the potential beneficial effect of glycine on the carbohydrate moieties of glycoproteins in an...

JOURNAL OF MEDICINAL FOODJ Med Food 7 (1) 2004, 108–113© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition

The Potential Beneficial Effect of Glycine on the Carbohydrate Moieties ofGlycoproteins in an Experimental Model of Alcohol-Induced Hepatotoxicity

Rajagopal Senthilkumar and Namasivayam Nalini

Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar, India

ABSTRACT Glycine is known to have a protective role against alcohol-induced liver damage. The aim of our study wasto evaluate the effect of glycine on liver and brain glycoproteins in alcohol-fed rats. Administering ethanol (7.9 g/kg of bodyof weight) every day to Wistar rats for 60 days resulted in significantly elevated levels of liver and brain hexosamine, fucose,and sialic acid and significantly reduced levels of total hexoses as compared with those of the control rats. Simultaneousglycine supplementation (0.6 g/kg of body weight) during the last 30 days of the experiment to rats given alcohol normalizedthe levels of hexosamine, fucose, and sialic acid and elevated the levels of total hexoses in the liver and brain significantlyas compared with unsupplemented alcohol-treated rats. Microscopic examination of alcohol-fed rat liver showed inflamma-tory cell infiltrates and fatty changes, which were reversed on treatment with glycine. Similarly, alcohol-treated rat braindemonstrated edema, which was markedly reduced on treatment with glycine. Thus glycine administration plays a significantrole in reducing the toxicity of ethanol.

KEY WORDS: � ethanol � fucose � glycine � hexosamine � sialic acid � total hexoses

INTRODUCTION

GLYCOPROTEINS ARE A FAMILY of complex proteins withcovalently bound oligosaccharides. The oligosaccha-

ride chain of glycoproteins encodes biological information.1

The molecular constituents of glycoproteins such as hex-osamines, sugars, and various derivatives of N-acetylneur-aminic acid are essential for maintaining the integrity of theextracellular matrix as well as the cellular content.2 Any im-pairment in the composition of these cellular macromole-cules can lead to degeneration of the matrix and loss of tissue function.3 Alcohol-induced impairment of hepaticglycoprotein secretion is known to be mediated by ac-etaldehyde in experimental rats with induced hepatic in-flammation.4 Such injury results in widespread alterationsin liver tissue, primarily due to increased deposition of con-nective tissue components, especially collagen and glyco-proteins.5

Glycine is a dietary nonessential amino acid that can bereadily synthesized from common metabolic intermediatesin all organisms. Glycine has multiple roles in many re-actions such as gluconeogenesis, purine, heme, and chloro-phyll synthesis, and bile acid conjugation.6 In an in vivo study of alcohol-induced liver injury using theTsukamoto–French model with a design where alcohol and

glycine were given together, glycine lowered the alcoholconcentration in the stomach and minimized liver damage.7

Glycine derivatives are also known to decrease considerablythe activation of lipid peroxidation in stress, reduce the du-ration of the alarm stage of the stress reaction, and limitstress damage to the heart.8 Glycine is said to activate chlo-ride channels in Kupffer cells, which hyperpolarizes the cellmembrane and blunts the intracellular Ca21 concentration,similar to its action in the neurons, and also decreases thelevels of superoxide ions from neutrophils via glycine-gatedchloride channels.9 Glycine prevents hepatic cancer and cer-tain melanomas in vivo by inhibiting angiogenesis and en-dothelial cell proliferation.10

Based on the ever-increasing list of the beneficial effectsof glycine, and the present-day need for an effective, eco-nomical, and simple strategy for inducing a reversal of liverinjury in chronic alcoholics, we planned the present study.Preliminary research in our laboratory showed that glycineprevents alcohol-induced erythrocyte lipid peroxidation inrats.11 Here we report the effects of glycine on liver andbrain glycoprotein levels in rats with alcohol-induced liverinjury.

MATERIALS AND METHODS

Animals

Male albino (Wistar strain) rats weighing 150–170 g wereobtained from the Department of Experimental Medicine,Raja Muthiah Medical College and Hospital, Annamalai

Manuscript received 22 July 2003. Revision accepted 28 October 2003.

