1.Lipotropic and anti cirrhotic effects of Disulphidesin rats fed high fat diet or Ethanol Thesis Submitted to Rajiv Gandhi University of Health SciencesFor the award ofDOCTOR OF PHILOSOPHY In Biochemistry[Medical Faculty]BySunanda M. Dept of Bio Chemistry, Dr.B.R.Ambedkar Medical College, Bangalore 45, Karnataka, India.

2. Declaration I here by declare that the matter embodied in thisthesis is the result of experiments carried out by me inthe Department of Biochemistry, Dr. B. R. Ambedkarmedical college, Bangalore under the guidance ofDr. R. T. Kashinath Prof. and Head, Department ofBiochemistry, Dr. B. R. Ambedkar medical college,Bangalore and has not been submitted for the award ofany degree, diploma, associate ship, fellowship etc ofany university or institute. Sunanda M.M.Sc. Dept of Bio Chemistry, Dr.B.R.Ambedkar Medical College, Bangalore 45, Karnataka, India. 3. Dr. B. R. Ambedkar Medical College,Kadugondanahalli, Bangalore-560045CertificateThis is to certify that the thesis entitledLipotropic and anti cirrhotic effects of DiSulphides in Rats fed high fat diet or Ethanolsubmitted by Sunanda M. to Rajiv GandhiUniversity Of Health Sciences (Medical faculty)Bangalore for the award of the degree of Doctor ofPhilosophy in Biochemistry is based on the resultsof the studies carried out by her under the guidanceand supervision of Dr. R.T. Kashinath M.Sc. Ph.D. The thesisor any part there of has not been submittedelsewhere for any other degree. Dr. G. Mohan, M. S. Principal Dr.B.R.Ambedkar Medical College, Bangalore. 4. Dr. B. R. Ambedkar Medical College,Kadugondanahalli, Bangalore-560045 Certificate This is to certify that the thesis entitledLipotropic and anti cirrhotic effects of DiSulphides in Rats fed high fat diet or Ethanolsubmitted by Sunanda M. to Rajiv GandhiUniversity Of Health Sciences (Medical faculty)Bangalore for the award of the degree of Doctor ofPhilosophy in Biochemistry is based on the resultsof the studies carried out by her under my guidanceand supervision. The thesis or any part there of hasnot been submitted elsewhere for any other degree.Dr. R.T. Kashinath M.Sc. Ph.D (Medical faculty).Prof and Head, Dept of Bio Chemistry,Dr .B. R. Ambedkar Medical College,Bangalore - 45 5. AcknowledgementsI wish to acknowledge my sincere thanks and indebtedness toDr. R. T. Kashinath, M.Sc. Ph.D. Professor and Head, Department ofBiochemistry, Dr B. R. Ambedkar Medical college, Bangalore for hisvaluable guidance, encouragement and continued support throughout thecourse of work. His kind counsel and patient understanding wereinvaluable in the preparation of this thesis.I wish to express my gratitude to Shri. M. K. Kempasiddayya LL. M.Trustee and Shri. B. Gurappaji, the then Chairman, Dr B. R. AmbedkarMedical College, Bangalore for encouraging me and allowing me topursue the course.I am extremely thankful to Dr. G. Mohan, M. S. Principal, Dr B. R.Ambedkar Medical College, Bangalore, for the support and help duringmy course of study.My thanks to madam Dr. A. Premarathna, Dr. R. Hemalatha, Dr.B. Ravi, Dr. T. M .R. Usha, Dr. G. Rajeshwari, Dr. C. D. Dayananda, Dr.B. Sudeer, Mrs. Sheema Ahamed, Mrs. T. M. Rashmi, Dr. Girish Desai, 6. Dr. B. Kala Suresh, Dr. Rama, Dr. Saroj Golia and all my other friends inthe college for their encouragement throughout my course.My sincere thanks to Dr. Y.A. Manjunath, Dr. D.S. VasudevaDepartment of pathology, Dr B. R. Ambedkar Medical College, Bangalorefor their valuable suggestions during my course..My special thanks to the technical staff Mrs. Darly Abharam, Mr. S.Swamy, Mrs. Violet, Mr. S. Shivalingaiah, Mrs. Ayesha, Mr. Nagaraj,Mr. Manjunath, Mr. Shivamahadeva, Mr. Ramesh, Mr. Devaraj andMrs. Valli for their help and assistance in maintaining experimentalanimals and to conduct experiments.I would like to extend my thanks to Mrs. Jayashri Rao, M.Lib.Sci. ChiefLibrarian who helped me to do the reference work.I remain ever grateful to my sister Mrs. Shobha Chinnobaiah andfamily, my brothers Mr. Prakash Adnur, Mr. Chandshekar Adnur,Babu Adnur and Girish Ramangoudar for their encouragement andmoral support throughout my course.I am grateful to my husband Mr.SreenivasMurthy and specialthanks to my son Sandeep Murthy who helped me in typing this thesis. Sunanda. M. 7. ContentsChapter - 1 IntroductionChapter - 2 Materials and Methods.Chapter - 3 ResultsTables, Graphs and FiguresChapter - 4 Discussions.Chapter - 5 ConclusionsChapter - 6 Summary Bibliography. 8. List of Abbreviations usedAbs AbsorbanceADH Alcohol dehydrogenaseAEG Aqueous Extract of GarlicALP Alkaline phosphotaseALT Alanine TransaminaseAMP Adenosine MonophosphateASTAspartate TransaminaseAR Grade or AnalarAnalytical reagent gradeBSABovine Serum AlbuminCampCyclic AMPDADS Diallyl DisulphideDPDS Dipropyl DisulphideEFA Esterfied fatty acidFFAFree fatty acidgGramHDL-CholesterolHigh Density Lipoprotein ChlosterolHEGHexane Methanol ExtractHLDHigh Lipid DietIU International UnitsLLitreLDHLactate DehydrogenaseLDLLow Density Lipoprotein 9. MEOS Microsomal Ethanol Oxidising systemm molmilli mole molmicro molem eq / l milli equivalents / litreml milli litre gram micro gramNAD Nicotin amide Adenine DinucleiotideNADPNicotin amide AdenineDinucleiotidePhosphate ( oxidized)NADHReduced Nicotin amide AdenineDinucleiotideNADPH Reduced Nicotin amide AdenineDinucleiotide Phosphate ( oxidized)NEFANon Esterfied Fatty AcidOAA Oxalo AcetateODOptical DensityODFROxygen Derived Free RadicalPPM Parts Per MillionSER Smooth Endoplasmic Reticulum-SH groupsSulphydryl groupsSI UnitsSystem international unitsTBARSThio barbituric acid reactive substanceUEFA Unesterfied Fatty AcidVLDL Very Low Density Lipoprotein 10. Chapter-1Introduction 11. 1.1. Plants and their medicinal usePlants have been the major source of drugs in the Indiansystem of medicine. The earliest reference of the medicinal plantsis found in Rig Veda (3500-1800 B.C). The important works ofmedicine of later period (1000-400 B.C) namely Charaka samhitaand Susruta samhita also give extensive description of variousmedicinal herbs. Apart from the written records, some knowledgeon the subject of medicinal plants has descended through times.Many other Asian and African countries have also reported themedicinal values of the plants and their extracts. Many medicinal plants, when subjected to scientificexperiments have yielded useful drugs, which could be taken up bythe modern system of medicine. One such plant is Ammi visanga.Decoction of the dried seeds of this plant is used as a diuretic andas an antispasmodic in renal colic in Mediterranean countries andAmerica. Investigation on this plant showed the active constituentto be Khellin which was found to be effective vasodilator with aselective action on coronary arteries (Anrep G. V. etal 1946). Thescattered information on the medicinal plants in India has beensystematically organized by Kirtikar and Basu in 1933 (Kirtikar K.H. etal 1933) and later by Chopra in 1956 (Chopra R. N. etal1956). These works cover an extensive list of medicinal plantsavailable in various parts of the country with their