in Liver Tissue
The effect of chronic alcohol ingestion on MDA content and the total TG in ALD mice are presented in
panels A and B of Figure 4. The content of hepatic TG and MDA in the ALD mice was significantly increased and matched the levels in the control mice, which suggested that the lipid metabolism of ALD mice was disorganized. Long-term consumption of alcohol increases TG levels in liver cells by greatly increasing TG synthetase activity, but alcohol metabolism can also influence the synthesis of apolipoprotein and increase the difficulty of transporting TG into the liver. Moreover, the significant increase in MDA content leads to the formation of Mallory corpuscles via cytoskeleton protein crosslinking, which leads to fibrosis or cirrhosis by activating Kupffer cells, which in turn promote the synthesis of collagen fibers (Niemelä et al., 2000). However, the hepatic TG content in all of the treatment groups was significantly reduced compared with that of the ALD mice. A significant reduction in hepatic MDA content was detected in the MWFL and HWFL mice. These findings indicated that WFL can help ALD mice maintain a balance between fat synthesis and fat metabolism. Effect of WFL on the GSH and Enzyme Activity in Liver Tissue
Superoxide dismutase can protect organisms, prevent aging, and treat diseases by catalyzing superoxide anions into H2O2 and O2 (Dean et al., 1997). Glutathione plays a key role in the process of protecting the liver from the ravages of alcoholic liver disease. Glutathione not only scavenges free radicals, but also removes drugs, chemicals, and reactive molecules from cells. Furthermore, GPx catalyzes GSH to become oxidized GSH by consuming H2O2 and LOOH. Therefore, the generation of hydroxyl radicals and lipid peroxidation is prevented, and the structure and function of cell membranes is protected. Ethanol becomes acetaldehyde via the catalytic action of alcohol dehydrogenase. Acetaldehyde can combine with glutathione, causing a decrease in GSH levels in the cytoplasm of liver cells, thereby reducing the ability of liver cells to eliminate free radicals (Cheng et al., 2006). Moreover, ethanol also interferes with Journal of Dairy Science Vol. 97 No. 7, 2014
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Figure 2. Effects of whey fermented liquid (WFL) on the liver and body. (A) Body weight; (B) liver-to-BW ratio. Results are expressed as the mean ± SD (n = 10). *P < 0.05 compared with the control group; #P < 0.05 compared with the alcoholic liver disease (ALD) group. LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dosetreated mice; HWFL = high-WFL dose-treated mice.
the function of carrier proteins on the mitochondrial membrane, reducing GSH levels in the mitochondria and causing the mitochondria to swell. In our study, the activity of SOD, GSH, and GPx were measured to analyze the antioxidant ability of the body. Superoxide dismutase, GSH, and GPx activity in the liver tissue is shown in panels A, B, and C, respectively, of Figure 5. Hepatic SOD, GSH, and GPx activity in Journal of Dairy Science Vol. 97 No. 7, 2014
Figure 3. Effects of whey fermented liquid (WFL) on the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity of normal and experimental mice. Data are expressed as means ± SD (n = 10). Values are significant compared with the following: control (*P < 0.05) and alcohol (#P < 0.05). ALD = alcoholic liver disease group; LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dose-treated mice.
the ALD mice were significantly reduced compared with those of the control mice. However, SOD, GSH, and GPx activity in all the treatment groups were significantly elevated compared with those of the ALD
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Figure 4. Effects of whey fermented liquid (WFL) on hepatic malondialdehyde (MDA) and triglyceride (TG) of normal and experimental mice. Data are expressed as means ± SD (n = 10). Values are significant compared with the following: control (*P < 0.05) and alcohol (#P < 0.05). ALD = alcoholic liver disease group; LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dose-treated mice.