Address reprint requests to: Dr. N. Nalini, Ph.D., Reader, Department of Biochemistry,Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamilnadu, India,E-mail: [email protected]

108

GLYCINE AND ALCOHOL-INDUCED HEPATOTOXICITY 109

University, Annamalainagar, India. The animals were main-tained in polypropylene cages in a controlled environment(22–24°C) under a 12-hour light/dark cycle. Standard pelletdiet (Hindustan Lever Ltd., Mumbai, India) and water wereprovided ad libitum. The animals were cared for as pre-scribed by the principles and guidelines of the Ethical Com-mittee for Animal Care of Annamalai University, in accor-dance with the Indian National Law on Animal Care andUse (Register Number 166/1999/CPCESA).12

Chemicals

Cysteine hydrochloride, phenol, glucosamine, and ace-tylacetone were purchased from Sigma Chemical Co. (St.Louis, MO). Periodic acid, sodium metaarsenate, thiobarbi-turic acid, and p-dimethyl aminobenzaldehyde were pur-chased from Ranbaxy (P) Ltd. (New Delhi, India). Ethanolwas obtained from Nellikuppam, Cuddalore District, Tamil-nadu, South India. Glycine was purchased from S.D. FineChemicals Ltd. (Mumbai). Other chemicals used were of an-alytical grade and were obtained from Central Drug House(New Delhi).

Experimental protocol

The animals were divided into four groups of 10 ratseach, and all were fed the standard pellet diet. Rats in groups1 and 2 received isocaloric glucose from a 40% glucose so-lution. Animals in groups 3 and 4 received 20% ethanol(2.5 mL in the morning and 2.5 mL in the afternoon) equiv-alent to 7.9 g/kg of body weight as an aqueous solution byintragastric intubation for 30 days as described earlier.13,14

At the end of this period the dietary protocols of group 1and 3 animals were unaltered. But, in addition, group 2 an-imals received glycine (0.6 g/kg of body weight) in distilledwater, and group 4 animals received glycine along with al-cohol every day by intragastric intubation for the next 30days.

Total duration of the experiment was 60 days, at the endof which some of the animals were fasted overnight, anes-thetized with an intramuscular injection of ketamine hy-drochloride (30 mg/kg of body weight), and sacrificed bycervical dislocation. Liver and brain were cleared of adher-ing fat, weighed accurately, and used for the estimations of tissue total hexoses,15 fucose,16 sialic acid,17 and hex-osamine.18 Tissue proteins were estimated by the method ofLowry et al.19

Histological analysis

The remaining animals were subjected to whole-body perfusion using normal saline and 10% formalin under lightether anesthesia. Brain and liver were removed and storedimmediately in 10% formalin. The tissues were subsequentlyembedded in paraffin, thinly sectioned using a microtome(5 mm), stained with hematoxylin and eosin, mounted inneutral disterene dibutyl phthalate xylene medium, and ex-amined by light microscopy.20

Statistical analysis

All the grouped data were evaluated statistically, and thesignificance of changes caused by the treatment was deter-mined using one-way analysis of variance followed by Dun-can’s Multiple Range Test by using SPSS version 9.05 forWindows.21 Results are presented as means 6 SD values of10 rats from each group. The statistical significance was setat P , .05.

RESULTS

The effect of administering glycine and alcohol on tissuetotal hexoses is shown in Fig. 1. The levels of total hexoseswere significantly lowered in alcohol-treated rats (group 3)(P , .05) as compared with those of the control rats (group1). Glycine supplementation at a dose of 0.6 g/kg of bodyweight along with alcohol (group 4) significantly elevatedthe liver and brain total hexoses as compared with those ofuntreated alcohol-supplemented rats (group 3). Glycine sup-plementation to control rats (group 2) did not produce anysignificant change in the concentration of total hexoses.

The effect of administering glycine and alcohol on tissuesialic acid is shown in Fig. 2. Liver and brain sialic acid lev-els were significantly higher in rats that received alcohol(group 3) as compared with those of the control rats (group1) (P , .05). Administering glycine at a dose of 0.6 g/kg ofbody weight to alcohol-treated rats (group 4) significantlyreduced the liver and brain sialic acid concentrations as com-pared with those of the untreated alcohol-supplemented rats.Glycine supplementation to control rats (group 2) did notproduce any significant change in the concentration of sialicacid.