reportedmedicinal values. Scientific studies and clinical trials haveconfirmed the medicinal properties of many of these plants. TheCentral Drug Research Institute, Lucknow, India has screenedaround three thousand plant materials for a wide variety of 12. chemotherapeutic and pharmacological activities of which manyhave been clinically confirmed (Dhar M. L. etal 1973, Bhakuni D.S. etal 1969, Dhawan B. N. 1977, Aswal B. S. 1984).1.2 Hypolipidemic effects of plants and their extractsAmong the various biological activities of the medicinalplants, the hypolipidemic activity has been the most commonlystudied one. Extracts of various plants have been shown to producehypolipidemia in normal experimental animals. Some of thecommonly studied plants are Allium sativum, Allium cepa,Momordica charantia, Ficus bengalensis, Coccinia indica etc.Active principles have been isolated from some of these plants.The plants belonging to the genus Allium group have beenextensively used as hypolipidemic agents in medicine forcenturies. Studies of products and essential oils of garlic and onionfor their physiological and therapeutic effects have been conductedsince early part of this century probably even before. But thesestudies were necessarily limited by the lack of knowledge aboutthe nature and inter relationship of the chemical components in thefresh tissue processed products or essential oils. The studies ofCavallito and Coworker (Cavallito etal 1985) and Stoll andSeeback (Stoll and Seeback 1951) may thus be seen as crucial inthe development of chemical and biochemical basis of therapeuticstudies.Table1.2 gives the detailed list of plants having hypolipidemicaction. 13. Table-1.2 List of some plants having hypolipidemic activity Name of the plant Fractions found to haveReferences hypolipidemic or hypoglycemic action1.Acacia catechu,Singh etal 1976Acacia sumo and SeedsAlbizza odoratissima2.Allium cepa Linn Petroleium ether andBramachari and Augusti(onion)ethanolic extracts and1961, Jain and vyas allicin 1974, Augusti 19733.Allium sativum LinnPetroleium ether, Diethyl Bramachari and Augusti(garlic) ether and ethanolic 1962, Jain and vyas extracts and allicin1975, Mathew, Augusti 19734.Bamusa Aqueous extract of theBapat etal 1969dendrocalamusleaves5.BougainvilleaEthanolic extracts of the Narayana etal 1984.Soectaoilisroots and aqueous extract of the leaves6.Coccinia indicaEthanolic extractsBramachari and Augusti 1963, Mukherjee etal 1972, Khan etal 19807.Eugenia JambolanaEthanolic extracts of Bramachari and Augusti, seeds Shroti etal 1963, Bansal etal 19818.Ficus bengalensisEthanolic and aqueous Joglekar etal 1963, 14. extracts of the bark Augusti 1975, Babu and bengalenosideMurthy1984.9.Ficus glomerrata Ethanolic extracts ofShrotri and Aiman 1960, bark Gupta 1964.10.Ficus religiosa Water extracts of bark Bramachari and Augusti1962.11.Gymnema Aqueous extractGupta 1963. svelvestre12.Melia uzadirachta Oil from the seeds and Pillai Shanthakumari nimbdin1981.13.Momordica Juice of the fresh fruit Sharma etal 1960, Charantia benzene and ethanolicChatterjee extract of the dried fruit 1963,Pugazhenthi and and charantinMurthy 1979,Leatherdale etal 1981.14.Pterocarpus Aqueous and ethanolicGupta 1963 Trivedi marsupium extract of the wood and1963. pterostilbena15.Trigonella foenum Trigonella and coumarin Shani etal 1974. graceum16.Vinca rosea Aqueous extracts of theShrotri etal 1963. leaves17.XanthiumA sulphur containing Kupiecki etal 1974. Strumariumhypoglycemic agent 15. 1.3. Garlic1.3.1. Chemistry and usesGarlic is used widely in food and pharmaceuticalpreparations in India. It is a member of allium species and itsbotanical name is Allium sativum Lin. The botanical word allium isderived from the celtic word, all means pungent and it betrays thepresence of a host of remarkable flavorants and odorants all ofthem having in common, one element, sulphur.Garlic has been used for its therapeutic effects such asantidiabetic, antioxidant, antiatherogenic, anticancer as well asfibrinolytic action for centuries. Biological actions of alliumproducts are ascribed to organo sulphur compounds having allylgroup (CH2=CH-CH2) or its isomer propenyl group (CH3-CH=CH-) (Itokayway etal 1973, J. L. Brewster etal 1990). Presence of theseorgano sulphur compounds is the characteristics of this genus. Following the innumerable claims about the miraculousmedicinal properties of garlic, scientific basis for these weresought much later. Thus, the important ingredients of garlic are sulphurcompounds, mainly sulphoxide and disulphide and are principallyresponsible for its medicinal properties.Various chemical contents present in garlic are given intable 1.3.1. 16. Table-1.3.1Table showing chemical constituents present in garlic ContentsChemical contents (g / 100)Moisture (%) 61.3-86.3Carbohydrate9.5-27.4Proteins 2.2-6.2 Ash0.6-1.5Energy (cal) 39-140Fat 0.2-0.3Bulk elements (mg / 100 g wet wt)Ca 50-90Mg 43-77Fe 2.8-3.9Al 0.5-3.9P390-460 Ba0.2-1.0K100-120Na 10-20Sulphur, Chlorine and trace elements (mg / 100 mg, wet wt)S65.0 Zn 1.8-3.1Cl 43.0Mn0.2-1.0B 0.3-0.6 Cr 0.3-0.5Cu0.02-0.03 Vitamins (mg/100mg wet wt)Thiamine0.25 Ascorbic acid5.0Pyridoxine Traces Retinol15.0Riboflavin0.08 Nicotinic acid0.5 17. 1.3.1.1. Carbohydrates of garlic Ananthakrishna and Ventakataraman (Ananthakrishna etal 1940.) determined various forms of carbohydrates of garlic. Apart from starch, reserve polysaccharides were chiefly made up of mannose, fructose and a non-reducing sugar. Srinivasn etal qualitatively estimated the water soluble carbohydrates in the bulbs of garlic. (Srinivasan etal 1953). 1.3.1.2. Lipids of garlic The garlic contains about 0.5% lipids on dry weight basis. Fractionation showed that garlic lipids comprised of neutral lipids, glycolipids and phospholipids, cholesterol, campesterol. The fatty acid compositions of total and component lipids have been determined.The presence of -sitosterol and sigma sterol have also been reported. The ethyl acetate extract of condensed garlic residue also yielded more than two saponins (Danata etal 1976).1.3.1.3. Amino acids and peptides of garlicAnanthakrishan and Venkataraman (Ananthakrishan etal 1940) reported the presence of appreciable amounts of lysine and histidine and 30.5% of total phosphorus as phytin in garlic. The alcoholic extract of garlic on paper chromatography showed the presence of major amino acids such as Alanine, Arginine, Aspartic acid. Aspargine, Histidine, Leucine, Methionine, Phenyl alanine, Proline, Serine, Threonine, Trytophan and Valine. These are present as per the WHO specifications. The occurrence of six new gamma-glutamyl peptides from root, bulb and aerial parts of garlic 18. were reported by Suzuki etal (Suzuki T. etal 1961) of which fourwere identified. Virtanen etal (Virtanen A. I. 1987) isolated at leastnine gamma-glutamyl peptides from garlic with gamma glutamyls-allyl cysteine and gamma-glutamyl s-propyl cysteine beingcharacteristics of garlic only. Anthocyanins, pectins, quercetin,flavonidesandsulphoxides are glycosides from garlic. Amino acid composition ofgarlic