Digestive Diseases and Sciences

, Volume 52, Issue 8, pp 2006–2014

Protective Effect of Soy Isoflavones and Activity Levels of Plasma Paraoxonase and Arylesterase in the Experimental Nonalcoholic Steatohepatitis Model

Authors

    • Department of Biochemistry and Clinical BiochemistryCollege of Medicine, Firat (Euphrates) University
  • Ibrahim Halil Bahcecioglu
    • Department of GastroenterologyCollege of Medicine, First (Euphrates) University
  • Kazım Sahin
    • Department of Animal NutritionCollege of Veterinary Medicine, Firat (Euphrates) University
  • Sevda Duzgun
    • Department of Biochemistry and Clinical BiochemistryCollege of Medicine, Firat (Euphrates) University
  • Suleyman Koca
    • Department of Internal MedicineCollege of Medicine, Firat (Euphrates) University
  • Funda Gulcu
    • Department of Biochemistry and Clinical BiochemistryCollege of Medicine, Firat (Euphrates) University
  • Ibrahim Hanifi Ozercan
    • Department of PathologyCollege of Medicine, Firat (Euphrates) University
Original Paper

DOI: 10.1007/s10620-006-9251-9

Cite this article as:
Ustundag, B., Bahcecioglu, I.H., Sahin, K. et al. Dig Dis Sci (2007) 52: 2006. doi:10.1007/s10620-006-9251-9
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Abstract

Nonalcoholic steatohepatitis (NASH) is characterized by diffuse fatty infiltration in the liver and ballooning degeneration and inflammation in hepatocytes. We aimed to study the protective effect of soy isoflavones on experimental NASH and their effects on plasma paraoxanese and arylesterase levels in rats. Twenty-eight male rats were divided into four groups: Group 1 (n=7) received an isocaloric normal diet for 8 weeks, Group 2 (n=7) was fed an isocaloric basal diet plus oral soy isoflavone for 8 weeks (100 mg/kg in diet), Group 3 (n=7) received a special diet that was methionine and choline deficient (MCD) and rich in fat for 8 weeks, and Group 4 (n=7) was fed a special diet that was MCD and rich in fat plus oral soy isoflavone for 8 weeks (100 mg/kg in diet). Blood samples were collected to measure plasma malondialdehyde (MDA), paraoxanese, and arylesterase and biochemical parameters. Tissue samples were duly taken for histopathological examination and measurement of tissue MDA levels. Plasma MDA levels were higher in Group 3 than in Groups 1, 2, and 4 (P <0.01, P <0.05, and P <0.05 respectively). Liver tissue MDA levels were also significantly higher in Group 3 compared to Groups 1, 2, and 4 (P <0.001, P <0.001, and P <0.05 respectively). A significant decrease was found in the plasma and liver tissue MDA levels in Group 4 compared to Group 3 (P <0.05 and P <0.05, respectively). The activity levels of plasma paraoxanase and arylesterase were significantly higher in Group 2 than in Groups 1 and 3 (P <0.05 and P <0.01, respectively). Also, the plasma paraoxanase and arylesterase levels were significantly higher in Group 4 compared to Groups 1 and 3 (P <0.05 and P <0.01, respectively). A significant reduction was observed in Group 4 in steatosis, inflammation, necrosis, and fibrosis compared to Group 3 (P <0.05 for each). We conclude that soy isoflavones seem to be effective in preventing liver damage by decreasing lipid peroxidation in the NASH model induced by a MCD diet. They stimulate and increase the activity of the antioxidative paraoxanase enyzme while decreasing the total cholesterol and triglyceride levels.

Keywords

Nonalcoholic steatohepatitisParaoxonaseArylesteraseSoy isoflavoneMethionine- and choline-deficient diet

Introduction

Nonalcoholic steatohepatitis (NASH) is characterized by diffuse fatty infiltration in the liver and ballooning degeneration and inflammation in the hepatocytes [1]. The experimental model of NASH has contributed to the understanding of its pathogenesis. There are three main models, which consist of genetically obese ob/ob mice, lipoatrophic mice, and experimental animals fed a methionine- and choline-deficient diet [MCD]. All models seem to support the hypothesis of multiple hits [2]. Steatohepatitis alcoholism is concomitant with increased oxidative stress in forms like jejuno-ileal bypass [3]. The MCD diet leads to a rise in oxidative stress in the experimental model [4], where oxidative stress causes secretion of pro-inflammatory cytokines and lipid peroxidation [5, 6]. Lipid peroxidation progresses to hepatocellular necrosis, and it initiates fibrogenesis [7]. It has been reported that the MCD diet model led to steatosis, chronic hepatocyte damage and inflammation, stellate cell activation, and development of fibrosis [8, 9]. Only steatosis develops in the ob/ob mice model [2], while with the MCD diet histopathological findings are similar to those for NASH in humans [8].