mice. Notably, SOD, GSH, and GPx activities in the HWFL group were significant elevated (increased 28.9, 69.2, and 64.3%, respectively). The results show that WFL can enhance the antioxidant capability of the body and reduce lipid peroxidation. Effects of WFL on the Liver Histopathology of ALD Mice
Chronic daily feeding of alcohol caused liver injury to mice, as determined by serum markers for liver damage and changes in hepatic histopathology. As shown in
Figure 5. Effects of whey fermented liquid (WFL) on the hepatic superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) activity of normal and experimental mice. Data are expressed as means ± SD (n = 10). Values are significant compared with the following: control (*P < 0.05) and alcohol (#P < 0.05). ALD = alcoholic liver disease group; LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dosetreated mice. Journal of Dairy Science Vol. 97 No. 7, 2014
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Figure 6. Effects of whey fermented liquid (WFL) on liver histopathology of alcoholic liver disease (ALD) mice using hematoxylin and eosin staining: (A) liver tissue of control mice; (B) ALD group; (C) low-WFL (LWFL) dose-treated group; (D) medium-WFL (MWFL) dose-treated group; (E) high-WFL (HWFL) dose-treated group. Color version available in the online PDF.
Figure 6, alcoholic foamy degeneration was found in the liver tissue of mice in the ALD group. The hepatic cells were swollen and the cytoplasm was full of fat foam. These pathological changes decreased after treatment with WFL. In the HWFL group, the hepatic cells of the treated mice had returned to a normal structure. The hepatic cells of treated mice in the MWFL group also displayed a certain degree of recovery. However, compared with the ALD group, the hepatocytes of treated mice in the LWFL group had not changed. These findings show that WFL could protect liver tissue. This may be due to the fact that WFL can reduce the production of free radicals, thereby allowing the restoration of hepatic tissue after damage caused by free radicals related to alcohol ingestion (Figure 6). Effects of WFL on Levels of CYP2E1 of ALD Mice
Cytochrome P450 2E1 is a major type of cytochrome CYP450 oxidase whose expression level is affected by many factors, and it plays an important role in detoxification and metabolism. After consuming alcohol, the metabolic substrate of ethanol leads to increased levels of CYP2E1 expression. As the level of CYP2E1 expression and metabolic substrate increases, a large number of free radicals, such as ROS, are produced (Gonzalez, 2005; Cederbaum et al., 2009). This change causes oxygen stress, lipid peroxidation, and biological membrane injury. Furthermore, ethanol oxidation by CYP2E1 does not produce ATP. This leads to an increase in the oxygen consumption of liver cells, which Journal of Dairy Science Vol. 97 No. 7, 2014
affects the normal metabolism of liver cells. Moreover, a high level of CYP2E1 in Kupffer cells increases the sensitivity of the liver to endotoxin damage and promotes hepatocyte apoptosis during the liver immune response (Cao et al., 2005). Therefore, it is important to study CYP2E1 levels. The position of CYP2E1 in the hepatic cells is shown in Figure 7A, and the CYP2E1 levels are shown in Figure 7B. Compared with the control group, the level of CYP2E1 in the liver tissue of ALD mice was significantly elevated by 63.64%. This showed that the increase in CYP2E1 expression, which was induced by ethanol, may be one of the mechanisms by which alcoholic liver disease causes damage. Meanwhile, CYP2E1 levels in LWFL, MWFL, and HWFL mice were reduced by 16.67, 33.34, and 44.45%, respectively, compared with mice in the ALD group. These results show that the protection of WFL against alcoholic fatty liver may be related to its ability to inhibit CYP2E1 expression, which contributes to the strong antioxidant capability of WFL. CONCLUSIONS
Chronic alcohol consumption increases lipid peroxidation and decreases antioxidant status, which then damages liver cells. However, these negative effects were reversed by WFL. Whey fermented liquid can lower CYP2E1 expression levels in alcohol-fed mice. Whey fermented liquid also lowered ALT, AST, TG, and MDA levels, and increased SOD, GSH, and GPx
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Technology Department of Ningbo University (Ningbo, China; 2012B82017; 2011C11017), and the K. C. Wong Magna Fund at Ningbo University. REFERENCES
Figure 7. Effects of whey fermented liquid (WFL) on levels of cytochrome P450 2E1 (CYP2E1) of alcoholic liver disease (ALD) mice: (A) immunohistochemistry (frozen sections) of liver tissue in which CYP2E1 was labeled with fluorescein isothiocyanate [FITC; green (light-colored) spots]; (B) Western blotting of CYP2E1 in liver tissue of 5 groups of mice. *P < 0.05 compared with the control group; #P < 0.05 compared with the ALD group. LWFL = low-WFL dose-treated mice; MWFL = medium-WFL dose-treated mice; HWFL = high-WFL dose-treated mice. Color version available in the online PDF.
