Introduction

Most risk factors involved in the causation of liver diseases are directly or indirectly due to the disturbances in lipid and lipoprotein metabolism (₁). Ethanol is a powerful inducer of hyperlipidemia both in animals and humans (₂). An increase in circulating triglycerides can be produced in fasting individuals after ingestion of ethanol for several hours (₃) as well as during administration of alcohol containing diets for several days (₄). The accumulation of fat in the liver acts as a stimulus for the secretion of lipoproteins into the blood stream and development of hyperlipidemia (₅).

Glycine is a dietary non-essential aminoacid that can be readily synthesized from common metabolic intermediates in all organisms. Glycine has multiple roles in many reactions such as gluconeogenesis, purine, haem and chlorophyll synthesis and bile acid conjugation (₆). Glycine lowers the rate of gastric emptying of ethanol resulting in the suppression of its absorption from the gastrointestinal tract (₇). In an in vivo study of ethanol induced liver injury using the Tsukamoto-French model with a design where alcohol and glycine were given together, glycine lowered ethanol concentration in the stomach and minimized liver damage (₈). Glycine derivatives are also known to decrease considerably the activation of lipid peroxidation in stress, reduce the duration of the alarm stage of stress-reaction and limit stress damage to the heart (₉). Glycine is said to activate chloride channels in kupffer cells, which hyperpolarizes the cell membrane and blunts intracellular Ca ²⁺ concentration. Similar to its action in the neurons, glycine also decreases the levels of superoxide ions from neutrophils via glycine gated chloride channels (₁₀). Glycine prevents hepatic cancer and certain melanomas in vivo by inhibiting angiogenesis and endothelial cell proliferation (₁₁). In addition, glycine given orally to schizophrenic patients to facilitate glutamatergic transmission at the level of N-methyl-D-aspartate receptor complex, improved their muscle stiffness and extra pyramidal symptoms (₁₂,₁₃).

On the basis of the ever-increasing list of the advantageous role of glycine, we planned the present study. Our aim was to elucidate the lipotropic property of glycine in a rat model with ethanol induced liver injury.

Materials and methods

Male albino rats weighing 150 to 170 g were procured from the Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University and were maintained in polypropylene cages in a controlled environment (22-24°C) under 12 h light and dark cycle. Standard pellet diet (Hindustan Lever Ltd., Mumbai, India) and water were provided adlibitum. The animals were cared for as per the principles and guidelines of the Ethical Committee for Animal Care of Annamalai University, in accordance with the Indian National Law on animal care and use (Register Number:166/1999/CPCESA) (₁₄).

Cholesterol, chromotropic acid, glycerol trioleate and sodium periodate were purchased from Sigma Chemical Company, St. Louis, MO, USA. Ferric chloride, albumin and sodium arsenite were purchased from Ranbaxy (P) Ltd., New Delhi, India. Ethanol was obtained from Nellikuppam, Cuddalore District, South India. Glycine was purchased from S.D. Fine Chemicals Ltd., Mumbai, India. Other chemicals used were of analytical grade and were obtained from Central Drug House, New Delhi, India.

The animals were divided into four groups and all were fed the standard pellet diet. Rats in groups 1 and 2 received isolcaloric glucose from a 40% glucose solution. Animals in groups 3 and 4 received 20% ethanol (2.5 ml in the forenoon and 2.5 ml in the afternoon) equivalent to 7.9 g kg-1 body weight as an aqueous solution by intragastric intubation for 30 days (₁₅,₁₆). At the end of this period the dietary protocol of group 1 and 3 animals were unaltered. But in addition, group 2 animals received glycine (0.6 g kg-1 body weight) in distilled water and group 4 animals received glycine along with alcohol every day by intra gastric intubation for the next 30 days. The study design followed is clearly represented below.

Figure 1

The total duration of the experiment was 60 days, at the end of which part of the animals were fasted overnight, anaesthetized with an intramuscular injection of ketamine hydrochloride (30 mg kg-1 body weight) and sacrificed by cervical dislocation. Blood was collected in heparinized tubes and processed for the determination of cholesterol and lipoproteins and the activity of lipoprotein lipase (LPL, EC. 3.1.1.34). Liver and brain were cleared of adhering fat, weighed accurately and used for lipid extraction. Lipids were extracted from tissues as described previously by Folch et al. (₁₇). Total cholesterol was estimated by the method of Sackett (₁₈). Serum triglycerides were estimated by the method of Foster and Dunn (₁₉).

High-density lipoprotein (HDL) was estimated in the supernatant after precipitating the serum. Very low density lipoprotein (VLDL) content was calculated as follows

Figure 2

The remaining animals were subjected to whole-body perfusion using normal saline and 10% formalin under light ether anaesthesia. Brain and liver were removed and stored immediately in 10% formalin. The tissues were subsequently embedded in paraffin, thinly sectioned using a microtome (5 m), stained with haematoxylin and eosin (H&E) and mounted in neutral disterene dibutyl phthalate xylene (DPX) medium and examined by light microscopy (₂₁).