Phytoestrogens are plant-derived diphenolic molecules, and they are similar to estradiol in function. The major elements of the phytoestrogen family are isoflavone, lignane, and cumestane. Of these, the most available and most studied one is isoflavone. The active agents of isoflavones are genistein and daidzein, and it has been demonstrated that these agents have selective estrogenic characteristics. The presence of estrogenic activities is associated with their binding onto estrogen receptors. This binding is mediated by the hydroxyl group on the built-in aromatic ring in the molecular structure [10, 11].

Flavonoids are plant-derived polyphenolic compounds [12]. They act through cellular GSH by inhibiting topoisomerases 1 and 2 and thyrosine kinase, which particularly facilitates an increase in genistein tumors, initiating apoptosis and thus inhibiting the formation of new capillary vessels required for tumor growth, decreasing oxidative stress, and directl removing oxidative radicals products [13]. It has been suggested that flavonoids are removers of major free oxygen radicals, interfering with the arachidonic acid cascade in various phases, particularly via cyclo-oxygenase or lipo-oxigenase in vitro and in vivo [1416].

It has been shown that F2-isoprostane levels, which are biomarkers of lipid peroxidation in the blood, in people who consume diets including isoflavone are low [17, 18]. Studies on rats have shown that flavonoids have an effect on plasma total lipid and LDL cholesterol levels, and they decrease cholesterol and triglyceride levels [19] and lipid levels both by reducing biliary acid and cholesterol absorption and increasing the insulin/glucagon rate and thyroid hormones [2023].

In this study, our objective was to examine the protective effect of soy isoflavones on experimental NASH in rats and their effects on the levels of paraoxanase enzymes, which are indicated to have antioxidative features, and to search for alterations in the NASH.

Methods

The study included 28 male rats, which were housed in cages inside a special room exposed to sunlight for 12 hr per day. The study was carried out in strict conformity with the ethical rules on standard experimental animal studies. The rats were divided into four equal groups: Group 1 (n=7) was fed an isocaloric normal diet for 8 weeks, Group 2 (n=7) received an isocaloric basal diet+oral soy isoflavone for 8 weeks (100 mg/kg in diet), Group 3 (n=7) was fed a special diet that was methionine and choline deficient (MCD) and rich in fat for 8 weeks, and Group 4 (n=7) received a special diet that was MCD and rich in fat + oral soy isoflavone for 8 weeks (100 mg/kg in diet) (see Table 1). The MCD diet was administered according to the literature [24]. Rats were sacrificed by decapitation at the end of 8 weeks. Blood samples were collected to measure plasma malondialdehyde (MDA), paraoxonase, and arylesterase and some biochemical parameters. Tissue samples were duly taken for histopathological examination and measurement of tissue MDA levels.
Table 1

Composition of experimental diets in the study (100 mg/kg diet)

 

Group 1a

Group 2b

Group 3c

Group 4d

Soy proteins (%)

20

20

20

20

Sucrose (%)

49.5

49.5

48.65

48.65

Olive oil (%)

14.5

14.5

14.5

14.5

Corn oil (%)

 6

 6

 6

 6

Cellulose powder (%)

 4

 4

 4

 4

Saline (%)

 2

 2

 2

 2

Vitamin premix in vitamin B12

 4

 4

 4

 4

 and choline deficient (%)

    

l-Cysteine

 0.3

 0.3

 0.3

 0.3

Methionine

 0.2

 0.2

Choline chloride

 0.35

 0.35

Soy isoflavone (100 mg/kg rat diet)

 Genistein (mg)

21.05/100

21.05/100

 Daidzein (mg)

47.36/100

47.36/100

 Glycitin (mg)

34.21/100

34.21/100

aControl + oral normal diet (n=7).bControl + oral soy isoflavone diet (n=7).cMCD + oral normal diet (n=7).dMCD + oral soy isoflavone diet (n=7).