protein, which showed hypolipidemic action, has beenreported earlier (Biju C. Mathew 1996). Flavonoids and sulfoxidesalso act as antioxidants. The active principles involved inantioxidant activity were considered to be s-alkenyl cysteinesulfoxides (Natto. S.1981) (alliins) and quercetin and its flavoneaglycone analogues (Pratt D. E. etal 1964).1.3.1.4. Other constituents of garlic Denjelak (Denielak Roman etal 1973) revealed theapplication of TLC in the determination of glucosinolates andsulphides of garlic. Rakhimber and coworker (Rakhimber I. R. etal1981) made use of TLC and four specific biological tests toidentify and confirm gibberlins A1 and A3 from germinating garlicbulbs.1.3.1.5. Allithiamine Perhaps the most exquisite property of garlic is its reactionwith thiamine to form allithiamine. Since the discovery ofallithiamine, many properties have been characterized (Watanabe 19. H. etal 1953). One of the striking properties of allithiamine is therapid permeability across the intestinal walls as evidenced byurinary excretion of thiamine after an oral administration ofallithiamine compared to thiamine hydrochloride in human subject. Since, the discovery of allithiamine various other derivativesof thiamine (thio and thiol) have been reported. The disulphidederivative of thiamine also exhibits growth inhibiting effects onsome cells and tissues (Hamaji M. etal 1966, Kazuo H. etal 1958).1.3.1.6. Volatiles of garlic Steam distillation of garlic cloves yields essential oil knownas garlic oil (0.1-0.2 %). Various chemical compounds present inthe oil are as follows.Composition of essential oil of garlic is as followsDiallyl disulphide60.0 %Diallyl trisulphide 29.0 %Diallyl tetrasulphide10.5 %Propyl disulphide6.0 %1.3.1.7. Allicin Raw garlic contains 0.4 % by weight of alliin which is s-allyl cysteine sulphoxide. On crushing the garlic an enzymeallinase convert alliin to allicin, which is responsible for garlic 20. smell. Allinase is also known as Alliin lyase or Alliin alkylsulphonate lyase[EC: 4,4,4,4.]. Cavallito and Baily (Cavallito etal1950) showed the production of allicin by enzyme acting on alliinfor the first time. Later, it was Stool and Seeback (Stoll A. etal 1951) whoisolated and determined the optimal conditions with regard to time,temperature, p and substrate specificity and also elucidated thebiosynthetic pathway for allicin. Allinase catalyses the conversionof allyl cysteine sulphoxide (alliin) to allyl-sulphinic acid whichspontaneously changes to diallyl thiosulphinate which on warming/ heating becomes diallyl disulphide (DADS). Goryachenkovademonstrated the stimulation of the enzyme allinaseactivity bypyridoxal phosphate. Subsequently, many workers reported thepresence of this enzyme in other allium species. Later it wasstudied in detail by Kupiecki and Virtanen (Kupiecki F. P. etal1913) and Schwimmer and Mazelis (Schwimmer S. etal 1963) inAllium cepa. Mazelis (Mazelis M. etal 1963) and Jacobson(Jacobson J. etal 1965) reported the presence of Allinase inBrassica species and Tulbaghia violanceae. Based on pH optima, Jacobson (Jacobson J. etal, 1965)classified Allinase into two categories with optimum pH 5.6 to 6.5(Allium satium) and pH 8.5 (Allium cepa). In 1968, Mazelies andCrew (Mazelies etal 1968) purified garlic allinase seven fold fromthat of homogenate and determined a number of characteristics.Allicin formed by the action of allinase on alliin was extractedfrom crushed garlic bulbs with water (Stoll A. and Seebeck 1951). 21. Biosynthetic path way for Allicin CH2=CH-CH2-S-CH2-CH-COOH | ONH2Allyl-L-Cysteinyl Sulphoxide (Alliin) CH3COCOOH (Pyruvic acid) Allinase NH3CH2=CH-CH2-SH O Allyl sulphinic acid (2molecules) 2H2O CH2=CH-CH2-S-S-CH2-CH=CH2 ODiallyl thio sulphinate (allicin) Warming / DistillationCH2 CH2||||CHCH | |CH2 S S CH2 Diallyl Disulphide (DADS) 22. Raghunandan Rao etal (Raghunandan Rao etal 1946) reported animprovised procedure for allicin extraction, its comparativestability in presence of blood and artificial gastric juice, itsactivation by artificial pancreatic juice and its enzyme inhibitingeffects on milk clotting activity of papain and amylolytic activityof alpha amylase. Srenivas Murthy etal reported (Srenivas Murthyetal 1960) the instability of allicin in aqueous extracts of garlicwhen stored at different temperatures. Allicin when heated orupon steam distillation looses oxygen and becomes a disulphidei.e. Diallyl disulphide (DADS). Allicin inhibits nearly allsulphydryl enzymes but very few non-sulphydryl enzymes whichwere associated with the presence of (-S-O-S-) group and not (-SO-), (-S-S-) or-S- groups. Such an enzyme inhibition by allicinwas prevented by reducing agents like cysteine or glutathione(Wills E. D. 1956).Therapeutic uses of garlic and its productsGarlic as a therapeutic agent has been found to cure variousdiseases of diverse etiology. The therapeutically active garlicpreparation with high content of alkyl sulphides and allicin wasmade by the enzyme decomposition of glucoside with otherchemicals, which stabilized the alkyl sulphides and allicin in theirnascent state, after allowing for 5 to 8 days for completefermentation. This preparation could also be stabilized by vacuumdrying (Spinka etal 1956).The enzymes inhibited by garlic and its fractions areenlisted in table 1.3.1.7. 23. Table 1.3.1.7Enzyme inhibiting activity of Garlic and its fractions. Enzyme inhibited Garlic ReferencesFractions1 Alcohol dehydrogenase AllicinWills 19562 Alkaline phosphataseAllicinWills 19563 Alpha amylase Garlic Suh. M. Yuna-Jah 19764 Beta amylaseAllicinRaghunandan Rao etal 19465 Choline estrase AllicinWills 19566 Fatty acid oxidase Garlic oilVanderhock etal 19807 GlyoxylaseAllicinWills 19568 HexokinaseAllicinWills 19569 Lactate dehydrogenase AllicinWills 195610 Papain AllicinRaghunandan Rao etal 194611 ProteaseGarlic extract Mamoru and Yoshika 197712 Succinate Dehydogenase AllicinWills 195613 Triose phosphate isomerase AllicinWills 195614 Trypsin Garlic extract Sumathi and Pattabhiraman15 Tyrosinase AllicinWills 1956, Agarwala etal 195216 Urease AllicinWills 195617 Xanthine oxidase AllicinWills 1956 24. 