Statistical Analysis

All the grouped data were evaluated statistically and the significance of changes caused by the treatment was determined using One Way Analysis of Variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT) by using 9.05 for windows (₂₂). Results are presented as means SD of ten rats from each group. The statistical significance was set at p<0.05.

Results

Effect of alcohol and glycine on body weight

Table 1 shows the average weight gained by the rats during the total experimental period of 60 days. The final body weight of alcohol-treated rats (Group 3) was significantly lower than those of the control animals (Group 1). Treatment with glycine along with alcohol (Group 4) improved the weight gain significantly. Control rats supplemented with glycine (Group 2) did not show any significant change in weight gain. The ratio of liver weight to the total body weight (final) showed a significant increase on alcohol treatment, and was lowered significantly on glycine supplementation.

Figure 3

Table1: Average weight gain by the animals during the experimental period of 8 weeks

Effect of alcohol and glycine on tissue cholesterol

Figure 1 highlights the concentration of cholesterol in the liver and brain of control and experimental animals. Cholesterol concentration was significantly high in the liver and brain of alcohol-treated animals (Group 3) as compared with those of the control rats (Group 1). Glycine supplementation (0.6 g kg ^-1 ) (Group 4) to alcohol-fed rats reduced the levels of cholesterol significantly as compared with the untreated alcohol-supplemented rats. Control rats supplemented with glycine (Group 2) did not show any significant change in the concentrations of liver and brain cholesterol.

Figure 4

Figure 1: Effect of alcohol and glycine on tissue cholesterol of the control and experimental rats

Effect of alcohol and glycine on serum cholesterol and lipoproteins

Effect of administering alcohol and glycine on serum cholesterol and lipoproteins are shown in figure 2. Serum cholesterol, LDL and VLDL levels were significantly higher and HDL levels significantly lower in rats that received alcohol (group 3) as compared with those of the control rats (group 1) (p<0.05). Treatment with glycine at a dose of 0.6 g kg ^-1 body weight to alcohol fed rats significantly decreased the serum cholesterol, LDL and VLDL levels and elevated the serum HDL levels (group 4) as compared with the untreated alcohol supplemented rats. Glycine supplementation to control rats (group 2) did not produce any significant change in the levels of serum cholesterol, LDL, VLDL or HDL.

Figure 5

Figure 2: Effect of alcohol and glycine on serum lipoproteins of the control and experimental rats

Effect of alcohol and glycine on tissue triglycerides

Effect of administering alcohol and glycine on tissue triglycerides is shown in figure 3. Liver and brain triglyceride levels were significantly higher in rats that received alcohol (group 3) as compared with the control rats (group 1) (p<0.05). Treatment with glycine at a dose of 0.6 g kg-1 body weight to alcohol fed rats significantly decreased the tissue triglyceride levels (group 4) as compared with those of the untreated alcohol supplemented rats. Glycine supplementation to control rats (group 2) did not produce any significant change in the level of triglycerides.

Figure 6

Figure 3: Effect of alcohol and glycine on tissue triglycerides of the control and experimental rats

Effect of alcohol and glycine on plasma LPL activity

Effect of administering alcohol and glycine on plasma LPL is shown in figure 4. The activity of LPL in plasma was significantly elevated in rats treated with alcohol (group 3) as compared with the control rats (group 1)(p<0.05). Glycine supplementation at a dose of 0.6 g kg ^-1 body weight to alcohol fed rats significantly reduced the activity of plasma LPL (group 4) as compared with those of the untreated alcohol supplemented rats. Glycine supplementation to control rats (group 2) did not produce any significant change in the activity of LPL.

Figure 7

Figure 4: Effect of alcohol and glycine on lipoprotein lipase in plasma of the control and experimental rats

Histopathological findings

The liver of alcohol-treated rats showed fatty changes of both macro- and microvesicular type and sinusoidal dilation were observed in all fields (Fig. 3). The liver of alcohol-treated rats which received 0.6 g kg ^-1 of glycine showed loss of individual hepatocytes by degeneration and the space were the cell had originally been appeared empty, but there was no evidence of fatty change (Fig. 4). The liver of control rats, which received 0.6 g kg ^-1 of glycine, showed only focal areas of fatty change. But not to the extent seen in the liver of rats treated with alcohol only (Fig. 2). Control liver demonstrated normal liver morphology (Fig. 1).

The brain tissue in alcohol-treated rats showed edema, which was not evident in rats treated with glycine (Fig. 7, 8). Brain tissue of control rats treated with glycine revealed a normal pattern (Fig. 5, 6).

Discussion

Alcohol is rich in calories and devoid of nutrients, thus contributing to accumulation of fat in the liver. On the other hand, alcohol is known to reduce the absorption of other foodstuff and nutrients from intestine (₂₃), which may be the cause for the decreased gain in the total body weight observed in our study. These results correlate with our previous findings (₂₄). Moreover, the ratio between liver weight and the total body weight showed a 3-fold decrease in alcohol-fed rats supplemented glycine than those of the unsupplemented alcohol-fed rats.