activity in alcohol-fed mice, thereby decreasing liver damage. In conclusion, WFL can protect the liver from alcohol-induced damage. This research provides some hope for treatment of alcohol-induced alcoholic hepatitis, fatty liver disease, and cirrhosis. ACKNOWLEDGMENTS
This work was supported by the Natural Science Funding of China (Beijing, China; 41276121) and Zhejiang Province (China; Z3110211), the Science and
Brody, E. P. 2000. Biological activities of bovine glycomacropeptide. Br. J. Nutr. 84:S39–S46. Cao, Q., K. M. Mak, and C. S. Lieber. 2005. Cytochrome P4502E1 primes macrophages to increase TNF-alpha production in response to lipopolysaccharide. Am. J. Physiol. Gastrointest. Liver Physiol. 289:G95–G107. Cederbaum, A. I., Y. Lu, and D. Wu. 2009. Role of oxidative stress in alcohol-induced liver injury. Arch. Toxicol. 83:519–548. Cheng, C., and T.-M. Pan. 2011. Protective effect of Monascus-fermented red mold rice against alcoholic liver disease by attenuating oxidative stress and inflammatory response. J. Agric. Food Chem. 59:9950–9957. Cheng, Z., M. Yan, D. Chai, B. He, H. Cheng, and Q. Liu. 2006. Experimental study of Radix notoginseng in resisting alcoholic fatty liver. China J. Traditional Chinese Med. Pharmacy 21:614–616. Cutler, R. G. 1991. Antioxidant and aging. Am. J. Clin. Nutr. 53:373S–379S. Dean, R. T., S. Fu, R. Stocker, and M. J. Davies. 1997. Biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324:1–18. Dey, A., and A. I. Cederbaum. 2006. Alcohol and oxidative liver injury. Hepatology 43:S63–S74. Frid, A. H., M. Nilsson, J. J. Holst, and I. M. E. Björck. 2005. Effect of whey on blood glucose and insulin response to composite breakfast and lunch meals in type 2 diabetic subjects. Am. J. Clin. Nutr. 82:69–75. Gholam, P. M., L. Flancbaum, J. T. Machan, D. A. Charney, and D. P. Kotler. 2007. Nonalcoholic fatty liver disease in severely obese subjects. Am. J. Gastroenterol. 102:399–408. Gonzalez, F. J. 2005. Role of cytochromes P450 in chemical toxicity and oxidative stress: Studies with CYP2E1. Mutat. Res. 569:101– 110. Ha, E., and M. B. Zemel. 2003. Functional properties of whey, whey components, and essential amino acids: mechanisms underlying health benefits for active people (review) . J. Nutr. Biochem. 14:251–258. Harman, D. 1994. Free-radical theory of aging. Increasing the functional life span. Ann. N.Y. Acad. Sci. 717:1–15. Hartmann, R., and H. Meisel. 2007. Food-derived peptides with biological activity: From research to food applications. Curr. Opin. Biotechnol. 18:163–169. Hou, Z., P. Qin, and G. Ren. 2010. Effect of anthocyanin-rich extract from black rice (Oryza sativa L. japonica) on chronically alcoholinduced liver damage in rats. J. Agric. Food Chem. 58:3191–3196. Kolankaya, D., G. Selmanoğlu, K. Sorkun, and B. Salih. 2002. Protective effects of Turkish propolis on alcohol-induced serum lipid changes and liver injury in male rats. Food Chem. 78:213–217. Korhonen, H., and A. Pihlanto. 2006. Bioactive peptides: Production and functionality. Int. Dairy J. 16:945–960. Lian, L.-H., Y.