1.3.2.1 Hypolipidemic effects of garlicAugusti and Mathew (Augusti etal 1973) first reported thatan aqueous extract of garlic has hypolipidemic action on normalrats. Later it was showed that allicin also reduces serum and tissuecholesterol and triglycerides (Augusti etal 1974).Mirhadi and others (Mirhadi etal 1991) reported thatsupplementation of garlic to rabbits, which were fed cholesterolrich diet suppressed the increased levels of cholesterol in plasma,aorta and liver. Total lipids, phospholipids and free fatty acids inaorta and liver. Gebhardt (Gebhardt etal 1991) found that cultureof rat hepatocytes on incubation with water-soluble extracts ofgarlic powder diminished cholesterol biosynthesis and its export into the medium. Pure alliin alone or after incubation with alliinase(that produces allicin) in concentration corresponding to itscontent in the extracts does not exert any inhibition. HMG COAreductase activity is significantly inhibited by garlic extracts(Diallyl Disulphide (DADS), a component of garlic extract). Fattyacid synthetase is the only enzyme, inhibited by alliin even athigher concentrations. Thus alliin does not seem to be of majorsignificance in the cholesterol biosynthesis (Kumar etal 1991).Bordia and Verma (Bordia etal 1978,1980) reported thatsupplementation of garlic to rabbits fed with a hypercholesteremicdiet showed significant decrease in LDL and VLDL andsignificant increase in HDL levels. In another report by Lau etal(Lau etal 1983) an increased HDL level was demonstrated in rats 25. fed freeze-dried garlic powder making up 2% of an atherogenicdiet.Jain (Jain 1978) compared the effects of garlic and onion inrabbits fed with a diet containing cholesterol at 0.5 % g / day. Thegroup supplemented with garlic juice equivalent to 0.25 g of garlic day showed approximately 20 % of the cholesterol level of thecontrol group after 16 weeks.Using garlic oil, Jain and Komar (Jain etal 1978) showedthat the cholesterol lowering effect was dose tested. Rabbits werefed with a diet of 2g day of cholesterol for 16 weeks.Administration of garlic oil at the dose of 0.25, 0.5 and 1g dayresulted in 6.2 %, 21 % and 30 % reduction in serum cholesterolrespectively.According to Benjamin Lau (Lau B. H. S. etal 1987) kyolic,an odour modified garlic product when given to patients havingelevated cholesterol level (220-440 mgdl) increased thecholesterol level and triglycerides level for the first two months,from the third month onwards a significant drop in serum lipidsbegan, by six months, normal levels of lipids reached in 65 % ofthe subjects. The initial rise in serum lipids may be due to the shiftof the lipid deposits from the tissues in to the blood stream (Changetal 1980, Bordia A. 1981, Chi M. S. 1982, Jain, R. C. 1975,Nakumura H. etal 1971, Krichevsky D. etal 1980). 26. On continued garlic consumption, the excess lipids werebroken down and finally excreted from the body. Similarly aninitial rise occurred with LDL VLDL before significant reductionfollowed while HDL steadily rose after the first month. Accordingto Krichevsky (Kritohevsky etal 1991) garlic can lower serumlipid levels in rats and significantly reduce the severity ofcholesterol induced atherosclerosis in rabbits. Qureshi etal(Qureshi A. A. etal 1983) reported that the odorless water solublecomponent of garlic was equally effective in lowering bloodcholesterol and triglycerides levels.Shoetan etal (Shoetan A. etal 1984) reported that whenalcohol mixed with garlic oil was fed to rats on a high fat diet noincrease of tissue lipids was observed.Severalanimal studies have demonstrated thatcomponents of garlic inhibit synthesis by liver cells (KritchevskyD. etal 1980, 1991, Qureshi A. A. etal 1983). On feeding garlic torats decreased the activity of several important enzymes involvedin the synthesis of lipids not only in the liver but also in adiposetissues such as fat pads (Chang etal 1980).1.3.2.2. Hypoglycemic effects of garlicLaland and Havre old (Laland P. etal, 1933) first reported onthe hypoglycemic effect of garlic. They extracted an ether soluble,steam volatile, alkaloid substance which when mixed with thedisulphides found in garlic and injected in to dogs and rabbitsshowed a hypoglycemic action. A hypoglycemic effect of garlic 27. was later confirmed by others (Brahmachari H. D etal 1962, Jainetal 1973, Chang M. L. W. etal 1980, Farva D. etal 1988).Another study by Wakunga Pharmaceutical Investigatorsshowed that liquid kyolic extract, (kyolic is an odorless garlicproduct from Japan) prevented the rise of blood sugar after oralloading of glucose in a standard glucose tolerance test (Nagai K.etal 1975). Zaman et al (Zaman Q. A. M. etal 1981) and Mahantaetal (Mahanta R. K. etal 1980) administered garlic oil and rawgarlic to human volunteers and found considerable blood sugarreductions.Blood sugar lowering effect of garlic was ascribed to allicinand its various sulphur containing compounds. The -SH groupcompounds are antagonistic to the action of insulin. Allicin andrelated disulphides remove these thiols from the system and thusspare some insulin from inactivation by thiols (Farva D. etal 1988,Mathow P. T. etal 1973).R1-S-S-R1 + 2R2 SHR1-S-S-R2 + R1-S-S-R2 + H2OAllicin ThiolsMixed disulphidesR1-S-S-R2 + R3 SH R1- S-S-R3 + R2 SH (Disulphide) R1 = C3H5 and R2 & R3 = aliphatic chains or protein in enzymeswith -SH group. 28. Rashiah (Rasiah S.V. etal 1985) demonstrated thehypoglycemic effect of garlic oil in experimental diabetes studies.All these findings illustrate that garlic is beneficial to diabetics.1.3.2.3. Anti tumor effects of garlic Garlic and its components significantly inhibited tumorformation (Mazelis M. 1968). Aqueous extract of garlic bulbsmarkedly suppressed the mutaganesis in both E.coil WP2 tryp-andE coli WP2 tryp. (Zhang etal 1989). Garlic and onion essential oilsand related compounds inhibited soybean lipoxygenase (Belmanetal 1989). According to Belman (Belman S. etal 1983) onion andgarlic oil inhibit tumor promotion. In cancer inhibition related studies comparison of effect ofgarlic with that of standard cancer drug, it was found that garlicshowed more positive results than BCG (Bacillus Calmette-Guerin), a live vaccine used to treat bladder cancer (Lau B. H. S.etal 1985). After five treatments of garlic extract, injected directlyinto the tumors of mice, no cancer cells were seen. In animal models, garlic compounds have been shown toinhibit chemical carcinogens and thus prevents various types ofcancer (Belman S. 1983, Wattenburg L. W.1983, Wargovich M. J.1987) Wattenburg L. W. has reviewed the anticarcinogenic activityof garlic and its principles. The allyl derivatives present in garliceffectively block the carcinogenic effects of many carcinogens.Western Reserve University reported that garlic extracts preventedtumor growth by inactivating SH compounds of tumor cells 29. (Weisberger A. S. etal 1957). All these studies show that garlichelps to inhibit tumor growth and can also enhance bodys ownimmune system.1.3.2.4. Antimicrobial action of garlicGarlic has been reported to posses a broad spectrum ofantimicrobial properties. Crude extract of garlic was found to beeffective against gram +ve and gram ve bacteria. It inhibited thegrowth of some bacterial cultures that were resistant to commonlyused antibiotics (Sharma A.D. etal 1977, Kumar A. Sharma etal1982).Garlic can be used as a prophylactic agent againstenterotoxigenic E.Coil induced diarrhoea (Sharma V. D. etal 1977,Kumar A. etal 1982). Reports showed that garlic extract exhibitedpromising antibacterial activity against several clinical strains ofStaphylococous, Escherichia and Pseudomonas Adetumbi etal(Adetumbi etal 1986) demonstrated that garlic extracts stop thegrowth of a yeast organism (Candia Albicans) by preventingformation of lipids in the membrane of these germs andobstructing the intake of oxygen. Garlic possesses an antimicrobialactivity against Coccidioides immitis, an opportunistic fungus thatmay be involved with AIDS (Adetumbi etal 1986).Tsai etal (Tsai Y. etal 