Table 2

Some biochemical parameters in the study groups

 

Group 1a

Group 2b

Group 3c

Group 4d

P

AST (U/L)

160±18.64

144±11.78**

229.5±19.09*

163.3±7.23

*P <0.05, Group 3 vs 1 and 4;**P <0.01, Group 3 vs 2

ALT (U/L)

74.8±23.02

52.4±9.36*

  114±12.72***

 88.7±5.98**

*P <0.05, Group 4 vs 2 and 3;**P <0.01, Group 2 vs 1 and 4;***P <0.001, Group 3 vs 1 and 2

Total biluribin (mg/dl)

0.34±0.09

0.57±0.21

 0.89±0.05***

 0.29±0.05*

*P < 0.05, Group 4 vs 2;**P < 0.01, Group 3 vs 2;***P < 0.001, Group 3 vs 1 and 4

Total cholesterol (mg/dl)

59.3±5.57

51.1±6.65*

 75.2±5.48**

 56.4±11.05*

*P <0.05, Group 4 vs 3; *P <0.05, Group 1 vs 2;**P <0.01, Group 3 vs 1 and 2

Triglyceride (mg/dl)

62.8±12.38

54.7±8.78

 74.5±5.67

 51.8±13.71

*P <0.05, Group 4 vs 3; *P < 0.05, Group 1 vs 2;**P <0.01, Group 3 vs 1 and 2

HDL (mg/dl)

25.7±9.15

39.2±10.6*

 19.1±5.39**

 28.4±5.21*

*P <0.05, Group 2 vs 1 and 4, Group 3 vs 4;**P <0.01, Group 3 vs 2

LDL (mg/dl)

10.1±2.63

13.9±3.01

 14.2±2.98

 11.2±3.32

NS

VLDL (mg/dl)

15.5±5.58

15.7±4.18

  5.3±2.87***

 11.2±3.56

***P <0.001, Group 3 vs 1 and 4

Total protein (g/dl)

 7.6±0.51

 7.9±0.63

  4.9±0.11*

  5.9±0.4

*P < 0.05, Group 3 vs 1 and 2

Albumin (g/dl)

 3.3±0.25

 3.5±0.37

  2.4±0.34*

 3.12±0.14

*P <0.05, Group 3 vs 1 and 2

Note. AST, aspartate aminotransferase; ALT, alanine amino transferase; ALP, alkaline phosphatase; GGT, γ-glutamyl transpeptidase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein.aControl + oral normal diet (n=7).bControl + oral soy isoflavone diet (n=7).cMCD + oral normal diet (n=7).dMCD + oral soy isoflavone diet (n=7).

Biochemical Parameters

The blood collected from rats was centrifuged, and the plasma samples were kept at –20°C until analysis. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), total cholesterol, high-density liporotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) were measured using Olympus kits and an Olympus AU 600 autoanalyzer (Olympus Corp., Japan).

Measurement of Plasma and Tissue MDA

Measurement of plasma MDA levels was carried out using the thiobarbituric acid method as modified by Satoh and Yagi [25]. The results were reported as nanomoles per milliliter. The MDA level of liver tissue was measured by the Ohkawa method, and results are reported as nanomoles per gram of tissue [26].

Measurement of Plasma Paraoxonase and Arylesterase

Plasma PON1 and plasma arylesterase levels were measured by a method modified by Ruiz et al. and Juretic et al. [27, 28].

Histopathological Examination

Liver tissue samples were kept in a 10% formalin solution, and paraffin blocks were prepared. The sections from blocks were examined after staining with hematoxylin/eosin and Masson Trichome, and histopathological examination was conducted by an expert pathologist. The percentage of steatotic cells was identified. In addition, steatosis of <33% of cells, 33–66% of cells, and >66% of cells was classified as grade I, grade II, and grade III, respectively [29]. Inflammatory cells were counted in 10 random fields at ×400 magnification, and the mean number of inflammatory cells per square millimeter was calculated by dividing that number by 10 [30]. At ×400 magnification, the foci of necrosis were counted in 10 random fields, and the mean necrotic foci per square millimeter was calculated by dividing that number by 10 [30, 31]. Ballooning degeneration was evaluated as absent/present.