The effect of administering glycine and alcohol on tissuehexosamine is shown in Fig. 3. The concentration of hex-osamine was significantly higher in the alcohol-treated rats(group 3) (P , .05) as compared with those of the controlrats (group 1). Glycine supplementation at a dose of 0.6 g/kgof body weight along with alcohol (group 4) significantlyreduced the liver and brain hexosamine levels as comparedwith those of the untreated alcohol-supplemented rats.

a60

ControlControl + GlycineAlcoholAlcohol + Glycine

50

40

Liver Brain

30

mg/

g tis

sue

20

10

0

a ba

a a ba

FIG. 1. Effect of glycine and alcohol on tissue total hexoses of con-trol and experimental rats. Data are mean 6 SD values of 10 rats fromeach group. Values not sharing a common letter differ significantly atP , .05 (Duncan’s multiple range test).

110 SENTHILKUMAR AND NALINI

Glycine supplementation to control rats (group 2) did notproduce any significant change in the concentration of hex-osamine.

The effect of administering glycine and alcohol on tissuefucose is shown in Fig. 4. The concentration of liver andbrain fucose was significantly elevated in alcohol-treatedrats (group 3) as compared with those of the control rats(group 1) (P , .05). Glycine supplementation to alcohol-fedrats (group 4) significantly decreased the liver and brain fu-cose concentrations as compared with those of untreated alcohol-fed rats. Glycine supplementation to control rats(group 2) did not produce any significant change in the con-centration of fucose.

Histopathological findings

Alcohol-treated rat liver section showed fatty changes ofboth macro- and microvesicular type and sinusoidal dilationin all fields (Fig. 5C). Glycine supplementation to alcohol-fed rats resulted in loss of individual hepatocytes by de-generation, and the space where the cell had originally beenappeared empty, but there was no evidence of fatty change(Fig. 5D). The liver of control rats that received glycineshowed only focal areas of fatty changes, comparatively toa lesser extent than the rats treated with only alcohol (Fig.5B). Control liver section demonstrated normal liver mor-phology (Fig. 5A).

Edema was noted in alcohol-treated rat brain, which wasnot evident in rats supplemented with glycine (Fig. 5G andH). Brain section of control rats treated with glycine revealeda normal pattern (Fig. 5E and F).

DISCUSSION

Glycoproteins are present on the surface of all cells, andsome are released into the bloodstream and other body flu-ids.22 Acetaldehyde, an intermediate in ethanol metabolism,is known to alter the hepatic glycoprotein content23 and se-cretion24 in acute alcohol-treated rats. A significant increasein serum glycoproteins and mucopolysaccharide content inrats with induced experimental hepatotoxicity was observedby Kawahara et al.25 Results of our present study also sug-gest significant alterations in the carbohydrate moieties ofglycoproteins in the liver and brain following ethanol toxi-city.

Significantly reduced levels of liver and brain total hex-oses in alcohol-fed rats were observed in our study, indi-cating an active degradation of hexose moieties of the gly-coprotein in alcohol-induced liver toxicity. These resultscorrelate with the previous findings, which showed de-creased hepatic total hexose levels in alcohol-fed rats.26

Renau-Piqueras et al.27 have reported that prenatal ethanolexposure reduces the concentrations of a-mannose, a-glucose-N-acetylneuraminic acid, and a-galactose residuesin the liver. Alcohol exposure also causes the Golgi cister-nae to disappear, which in turn can result in significant al-terations in the glycosylation of hepatic protein. Glycinesupplementation to alcohol-fed rats significantly elevatedthe liver and brain total hexoses to near those of the controlrats.