1985) reported that garlic has antiviralactivity against influenza virus and Herpes simplex virus.Benjamin Lau found that human immunodeficiency virus (HIV) orAIDS did not grow well in the presence of garlic in tissue culture 30. (Lau B. H. S. 1988). Garlic was also used for treating leprosy(Chaudary D. S. etal 1962) and diarrhoea caused by Entamoebahistolytica (Varon S. 1987).1.3.2.5. Anti platelet aggregation effects of garlicPlatelet aggregation is a normal response to bleeding orvascular injury. Excessive platelet aggregation is undesirable inindividuals with thrombosis tendencies. Bordia (Bordia A. 1978)showed that platelet aggregation induced by ADP epinephrine orcollagen could be inhibited by an in vitro addition of garlic oil in adose related manner.Makheja and Baily (Makheja A. N. etal 1990) reported thatfrom garlic and onion three anti platelet constituents wereidentified namely adenosine, allicin and paraffinic polysulphides.The antiplatelet effects of garlic and onion ingested are attributablemore to adenosine than to allicin and paraffinic polysulphides.Inhibition of platelet aggregation in vitro has been reported bymany investigators using garlic oil (Bordia A. 1975, Boulin D. J.1981 Makheja A. N. etal 1979, Vanderhock J.Y. 1980) and anaqueous extract of garlic (Srivastava K. C. 1984, Mohammed S. F.etal 1986, Srivastava K.C.1986).A compound with potent antithromobotic activity wasisolated by Apitz Castro etal (Apitz Castro etal 1986) in 1983 andlater identified and synthesized by Block etal (Block etal 1984). Acompound with the empirical formula C9H14S3O was named,Ajoeneafter the Spanish word ajo for garlic, which is a 31. decomposed and rearranged form of allicin that is present in agedgarlic.Commercially Ajoene a product of garlic is available(Apitz Castro etal 1986, Block E. 1984).. 3C3H5-S-S-C3 H5 2C3 H5-S-CH2-CH=CH-S-S-C3H5O -H2OO (Allicin) (Ajoene)1.3.2.6. Effects of garlic on blood coagulation parametersThrombosis is believed to be one of the precipitating factortriggering myocardial infarction and stroke. A high percentage ofpatients with acute myocardial infarction had either low ordefective fibrinolytic activity (Sassa H. etal 1975) and increasedlevels of plasma fibrinogen and shortened blood coagulation time.Bordia and Bansal (Bordia etal 1973) reported in 1973 thatfresh garlic juice or equivalent essential oil extract prevented thefat induced increase in plasma fibrinogen and decrease incoagulation time and fibrinolytic activity. Bordia etal (Bordia A.K. etal 1997) compared the effects of garlic oil (extract from 1ggarlic kg body weight) on serum fibrinolytic activity in patientswith myocardial infarction. Patients with previous myocardialinfarction showed 83 % increase in fibrinolytic activity overcontrol patients. Patients with acute myocardial infarction fed withgarlic oil showed an increase in fibrinolytic activity of 63 % to95.5 %. 32. Chutani and Bordia (Chutani S. K. etal 1981) found thatwhen raw or fried garlic (30g day) was administered to 20patients with ischaemic heart disease for four weeks, theirfibrinolytic activity increased significantly.Bordia etal (Bordia etal 1985) compared essential oils ofonion and garlic with the antilipidemic agent clofibrate. Essentialoil of both onion and garlic reduced the elevated serum cholesteroland blood coagulability in cholesterol fed rabbits (0.2g kg day).Garlic and onion proved more effective than clofibrate and garlicappeared superior to onion. These findings definitely enlist garlicas a source of therapeutic agents.Hasenberg etal (Hasenberg J. etal 1988) found that intake ofdried garlic by hyperlipoproteinemic patient decreased fibrinogenand fibrino peptide (Adoga etal 1991). It is also reported that garlicoil when added in the diet of Streptozocin (STZ) induced diabeticrats, reduced the hypercoagulable state of their blood, there byminimizing the risk of intravascular clotting abnormalities seen indiabetes mellitus.1.3.2.7. Effects of garlic on blood pressureHypotensive effects of garlic extract were described as earlyas 1921 by Leoper and Debrayl (Leoper M. etal 1921). Accordingto Bolton etal (Bolton etal 1985) garlic has been used for treatinghypertension in China and Japan for centuries and is recognizedofficially for this purpose by the Japanese Food and Drug 33. Administration. Malik etal (Malik etal, 1981) also reportedhypotensive effects of garlic in dogs.The hypotensive effect of garlic has been postulated to bedue to a prostaglandin formed by the action of unsaturatedpolysulphides (viz.ajoene) formedfrom allicin. Bulgarianresearcher Pettov (Petkov V. etal 1953, 1981) conducted extensivestudies involving both animals and humans in an attempt todemonstrate the effects of garlic on high blood pressure. Hereported that the garlic stored for seven to twelve months, theblood pressure lowering activity was significantly increased.Petkov summarized that storage enabled certain enzyme processesto release the active components of garlic.A recent study by the Chinese Co-operative Group(Zheziang 1986) on 70 hypertensive patients who were given theequivalent of 50 g of raw garlic a day. As per the study, 33 of thesubjects showed a marked lowering of blood pressure, 14 showedmoderate reductions in blood pressure, with an overall success rateof 61.7 %.Another study using essential oil of garlic conducted at thePeoples Experimental Academy of Health of Zheziang provincedemonstrated similar results in lowering of blood pressure. (BordiaA. K. 1991) 34. 1.3.2.8. Other therapeutic effects of garlicBenjamin Lau (Lau B. H. S. 1991) conducted experimentsto find out the effect of garlic on stress. In his experiments twogroups of mice were employed, one group on a regular laboratorydiet and the other on the regular diet plus 25mg / day / mouse of aspecial garlic powder (Wakunaga Pharmaceutical Preparation,Japan). At the end of one week the levels of blood corticoid thestress hormone, secreted by the body under stress weremeasured. The mice on a regular diet had an average bloodcorticoid level of 500 ng / ml, while those who took supplementshad a dramatically lower level of 100 ng / ml. 1.3.4. Toxic effects of garlicThe overuse or misuse of either oils or aqueous extracts ofgarlic often produces certain undesirable effects. As compared tothe studies conducted on the benefits of garlic there are relativelyfew studies on the toxicity of the garlic.Further Osamu Imada etal (Osamu Imada etal 1990) showedthat raw garlic causes extensive edema, bleeding and ulceration offore-stomach mucosa, reduction of red blood cells count andhematocrit and increase of reticulocytes and anemia (Joseph P. K.etal 1989).On ingestion of garlic to nursing mothers their milk getsodour of garlic. Some people have allergic reaction like dermatitisor asthma to fresh garlic or onion. 