Sections from paraffin blocks were stained with Masson Trichrome and examined at ×40, ×100, ×200, and ×400 magnifications. At ×400 magnification, the central vein was focused on, and the images randomly taken from 10 fields were evaluated by Image Analysis, a specially designed software ware program [32]. For each sample, fibroticfields per millimeter square are reported.

Statistical Evaluation

The study data are reported as mean±standard deviation. For group comparisons, Kruskal-Wallis one-way analysis of variance was used; for dual comparisons, Mann-Whitney U test. Also, Pearson Spearman correlation tests were used for some parameters. Statistical evaluations were performed using the SPPS 11.0 software program.
Table 3

Levels of malondialdehyde, paraoxanase, and arylesterase

 

Group 1a

Group 2b

Group 3c

Group 4d

P

Plasma MDA (nmol/ml)

1.42±0.31

1.81±0.43*

4.94±0.91**

3.61±0.33*

*P <0.05, Group 3 vs 2 and 4, Group 4 vs 1;**P <0.01, Group 3 vs 1

Liver MDA (nmol/g tissue)

54.45±6.05

51.62±13.21

116.87±15.28**

90.34±10.41*

*P <0.05, Group 4 vs 1, 2, and 3;**P <0.01, Group 3 vs 1 and 2

Plasma paraoxanase (U/L)

144.56±12.38*

179.81±26.32

125.56±11.31**

157.01±22.43

*P <0.05, Group 1 vs 2 and 4;**P <0.01, Group 3 vs 2 and 4

Plasma arylesterase (U/L)

30.24±2.81*

35.64±2.31

27.73±4.45**

37.46±6.54

*P <0.05, Group 1 vs 2 and 4;**P <0.01, Group 3 vs 2 and 4

aControl + oral normal diet (n=7).bControl + oral soy isoflavone diet (n=7).cMCD + oral normal diet (n=7).dMCD + oral soy isoflavone diet (n=7).

https://static-content.springer.com/image/art%3A10.1007%2Fs10620-006-9251-9/MediaObjects/10620_2006_9251_Fig1_HTML.gif
Fig. 1

The level of liver tisssue malondialdehyde. *P < 0.05, MCD plus soy isoflavone diet (Group 4) vs control plus normal diet (Group 1), control plus soy isoflavone diet (Group 2), and MCD plus normal diet (Group 3).**P < 0.01, MCD plus normal diet (Group 3) vs control plus normal diet (Group 1) and control plus soy isoflavone diet (Group 2)

Results

Biochemical parameters obtained at the end of the study are reported in Table 2. The AST and ALT values were significantly increased in Group 3 compared to the control group (P <0.001 and P <0.05 respectively), while in Group 4, which received the MCD diet including soy isoflavone, the AST and ALT levels were significantly decreased compared to those in Group 3 (P <0.05 and P <0.001, respectively).

Plasma cholesterol and triglyceride levels were significantly higher in Group 3 compared to Groups 1, 2, and 4 (P <0.01, P <0.01, and P <0.05, respectively). The difference between Group 1 and Group 2 was significant (P <0.05). HDL cholesterol levels were significantly lower in Group 3 compared to Groups 2 and 4 (P <0.01 and P <0.05, respectively). No significant difference was found in LDL cholesterol levels (P>0.05). Plasma MDA levels were higher in Group 3 compared to Groups 1, 2, and 4 (P <0.01, P <0.05, and P <0.05, respectively). Liver tissue MDA levels were significantly higher in Group 3 compared to Groups 1, 2, and 4 (P <0.001, P <0.001, and P <0.05, respectively). In Group 4, there was a significant decrease in plasma and liver tissue MDA levels compared to Group 3 (P <0.05 and P <0.05, respectively).
https://static-content.springer.com/image/art%3A10.1007%2Fs10620-006-9251-9/MediaObjects/10620_2006_9251_Fig2_HTML.gif
Fig. 2