Sialic acid is widely distributed in mammals and occursas a terminal component at the nonreducing end of the car-bohydrate chains of glycoproteins and glycolipids. Sialic

a


35

20

25

30

Liver Brain

15

mg/

g tis

sue

10

5

0

a

b

b

aa a a FIG. 2. Effect of glycine and alcohol on tissue sialic

acid of control and experimental rats. Data are mean 6SD values of 10 rats from each group. Values not shar-ing a common letter differ significantly at P , .05 (Dun-can’s multiple range test).

a

160


120

140

Liver Brain

100

mg/

g tis

sue

60

80

20

40

0

a

b

a

a a

b

a

FIG. 3. Effect of glycine and alcohol on tissue totalhexosamine of control and experimental rats. Data aremean 6 SD values of 10 rats from each group. Valuesnot sharing a common letter differ significantly at P ,.05 (Duncan’s multiple range test).


acid has been implicated in a number of phenomena, in-cluding secretion and transport processes, tumor antigenic-ity, and viral receptor.28 Chronic alcohol exposure signifi-cantly elevated the liver and brain sialic acid levels in our

present study. Kaur29 noted that acute ethanol administra-tion elevated the increase in levels of brain sialic acid, a keycomponent of gangliosides and glycoproteins. Though theexact cause of the increase in hepatic and brain total sialicacid content on alcohol supplementation is not known, var-ious theories attribute it to impairment of Golgi complexfunction,30 decreased activity of glycoprotein glycosyl-transferases,31 and increased activity of dolichol kinase.32

In this context, Petreu and Vesterberg33 and Ghosh et al.34

have shown that chronic alcohol misuse alters the N-acetyl-neuraminic acid and hexosamine content of transferrin, re-sulting in increased serum concentration of sialic acid-defi-cient transferrin and accumulation of sialic acid in the liver.Liver and brain levels of fucose were also significantly el-evated in alcohol-fed rats. This may be due to an increasein liver fibronectin, which has a core fucose residue, whereasplasma fibronectin contains sialic acid residues. In this re-gard, both liver fibronectin and plasma fibronectin have beenreported to increase in alcohol-induced liver toxicity.35

On glycine administration we observed near-normal lev-

a

35


30

25

Liver Brain

20

5

mg/

g tis

sue

15

10

0

a

b

a

a a

b

a

FIG. 4. Effect of glycine and alcohol on tissue fucose of control andexperimental rats. Data are mean 6 SD values of 10 rats from eachgroup. Values not sharing a common letter differ significantly at P ,.05 (Duncan’s multiple range test).

FIG. 5. A: Liver of control rat. Hematoxylin andeosin, 310. B: Liver of control rat treated withglycine. Hematoxylin and eosin, 310. C: Liver ofalcoholic rat. Arrows indicate fatty changes ofmacrovesicular type (open arrow), microvesiculartype (curved open arrow), and sinusoidal dilation(open arrow with bar). Hematoxylin and eosin,310. D: Liver of alcoholic rat treated with glycine.The open arrow indicates hepatocyte drop out; fattychanges were markedly reduced. Hematoxylin andeosin, 310. E: Brain of control rat. Hematoxylinand eosin, 310. F: Brain of control rat treated withglycine. Hematoxylin and eosin, 310. G: Brain ofalcoholic rat shows edema. Hematoxylin and eosin,310. H: Brain of alcoholic rat treated with glycine.Edema is markedly reduced. Hematoxylin andeosin, 310.

A B

D

F

HG

E

C

112 SENTHILKUMAR AND NALINI

els of liver and brain sialic acid as well as fucose in alco-hol-fed rats. Glycine is known to lower the rate of gastricemptying of ethanol, thereby minimizing liver damage,36

and glycine may also directly prevent acetaldehyde, themetabolic product of alcohol, from inducing changes in thecarbohydrate moieties of glycoproteins, thereby protectingthe structural and functional integrity of liver from the ad-verse consequences of alcohol.37

Significant pathomorphological alterations in the liverand brain were observed in alcohol-treated rats. Thesechanges can alter the properties of a cell. The microscopicchanges observed in the liver of alcohol-treated rats werepredominantly in the centrilobular region. The hepatic dam-age observed may be partially attributed to cytochrome P450-generated metabolic cytochrome P450-dependent enzyme ac-tivities in liver that tend to be present at their greatestconcentration near the central vein, and lowest near the pe-ripheral sites.38 Supplementing glycine to alcohol-fed ratsreduced the fatty change and improved the histomorphologyof the liver.

Microdysplasia and spongioform changes have beendemonstrated in the hypothalamic and thalamic regions ofthe brain of alcohol-treated rats.39 These are indicative oflocal brain development disorders. In the present study, weobserved edema in the brain of alcohol-fed rats, which wasreversed on treatment with glycine.