35. Benjamin Lau and co-workers (Lau B. H. S. 1991)incubated human lymphocytes in kyolic or fresh garlic extract. Forcomparisons they also set up tests using L-cysteine, a compoundused to protect against radioactivity. They found that L-cysteineenjoyed significant protection. It was also found that the freshgarlic extract was quite toxic for lymphocytes in their test system.This may be due to the cytotoxicity of allicin in fresh garlic. Allthe cells were dead after incubating with fresh garlic for 24 hours.Studies have been conducted by others showing that too muchfresh garlic can have harmful side effects.The administration of Diallyl disulphide and S-allyl-L-cysteine at sub acute levels caused hematological disorders,increased serum transaminases and alkaline phsophotase and bloodurea levels. It was observed when 100 mg / Kg body weight garlicoil fed to 24 hours fasted rats, died of pulmonary edema (Joseph P.K. etal 1989). The negative as well as positive effects of alliummay be due to the action of their principles on thiol group systems.Excessive reactions may retard many enzymes and there by retardprotein synthesis.Further studies conducted by Kashinath (Kashinath R. T.1993 established that the toxic effects of garlic may be due to theproduction of acrolein, a metabolite of DADS which is a toxicproduct (Reynold E. F. 1993).Therefore only customary amounts of these species may beused as part of diet, salads and pickles (Augusti P.K. 1996). 36. The studies of Dr.Osamu Imade (Osamu Imade etal 1990)indicated that the orla LD (50) values (mg/kg body weight) inmices for various garlic components were as follows.LD (50) values for various garlic componentsGarlic componentsLD (50) Values (mg/Kg Body) weight)MalesFemalesAllicin 309363Diallyl disulphide 145 130S- ally mercapto cysteine600 922Diallyl sulphide 20291089S-allyl889093901.3.3. Other garlic preparationsThere are a number of preparations of garlic other than thetherapeutic ones. As garlic is widely used as a flavoring agent,essentially powdered and stable garlic preparations are preferredfor storage. The current trend is to produce deodorized garlicpreparations for cosmetics, metal detoxications etc.Table 1.3.3 summarizes the various garlic preparations withtheir possible uses. 37. Table1.3.3 Non-therapeutic garlic preparations and their usesPreparationApplicationReferences1.Stable garlic powder As a spice for flavourPruthi etal 19592.Deodorised garlic -do- Yamamato 19603. -do- -do- Chrobe, 19724. -do- -do-Hauro, 19735. -do- -do- Askazu, 19786.Stable garlic -do- Spinka and PreparationStampler 19527.Sake Deodorised garlic wineIuemon 19708.Garlic wineGarlic odour free wineKemichi Goku 19779.Hair dressing Stop of hair loss,Ameroetta 1967re growth andrevitalization of hair. 38. 10.Garlic preparations To keep the nailsMadeleine and for Strengtheningflexible from hardeningMavala 1968 of nails.with the contact offoreign material.11.Metal stabilizerFor decomposition ofTolok etal 1972(Garlic waste)Nickel and Cobaltproduct12.Cooking oil To prepare flavourSuzuki, 1973 enhanced fried vegetables.13.Cosmetic packPrevents wrinklingMasao 1977 dis-colourlisation of skin.14.Transparent aqueous Cosmetics, flavourToshio etal 1977garlic solutions and therapeutics.15.Garlic To prepare off flavouredCarl etal 1969condimentsfree condiments forsalad dressing.16.A formulationTo mask the strongJoii O. etal 1979 mutton odour. 39. 1.4. Ethanol metabolism1.4.1. Ethanol and its metabolic effectsEthanol or ethyl alcohol (C2H5OH) generally known asalcohol is commonly used organic solvent in the laboratory.Although a trace amount of ethanol can be synthesizedendogenously (McManus etal 1966, Iwata K.1972) ethanol isprimarily exogenous compound, which can be readily absorbedfrom gastro intestinal tract [GIT]. Only 2-10 % of the absorbeddose can be eliminated through the kidneys and lungs, the restmust be oxidized in the body and most of this occurs in the liver.The liver is the only organ capable of metabolizing significantamount of ethanol and magnitude of gastrointestinal ethanolmetabolism has been reported to be negligible. Since liver is themajor organ of ethanol induced toxicity many of the derangementsof hepatic functions have been attributed to the products of ethanoloxidation rather than to ethanol perse. (Liber C. S. 1984, Orrego H.1981, Sorrel M. E. etal 1979).Although ethanol perse has been shown to alter membranefluidity and in this case impaired function in a variety of organs,the susceptibility of the liver to the toxic action of ethanolindicates that metabolism of ethanol in this organ likely plays akey role in alcoholic liver injury (Rubin E. etal 1982).Despite the vast number of studies reported concerning theeffects of ethanol on liver the mechanism by which ethanolexhibits its hepato toxic effects is still an enigma. The following 40. mechanisms are proposed to explain the pathogenesis of alcoholicliver injury. Most of the absorbed ethanol is degraded by oxidativeprocess primarily in the hepatocytes first to acetaldehyde and thento acetate.CH3CH2OH CH3CHOCH3COOH(Ethanol) (Acetaldehyde)(Acetic acid) The acetaldehydeformed, covalently binds tohepatocellular macromolecules especially to proteins and there byalters hepatocellular structure and function ultimately resulting inthe liver injury (Liber C. S. 1984, Orrego H. 1981).The hepatocytes contain three main pathways for themetabolism of ethanol, each located in a different sub cellularcompartment. They are,1.Alcohol dehydrogenase pathway of the cytosol or soluble fraction of the cell.2.Microsomal ethanol oxidizing system (MEOS) located in theendoplasmic reticulum, and3.Catalase degradative route located in peroxisomes.Major amount of ethanol is oxidized to acetaldehyde in theliver by alcohol dehydrogenase (ADH). Catalase and mixedfunction dehydrogenase - microsomal ethanol oxidizing system, 41. MEOS, which accounts for about 25% of ethanol oxidation[Racker etal 1949].1.4.1.1. Alcohol dehydrogenase - [ADH] [EC: 1.1.1.1.]ADH is a major and rate limiting step for alcoholmetabolism, although alternate pathway exists. ADH is an NAD+dependent enzyme of the cell sap (cytosol) with an optimum pH of10-11 and catalyses the conversion of ethanol to acetaldehyde. InADH mediated oxidation of ethanol, hydrogen is transformed fromsubstrate to cofactor Nicotinamide Adenine Dinucleotide [NAD+]resulting in the conversion of its reduced form NADH. AlcoholCH3CH2OH + NAD+ CH3CHO +NADH +H+ dehydrogenaseThis results in the generation of reducing equivalent in thecytosol in the form of NADH and H+ with concomitant depletionof NAD+. This depletion of NAD+ can have profound effects onintermediary metabolism and is responsible for some of themetabolic consequences of alcohol intoxication (Jatlow P. 1980).During metabolism of alcohol, NADH is accumulated and NAD+is depleted resulting in lactate not being oxidized to pyruvate andlatter accumulates (Jatlow P. 1980).ADH is a heterodimer with multiple forms which arise fromthe association of different types of subunits, namely , and .The chain is mutated in some individuals [1 / 2] and mutationrate is more in Orientals. In Orientals 85 % are 2, so ethanol israpidly converted to acetaldehyde. In Whites this is minor i.e. 5-10 42. %, and it has optimum pH of 8.5. The racial difference in degree ofsusceptibility to