Plasma paraoxonase activity in the study groups. *P < 0.05, control plus normal diet (Group 1) vs control plus soy isoflavone diet (Group 2) and MCD plus soy isoflavone diet (Group 4).**P < 0.01, MCD plus normal diet (Group 3) vs control plus soy isoflavone diet (Group 2) and MCD plus sohy isoflavone diet (Group 4)

https://static-content.springer.com/image/art%3A10.1007%2Fs10620-006-9251-9/MediaObjects/10620_2006_9251_Fig3_HTML.gif
Fig. 3

Plasma arylesterase activity in the study groups. *P < 0.05, control plus normal diet (Group 1) vs control plus soy isoflavone diet (Group 2) and MCD plus soy isoflavone diet (Group 4).**P < 0.01, MCD plus normal diet (Group 3) vs control plus soy isoflavone diet (Group 2) and MCD plus soy isoflavone diet (Group 4)

The activity levels of plasma paraoxanase and arylesterase were significantly higher in Group 2 compared to Groups 1 and 3 (P <0.05 and P <0.01, respectively). Also, the plasma paraoxanase and arylesterase levels were significantly higher in Group 4 compared to Groups 1 and 3 (P <0.05 and P <0.01, respectively). Among the treatment groups, a significant negative correlation was found between plasma MDA levels and plasma PON1 levels in Group 3 (fed on MCD + normal food) and Group 4 (fed on MCD + soy isoflavone) (r=–0.658, P <0.05, and r=–0,737, P <0.01, respectively). The levels of liver MDA plasma paraoxanase and arylesterase are reported in Table 3 and 13.

Regarding the histopathological changes which were observed in the liver tissue in Group 3 (fed the MCD) during histopathological examinations of the study groups, steatosis, inflammation, necrosis, and fibrosis decreased significantly in Group 4 (fed the MCD + soy isoflavone) (P <0.05 for each). The histopathological findings are shown in Table 4 and Figs. 4a–c.
Table 4

Histopathological findings in the study groups

 

Group 1c

Group 2d

Group 3e

Group 4f

Steatosis (Grade)a

 1.83±0.41

 1.16±0.40*

İnflammation (Hücre/mm2)

26.83±3.92

20.04±2.01*

Necrosis (foci/mm2)

 1.30±0.31

 0.81±0.21*

Fibrosis (%)b

17.53±3.34

14.01±1.78*

*P < 0.05, Group 3 vs 4.aThe percentage of steatotic cells was identified. Steatosis of <33%, 33–66%, and >66% of cells was classified as grade I, grade II, and grade III, respectively.bAt ×400 magnification, the central vein was focused on, and the images randomly taken from 10 fields were evaluated by Image Analysis, a specially designed software program. For each sample, fibrotic fields per square millimeter were identified.cControl + oral normal diet (n=7).dControl + oral soy isoflavone diet (n=7).eMCD + oral normal diet (n=7).fMCD + oral soy isoflavone diet (n=7).

Discussion

Some patients with NASH may progress to liver cirrhosis [33, 34]. Thus, prevention of progression and reversibility of the condition are very important. Increased oxidative stress plays a major role in the pathonegenesis. Increased reactive oxygen products lead to lipid peroxidation of cellular membranes and secretion of proinflammatory cytokines [3, 33, 35].

Antioxidants like probucol, vitamin E, and carotenoids with antioxidative features inhibiting lipid peroxidation and having a lipophilic character have been studied in liver models resulting from various factors, and they are well known [33, 36]. Among the many compounds with a polyphenolic structure in the plant world, flavonoids are probably the most important group [68]. Several studies have shown that flavonoids also modulate lipid peroxidation, thrombosis, and carcinogenesis [37]. Flavonoids demonstrate their antioxidative effect on OH and H2O2 radicals and on lipid peroxidation products mediated by their peroxy radicals [38, 39].

In our study, we found significant increases in plasma and liver tissue MDA levels of rats with NASH, experimentally induced by the administration of an MCD diet, compared to the control group and the control+soy isoflavone diet group. We also observed that in the group fed the MCD diet along with soy isoflavone, there were significant reductions in the increased plasma and tissue MDA levels (P < 0.05 and P < 0.05 respectively), and administration of soy isoflavone had an inhibitive effect on lipid peroxidation. Lee et al. [40] reported that the increased plasma and liver tissue MDA levels in rats on a cholesterol-rich diet were decreased by the administration of naringenin, which is a soy isoflavone. In an alcohol-dependent study, Seo et al. [41] stated that naringenin decreased high cholesterol levels but increased HDL cholesterol levels. It has also been reported that antioxidant enzyme levels changed due to alcohol-induced oxidative stress and oxidation of LDL cholesterol, and particularly when there was a reduction in hepatic SOD activity, administration of naringenin caused increased hepatic SOD activity, thus naringenin is an indirect superoxide scavenger [14].