Control rats were also supplemented with glycine to ex-amine the role of glycine per se under controlled conditions,and to evaluate statistically the extent of benefit it offers inalcohol-induced hepatotoxicity. The data did not show anysignificant effect on tissue glycoproteins when glycine wasadministered.

CONCLUSIONS

The above data indicate that glycine administration mayplay a significant role in reducing the toxicity of ethanol andmaintaining the integrity of the parenchymal cells, which isreflected by the near-normal levels of liver and brain totalhexoses, fucose, hexosamine, and sialic acid. Significant im-provements in the histopathological changes observed in theliver and brain of alcohol-supplemented rats treated withglycine further emphasize the protective role of glycine.

ACKNOWLEDGMENTS

The financial assistance from R.D. Birla Endowment,Mumbai, India, is gratefully acknowledged.

REFERENCES

1. Murray RK: Glycoprotein. In: Harper’s Biochemistry, Vol. 25(Murray RK, Granner DK, Mayes PA, Rodwell VW, eds.), LangeMedical Publications, Stamford, CT, 1996, pp. 648–666.

2. Ross R, Bornestein P: Elastic fibres in the body. Sci Am1971;224:44–52.

3. Koettgen G, Baver CH, Ellerding V, et al.: Determination and dif-ferentiation of lectin glycoconjugate interaction by laser nephlom-etry. Hepatol Rap Lit Rev 1980;9:11.

4. Volentine GD, Ogden KA, Kortje DK, Tuma DJ, Sorrell MF: Roleof acetaldehyde in ethanol induced impairment of hepatic glyco-protein secretion in the rat in vivo. Hepatology 1987;7:490–495.

5. Koeller KM, Wong CH: Synthesis of biologically active gly-copeptides. J Clin Chem Soc 1999;46:654.

6. Mabrouk GM, Jois M, Brosnan JT: Cell signalling and the hor-monal stimulation of the hepatic glycine cleavage enzyme systemby glucagon. Biochem J 1998;330:759–763.

7. Iimuro Y, Bradford BU, Forman DT, Thurman RG: Glycine pre-vents alcohol-induced liver injury by decreasing alcohol in the ratstomach. Gastroenterology 1996;110:1536–1542.

8. Malyshev VV, Oshchepkova OM, Seminskii IZh, Nefedova TV,Morozova TP: [The limitation of lipid hyperperoxidation and theprevention of stressor damage to the heart by glycine derivatives].Eksp Klin Farmakol 1996;59:23–25.

9. Wheeler M, Stachlewitz RF, Yamashina S, Ikejima K, MorrowAL, Thurman RG: Glycine-gated chloride channels in neutrophilsattenuate calcium influx and superoxide production. FASEB J2000;14:476–484.

10. Ikejima K, Iimuro Y, Forman DT, Thurman RG: A diet contain-ing glycine improves survival in endotoxin shock in the rat. AmJ Physiol 1996;271:G97–G103.

11. Senthilkumar R, Sengottuvelan M, Viswanathan P, Nalini N: Pro-tective effect of glycine supplementation on the levels of lipidperoxidation and antioxidant enzymes in the erythrocyte of ratswith alcohol induced liver injury. Cell Biochem Funct 2003;21:1–6.

12. National Institutes of Health: Guide for the Care and Use of Laboratory Animals, Office of Science and Health Reports,DRR/NIH, Bethesda, MD, 1985.

13. Kumar RS, Ponmozhi M, Viswanathan P, Nalini N: Effect of Cas-sia auriculata leaf extract on lipids in rats with alcoholic liver in-jury. Asia Pac J Clin Nutr 2002;11:157–163.

14. Senthilkumar R, Ponmozhi M, Viswanathan P, Nalini N: An-tioxidant activity of Cassia auriculata leaf extract in rats with al-coholic liver injury. J Nutr Biochem 2003;14:452–458.

15. Dubois M, Gilles KA, Hamilton JK, et al.: Colorimetric methodfor the determination of sugars and related substances. AnalBiochem 1956;28:350–356.

16. Dische Z, Shettle LB: A specific color reaction of methyl pen-toses and spectrophotometric determination. J Biol Chem 1948;175:596–603.