alcohol intoxication is attributed to theisoenzymesof Alcoholdehydrogenaseand Aldehydedehydrogenase, which catalyses further oxidation of acetaldehydeto acetate. Aldehyde dehydrogenase CH3CHO CH3COOH(Acealdehyde) NAD+NADH + H+ (Acetic acid)Oxidation of ethanol mainly by ADH in vivo was supportedby pyrazole, a potent inhibitor of ADH, which reduces ethanoloxidation in vivo (Lester D. etal 1968). ADH normally accountsfor the bulk of ethanol oxidation at low blood ethanol levels, butnot necessarily at high ethanol levels or during long-term use ofalcohol.ADHlevel isreduced in chronic alcoholics.ADH has broad specificity as it can oxidize methanol, retinol,dehydrogenation of steroid, and omega oxidation of fatty acids(Lieber C. S. 1982).It is generally recognized that the liver is the main site ofethanol metabolism although gastrointestinal metabolism isreported which is negligible (Lin G. W. etal 1980). However whenalcohol is ingested in moderate amounts, a notable fraction (about20%) (Caballeria J. etal 1987) does not enter the systemiccirculation and is oxidized mainly in the stomach. (Julkunen R. J.etal 1985, DiPadova C. etal 1987). Thus gastric ethanolmetabolism appears to decrease the bioavailability of ethanol andmay represent a barrier to the penetration of ethanol, therebymodulating its systemic effects and potential toxicity. After long 43. term ethanol consumption, much of that barrier is lost, an effectthat may be due in part to diminished ADH activity (DiPadova C.etal 1987). This gastric barrier is low in women, therebycontributing to their increased susceptibility to ethanol.1.4.1.2. Microsomal ethanol oxidizing system [MEOS]This microsomal enzyme is in smooth endoplasmicreticulam [SER], proved by morphological observation in rats.Ethanol feeding resulted in proliferation of smooth endoplasmicreticulum (SER), which is a part of microsomal fraction (Iseri O.A. 1964, Lieber etal 1966).Ethanol +O2+NADPH + H+ Acetaldehyde +2H2O + NADP+This proves that in addition to its oxidation by ADH in thecytosol, ethanol may also be metabolized by microsomes. TheMEOS requires NADPH or NADPH generating system andoxygen. Its availability is optimum at physiological pH i.e. 7.4. Therate of ethanol oxidation by this is ten times higher. MEOS systemrequires higher conc. of ethanol for half maximum activity (LieberC. S. etal 1972). MEOS is an inducible enzyme, associated withchronic alcohol, drug metabolism such as cyto-p450 reductase,cyto-p450.Ethanol increases the activity of variety of hepaticmicrosomal enzymes, which metabolizes other drugs. Theconverse also occurs, administration of variety of drugs results inincreased MEOS activity (Lieber C. S. 1972). These changes 44. render the alcoholic more resistant to the effects of many commonsedative like barbiturates, resulting in the necessarily for largerthan normal dose when sedation is required. Competition betweenalcohol and for oxidation by cyto-p450 causes depression of drugmetabolism. However, ethanol and barbiturates are consumedsimultaneously competitive inhibition for those enzymes results ina reduction in clearance (Lieber C. S. 1974, Kater etal 1969) andabnormally high blood levels. MEOS utilizes cyto-p450 as anelectron carrier. This system becomes prominent at higher alcoholconcentration.Microsomal induction affects total body energy metabolism.Alcoholics given ethanol in addition to normal diet did not gainweight (Lieber C.S. etal 1965, Piroda R.C. etal 1972). Onepostulated mechanism for this apparent energy wastage isoxidation without phosphorylation by MEOS. Indeed, ethanoloxidation to acetaldehyde by ADH is associated with thegeneration of NADH, a high energy compound. But when ethanolis oxidized by MEOS, a high energy compound NADPH is usedand no high energy compound is formed, the reaction onlygenerates heat, to the extent that calorigenesis exceeds the needsfor thermoregulation which is a energy wastage.1.4.1.3. Catalase [EC 1.11.1.6]The enzyme catalase is capable of oxidizing ethanol in vitroin presence of H2O2, a generating system shown by Keilin etal(Keilin 1945). 45. CatalaseEthanol + H2O22H2O + AcetaldehydeIt plays minor role in ethanol metabolism in vivo, becauseits activity depends on H2O2 production, which has been shown tobe low under normal circumstances in liver. Hepatic microsomescontain an enzyme, NADPH oxidase, which in presence ofNADPH and O2 generates H2O2 addition of catalase to this systemallows it to oxidize ethanol (Gillette 1957).Catalase resides primarily in the micro bodies, which areseparated with mitochondrial fraction. Other organelles howeverincluding the microsomes contain traces of catalase. Thus, thecombination of H2O2 generation from NADPH oxidase andcatalase could account for microsomal oxidation (Roach etal 1969,Tephly etal 1969). It appears that slow rate with which H2O2 canbe generated from NADPH oxidase or xanthine oxidase preventscatalase from contributing to more than 2 % of the in vivo ethanoloxidation. The NADPH oxidase activity increases upon ethanolfeeding. As a result catalase activity remained unchanged (Lieberand DeCarli 1970). MEOS+CH3CH2OH+NADPH+H +OCH3CHO +NADP+2H2O NADPH Oxidase +NADP + O2 NADP+ + H2O2+ CatalaseCH3CH2OH +H2O2CH3CHO + 2H2O 46. Chronic ethanol consumption results in an enhanced rate ofethanol disappearance from blood. This is not due to the increasein ADH or catalase activity but it is due to significant change inMEOS. Increased rate of alcohol metabolism following ethanolconsumption contributes in addition to the central nervous systemadaptation to the increased tolerance to ethanol which alcoholicsare known to develop. The product of these varied oxidation pathway isacetaldehyde. It appears that either ethanol or acetaldehyde but notnecessarily both, induce various organ disorders and biochemicalalterations. For example, the fetal alcohol syndrome appears to bean ethanol effect independent of acetaldehyde (Mathions P. R etal1982) where as liver fibrosis and collagen formation is moreclosely associated with acetaldehyde than ethanol (Mendenhall C.L. 1981). The increase in NADH with concomitant decrease inNAD+ associated with ADH activity may also produce sequentialmetabolic changes with clinical consequences.1.4.2. Biochemical and metabolic effects of ethanol The oxidation of ethanol results in the transfer of hydrogento NAD+, which is reduced to NADH. This excess reducingequivalent of NADH reflects shift in redox potenicial of thecytosol and change in ratio of some metabolites such as pyruvateand lactate, as measured by changes in the lactate and pyruvateratio. 