In our study, we observed that administration of soy isoflavone had a lowering effect on total cholesterol and triglyceride levels in the plasma lipid profile, and hypertriglyceridemia occurred in rats on the MCD diet together with increased plasma cholesterol levels. However, we found a significant reduction in the levels of triglycerides and total cholesterol by the administration of soy isoflavones. Several researchers have reported that the antioxidant defense systems are reduced depending on the hyperlipidemia or abnormal lipid profile, and particularly lipid peroxide products increase and toxic intermediate products occur along with decreased antioxidant enzyme levels [12, 41]. Studies have shown that administration of soy isoflavone has a specific regulating effect on triglyceride levels in rats [19].

Flavonoids may activate cellular oxygenases (NADPH oxidase or cellular antioxidants), reducing oxidative stress. They demonstrate such effects via enyzmes like SOD catalase [42]. PON1 is a calcium-dependent esterase contained in plasma high density lipoproteins. At the same time, it protects plasma lipoproteins, mainly LDL cholesterol, from oxidation via free oxygen radicals. Therefore, nutrition via a diet including soy isoflavone is very important for both stimulation of antioxidative enyzmes and their effects on lipid levels [43, 44].

PON1 lipid peroxides and potent oxidant structures like H2O2 can be used as substrates in enzymatic reactions. H2O2 is a major ROS product, particulary generated in the endothelium and the artery wall during atherogenesis, and it may cause generation of more potent oxidative products by leading to LDL oxidation under ROS. Therefore, PON1's ability to hydrolyze H2O2 plays a crucial role particularly in the removal of potent antioxidant structures [40]. In studies on atherosclerosis, which increases in parallel with obesity and weight gain, it has been reported that leptin levels decreased plasma paraoxanase levels and resulted in oxidative stres. At the consequence of constitution of oxidative stres, proatherogenic structures build [43].
https://static-content.springer.com/image/art%3A10.1007%2Fs10620-006-9251-9/MediaObjects/10620_2006_9251_Fig4_HTML.gif
Fig. 4

(a) Section from normal rat liver. (HE; original magnification, ×200.) (b) Ballooning degeneration and steatosis from MCD. (HE; original magnification, ×200.) (c) Liver tissue after treatment with soy isoflavone in experiment nonalcoholic steatohepeatitis. (HE; original magnification, ×200)

In our study, we observed that the levels of PON1, which is mostly found in association with HDL lipoproteins and is known as an antioxidant enzyme particularly hydrolizing lipid peroxides, and the levels of plasma arylesterase decreased in the MCD diet group, and as a result of administration of soy isoflavone, those enzymes caused a significant increase in the plasma levels and stimulated them. It was also shown that increased MDA levels in the MCD diet group had a reverse correlation with plasma paraoxonase levels and stimulated paraoxonase enyzme in the soy isoflavone group significantly reduced MDA levels.

Lipid peroxidation in the MCD diet model is associated with inflammation, hepatic stellate cell activation, and fibrosis [45]. Lipid peroxidation progresses to hepatocellular necrosis, and it initiates fibrogenesis [8]. Permanence of the damaging factor will lead to progression of the disease. We found a reduction in inflammation, necrosis, and fibrosis in rats receiving soy isoflavone. All these effects can be explained by the powerful antioxidative characteristics of soy isoflavones.

These results demonstrate that soy isoflavones have antioxidative activity and a lowering effect on the total cholesterol and triglyceride levels in experimental NASH as well as stimulating antioxidative paraoxanase enzyme, at the same time they have a lowering effect on the increased oxidative products. Based on these results, it seems that soy isoflavones have a protective role in preventing liver damage in NASH. This issue requires further studies.

Acknowledgment

Vitamin B12, methionine- and choline-deficient diet, and choline chloride were supplied by Farmövert A.Ş

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