17. Warren L: The thiobarbituric acid assay of sialic acid. J Biol Chem1959;234:1971–1976.

18. Winzler RJ: Determination of serum glycoproteins. MethodsBiochem Anal 1958;2:279–331.

19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein mea-surement with the Folin phenol reagent. J Biol Chem 1951;193:265–275.

20. Carson FL: Fixation, processing, instrumentation, routine stain-ing. In: Histotechnology. A Self-Instructional Text, Vol. 4 (Car-son FL, ed.), ASCP Press, Stamford, CT, 1990, pp. 1–67.

21. Duncan BD: Multiple range test for correlated and heteroscedas-tic means. Biometrics 1957;13:359–364.

22. Hirschberg CB, Snider MD: Topography of glycosylation in therough endoplasmic reticulum and Golgi apparatus. Annu RevBiochem 1987;56:63–87.

23. Phillips PA, McCarroll JA, Park S, et al.: Rat pancreatic stellatecells secrete matrix metalloproteinase: implications for extracel-lular matrix turnover. Gut 2003;52:275–282.

24. Casini A, Ceni E, Salzano R, et al.: Regulation of undulin syn-


thesis and gene expression in human fat-storing cells by ac-etaldehyde and transforming growth factor-beta 1: comparisonwith fibronectin. Biochem Biophys Res Commun 1994;199:1019–1026.

25. Kawahara H, Matsuda Y, Tsuchishima M, Wang XE, Takada A:Effects of ethanol and acetaldehyde on the maturation of hepaticsecretary glycoproteins. Alcohol Alcohol Suppl 1993;1A:29–35.

26. Rajakrishnan V, Menon VP: Effect of curcumin on carbohydratemoieties of glycoprotein in alcohol induced hepatotoxicity. JHerbs Spices Med Plants 2002;9:19–28.

27. Renau-Piqueras J, Guasch R, Azorin I, Segui JM, Guerri C: Pre-natal ethanol exposure affects galactosyltransferase activity andglycoconjugates in the Golgi apparatus of fetal rat hepatocytes.Hepatology 1997;25:343–350.

28. Emmetot P: Biochemical properties of normal and neoplastic cellsurface. Eur J Cancer 1995;9:319–333.

29. Kaur J: Chronic ethanol feeding affects intestinal mucus lipid com-position and glycosylation in rats. Ann Nutr Metab 2002;46:38–44.

30. Guasch R, Renau PJ, Guerri C: Chronic ethanol consumption in-duces accumulation of proteins in the liver Golgi apparatus anddecreases galactosyl transferase activity. Alcohol Clin Exp Res1992;16:942–946.

31. Schachter H: Enzymes associated with glycosylation. Curr OpinStruct Biol 1991;1:755.

32. Strous GJ, Dekker J: Mucin type glycoproteins. CRC Crit RevBiochem Mol Biol 1992;27:57.

33. Petreu S, Vesterberg O: Separation of different forms of trans-ferrin by isoelectric focusing to detect effect on the liver by xeno-biotics. Electrophoresis 1999;10:600–604.

34. Ghosh P, Hale EA, Lakshman MR: Plasma sialic acid index ofapolipoprotein J: a new alcohol intake marker. Alcohol 2001;25:173–179.

35. Ruoslahti E, Engvall E, Hayman, EG: Fibronectin: current con-cepts of its structure and functions. Collagen Relat Res 1981;1:95–128.

36. Akao T, Kohashi K: Inhibitory effect of glycine on ethanol ab-sorption from gastrointestinal tract. Biol Pharmacol Bull 1995;18:1653–1656.

37. Iimuro Y, Bradford BU, Yamashina S, et al.: The glutathione pre-cursor L-2-oxothiazolidine-4-carboxylic acid protects against liverinjury due to chronic enteral ethanol exposure in the rat. Hepa-tology 2000;31:391–398.

38. Peiffer J, Majewski F, Fischbach H, Bierich JR, Volk B: Alcoholembryo- and fetopathology. Neuropathology of 3 children and fe-tuses. J Neurol Sci 1979;41:125–137.

39. Sarkar SN, Chattopadhyay SK, Majumdar AC: Subacute toxicityof urea herbicide, isoproturon, in male rats. Indian J Exp Biol1995;33:851–856.

the potential beneficial effect of glycine on the carbohydrate moieties of glycoproteins in an...

Documents