-L. Wu, S.-Z. Song, Y. Wan, W.-X. Xie, X. Li, T. Bai, B.-Q. Ouyang, and J.-X. Nan. 2010. Gentiana manshurica Kitagawa reverses acute alcohol-induced liver steatosis through blocking sterol regulatory element-binding protein-1 maturation. J. Agric. Food Chem. 58:13013–13019. Lieber, C. S., and L. M. DeCarli. 1970. Hepatic microsomal ethanoloxidizing system: In vitro characteristics and adaptive properties in vivo. J. Biol. Chem. 245:2505–2512. Lin, M.-Y., and F.-J. Chang. 2000. Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708 and lactobacillus acidophilus ATCC 4356. Dig. Dis. Sci. 45:1617–1622. Marklund, S., and G. Marklund. 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47:469–474. Journal of Dairy Science Vol. 97 No. 7, 2014
10
Zhao et al.
Niemelä, O., S. Parkkila, R. O. Juvonen, K. Viitala, H. V. Gelboin, and M. Pasanen. 2000. Cytochromes P450 2A6, 2E1, and 3A and production of protein-aldehyde adducts in the liver of patients with alcoholic and non-alcoholic liver diseases. J. Hepatol. 33:893–901. Park, H.-Y., Y. Park, Y. Lee, S. K. Noh, E.-G. Sung, and I. Choi. 2012. Effect of oral administration of water-soluble extract from citrus peel (Citrus unshiu) on suppressing alcohol-induced fatty liver in rats. Food Chem. 130:598–604. Raza, H., S. K. Prabu, M.-A. Robin, and N. G. Avadhani. 2004. Elevated mitochondrial cytochrome P450 2E1 and glutathione Stransferase A4-4 in streptozotocin-induced diabetic rats tissue-specific variations and roles in oxidative stress. Diabetes 53:185–194. Scarpellini, M., D. Mora, S. Colombo, and L. Franzetti. 2002. Development of genus/species-specific PCR analysis for identification of Carnobacterium strains. Curr. Microbiol. 45:24–29. Shimada, K., K. Fujikawa, K. Yahara, and T. Nakamura. 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem. 40:945–948. Smirnoff, N., and Q. J. Cumbes. 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28:1057–1060.
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Takemura, G., T. Onodera, and M. Ashraf. 1992. Quantification of hydroxyl radical and its lack of relevance to myocardial injury during early reperfusion after graded ischemia in rat hearts. Circ. Res. 71:96–105. Tipoe, G. L., E. C. Liong, C. A. Casey, T. M. Donohue, P. K. Eagon, H. So, T. M. Leung, F. Fogt, and A. A. Nanji. 2008. A voluntary oral ethanol-feeding rat model associated with necroinflammatory liver injury. Alcohol. Clin. Exp. Res. 32:669–682. Vidali, M., M.-F. Tripodi, A. Ivaldi, R. Zampino, G. Occhino, L. Restivo, S. Sutti, A. Marrone, G. Ruggiero, E. Albano, and L. E. Adinolfi. 2008. Interplay between oxidative stress and hepatic steatosis in the progression of chronic hepatitis C. J. Hepatol. 48:399–406. Wang, G., M. Liu, X. Jiang, L. Ming, and Q. Xie. 2002. The value of gamma-glutamyltransferase in the diagnosis of chronic hepatitis B. Zhonghua Gan Zang Bing Za Zhi 10:120–122. WHO (World Health Organization). 2011. Global status report on alcohol and health. WHO, Geneva, Switzerland. Williams, R. 2006. Global challenges in liver disease. Hepatology 44:521–526.