Ethanol causes a significant change in the metabolism of lipids and lipoproteins. The simultaneous production of steatosis and hyperlipidemia has a direct bearing on the pathogenesis of alcoholic fatty liver. Our data shows simultaneous fatty liver development and increased lipoprotein production after chronic feeding of ethanol. The accumulation of fat in the liver on chronic alcohol intake acts as a stimulus for the secretion of lipoproteins into the blood stream and the development of hyperlipidemia (₂₅). Previous studies have shown that the serum and tissue cholesterol levels increase with alcohol consumption (¹⁵). Our results also correlate with the above findings. Moreover, decreased fatty acid oxidation in the liver or increased fatty acid synthesis or both would increase the availability of substrate for lipoprotein synthesis. Such increased availability of substrate in the liver itself has to be postulated, since we found accumulation of lipids in both liver and blood, without evidence that ethanol enhances the clearance of lipoproteins from the liver or affects lipid absorption or peripheral utilization (₂₇). Katan et al (₂₈) and Sugiyama et al (₂₉) have demonstrated the hypocholesterolemic effect of glycine. Our results correlate with the above findings. The cholesterol lowering effect of glycine may be due to the increased conversion of cholesterol to bile acids especially as glycocholic acid.

Plasma LPL is an important enzyme responsible for the hydrolysis of triglycerides present in chylomicrons and VLDL (₃₀). Significantly low activity of plasma LPL in alcohol fed rats, as seen in our study, can cause the accumulation of triglycerides and hydrolysis of VLDL. The hypertriglyceridemia noticed in the alcohol supplemented rats, was not observed when the rats were simultaneously treated with glycine. Triglyceride lowering effect of glycine may be attributed to both enhanced peripheral tissue clearance, and increased plasma LPL activity.

Lipoproteins are chemically modified by oxidation. These oxidised or modified lipoproteins do not react with LDL receptors, leading to esterification of cholesterol and conversion of macrophages to foam cells, thereby contributing to the hyperlipidemia observed on alcohol consumption (₃₁). Serum VLDL and LDL concentrations were significantly higher in rats fed alcohol than in controls but on glycine supplementation both VLDL and LDL levels were restored to near normal levels. Glycine a small ampipathic aminoacid might change the membrane structure and alter the availability of phospholipids as substrate for the biosynthesis of lipoproteins (₃₂). The unaltered LDL levels observed on treatment with glycine may also be due to the optimal activity of plasma LPL observed in these rats.

HDL is considered to be a beneficial lipoprotein (₃₃) and has a negative effect on the development of fatty liver. HDL helps in scavenging cholesterol from the extrahepatic tissues in the presence of lecithin cholesterol acyl transferase (LCAT) and brings it to the liver. In our study, the HDL concentration in serum was significantly lower in rats receiving alcohol than in control rats. But on supplementing glycine along with alcohol, the elevated serum HDL levels in alcohol treated rats can be attributed to decreased plasma LPL and LCAT activity in these rats. The high serum HDL concentrations in rats on glycine supplementation as compared to the alcohol fed rats may be due to delayed clearance and synthesis of HDL constituents. In this context, Nikkila et al (₃₄) have shown that elevated activity of plasma LPL leads to a rise in HDL production and reduction in LDL constituents.

Significant pathomorphological alterations in the liver and brain were observed in alcohol treated rats. These changes can alter the properties of a cell. The microscopic changes observed in the liver of alcohol treated rats were predominant in the centrilobular region. Hepatic damage observed may be partially attributed to cytochrome-P450 generated metabolic cytochrome-P450 dependant enzyme activities in liver that tend to be present at their greatest concentration near the central vein, and lowest near the peripheral sites (₃₅). Supplementing glycine to alcohol treated rats reduced the fatty change and improved the histomorphology of the liver.

Microdysplasia and spongioform changes have been demonstrated in the hypothalamic and thalamic regions of the brain of alcohol treated rats (³⁶). These are indicative of local brain development disorders. In the present study, we observed edema in the brain of alcohol treated rats, which was reversed on treatment with glycine.

Control rats were also treated with glycine to examine the role of glycine per se under controlled conditions, and to evaluate statistically the extent of benefit it offers in alcohol induced hepatotoxicity. The data did not show any significant effect on serum lipoproteins and tissue lipids when glycine was administered.

Conclusion

The above data indicate that glycine administration has a hypolipidemic effect in an animal model of alcohol induced hyperlipidemia. Significant improvements in the histopathological changes observed in the liver and brain of alcohol supplemented rats treated with glycine correlate with our biochemical findings emphasising the protective role of glycine.

Acknowledgement

Correspondence to

Dr. N.Nalini, M.Sc., Ph.D., Reader, Department of Biochemistry, Annamalai University, Annamalainagar – 608 002, Tamilnadu, India. Fax: 91- 4144-238343. E-mail: nalininam@yahoo.com