47. The altered redox state, in turn is responsible for a variety ofmetabolic abnormalities. The redox changes associated withoxidation of ethanol results in increased lactate level resultingfrom either decreased hepatic utilization of lactate derived fromextra hepatic tissues or depending on the metabolic state of liverincreased hepatic lactate production. As a consequence lactatelevel rises in blood resulting in hyperlacticidemia and lacticacidosis (Daughaday W. H. etal 1962).In addition to acidosis the hyper lactiacdemia also hasclinically significant effects on uric acid metabolism. The rise inblood lactate decreases urinary uric acid out put, which leads to anincrease in uric acid level, i.e. hyperuricemia (Lieber etal 1962).Various hepatotoxic agents result in an increased breakdown ofliver nucleoproteins and enhance the release of uric acid into theblood. Both hyper uricemia and hyper lactacidemia play a role inthe aggravation or precipitation of gouty attacks traditionallyassociated with alcoholism.Enzyme activities related to ethanol oxidation and drugmetabolism are frequently altered these changes may be producedeither directly by enzyme induction or suppression or indirectly bythe shift in reducing equivalents NAD+ / NADH.1.4.2.1. Effects of ethanol on carbohydrate metabolismIn vitro ethanol has been shown to inhibit the activetransport of D-glucose (Chang T. etal 1967) into intestinal cells.Ethanol impairs galactose utilization by inhibiting its conversion to 48. glucose by UDP galactose 4-epimerase (Isselbacher K. J. 1961) asthis reaction is NAD+ dependent and is impaired by NADHgenerated from ethanol. Gluconeogenesis is similarly impaired byethanol by variety of mechanisms. The principle amino acidglutamate, entering gluconeogenetic pathway after deamination isconverted to -ketoglutarate due to Glutamate dehydrogenase[GDH] reaction or other compounds of citric acid cycle (Friden C.1959).GlutamateGlutamate + NAD+ Ketoglutarate + NADH + H+ dehydrogenaseThis reaction is opposed by the oxidation of ethanol. Thusdecrease in the citric acid cycle activity from these precursors.Glutamate dehydrogenase is NAD+ dependent enzyme, theavailability of NAD+ is reduced due to ethanol oxidation by ADHreaction (Madison L. L. etal 1967). Increased NADH producesdissociation of glutamate dehydrogenase. The excess of NADH byethanol also favors lactate formation and retards its conversion topyruvate for gluconeogenetic pathway.Lactate Pyruvate +NADH + H+ Lactate + NAD+ dehydrogenaseEthanol promotes glycerol-lipid formation and impairsamino acid transport there by availability of the amino acid isdecreased, and hence reduced gluconegenesis. The NADH 49. generated by ethanol oxidation are shuttled into the mitochondria,supplant, the citric acid cycle as a source of Hydrogen, there bycitric acid cycle activity is affected. Increased NADH / NAD+slows the reactions of the citric acid cycle, which require NAD+.Moreover, the redox change associated with ethanol oxidationdecreases hepatic oxaloacetate (Williamson etal 1969) theavailability of which controls the activity of citrate synthase.Under these conditions, mitochondria will utilize the hydrogenequivalents from the ethanol oxidation rather than oxidize twocarbon fragments derived from fatty acids, which normallyrepresent the main mitochondrial fuel (Fritz 1961).By combination of these and possibly other metabolicblocks both in extra and intra mitochondrial compartments ofhepatocytes, ethanol contributes to decreased gluconeogenesis,which in turn cause alcoholic hypoglycemia in individuals whoseglycogen stores are already depleted or who have pre existentabnormal carbohydrate metabolism (Arky R. A. etal 1966). In addition to hypoglycemia, hyperglycemia has also beendescribed. Pancreatites that occurs in alcoholic could play a role.The increase in circulating catecholamines observed after ethanolabuse could also result in hyperglycemia. The mechanism ofhyperglycemia is still obscure (Lieber C. S. 1972).Typically the storage of glycogen in the liver is alsodiminished. This results from poor dietary intake and liver diseaseso frequently associated with chronic alcoholism. 50. 1.4.2.2. Effects of ethanol on protein metabolismThe abnormal redox state affects protein metabolism andprotein function. Inhibition of protein synthesis has been observedafter addition of various preparations invitro (Jeejeebhoy K. etal1975). Ethanol administration increases glutamate level in the liverdue to the decreased activity of glutamate dehydrogenase becauseof low level of NAD+ in the tissue (Madones J. 1963).Further, increasedNADH producesdissociation ofglutamate dehydrogenase into inactive subunits (Frieden 1959) anddecreases the availability of alpha ketoglutarate necessary fortransamination before its conversion into glucose. Thus synthesisof other amino acid in the liver is affected. The availability ofamino acid for protein synthesis is altered because ethanoldecreases Na+, K+, and Mg++ ATPase activity necessary for theactive transport with concomitant suppression of neutral aminoacid (Israel Y. etal 1963, 1968).Not all the proteins are necessarily affected, synthesis ofconstituent protein of fibrous tissue, mainly collagen may infact beincreased. This may be due to increased synthesis or decreaseddegradation or both. Thus, the process of formation or degradation of collagen inliver is complex. Increased collagen synthesis may be observed byincreased activity of hepatic peptidyl proline hydroxylase leadingto increase incorporation of proline into hepatic collagen in ratliver slices (Feinman L. 1972). 51. However during early stage of alcoholic liver injury (MakerA. B. etal 1970) increased activity of collagenase is observed,subsequently collagenase activity may decrease contributing to theaccumulation of collagen (Maker A. B. etal 1968) on the otherhand, ethanol consumption may increase, the tissue lactateresulting in increased peptidyl proline hydroxylase activity both invivo (Green H. etal 1964) and invitro (Lindy S. etal 1971).1.4.2.3. Effects of ethanol on lipid metabolismDue to ethanol metabolism there is increased NADH /NAD+ ratio which results in accumulation of lipids in most tissuesin which ethanol is metabolized. The mechanism is multifactorialresulting from both increased accumulation and decreased removalof lipid. The ingestion of ethanol with a fatty meal greatlyincreases uptake of fat into chyle (Mendenhall C. L. etal 1974)with an accompanying increase in both hepatic and intestinallymph flow (Baraona E. etal 1975). Lipid synthesis is alsoaccelerated. The increase in NADH/NAD+ ratio results inenhanced fatty acid synthesis (Lieber C. S. etal 1959) possiblyinvolving fatty acid elongation system of outer mitochondria. Inliver, the increased NADH / NAD+ ratio raises the concentration of-glycerol phosphate (Nikkila E. A. etal 1963) which favorsaccumulation of hepatic triglyceride by trapping fatty acids(Johnson D. 1974). Ethanol consumption enhances the activity ofhepatic microsomal -glycerophosphate acyltranferase (Joly etal1971) and phosphotidate phospho hydrolase, which is a rate-limiting enzyme in hepatic triglyceride synthesis (Savolainen M. J.1978). It is reported that increased quantity of phospholipid 52. content of the liver is due to increased activity of enzymes namelycholine phospho transferase and phosphotidyl ethanolaminemethyl transferase. Moreover, if reduction of oxaloacetate tomalate is coupled with oxidation of ethanol, according to themetabolic scheme, enhanced NADH may result in increasedNADPH. Theoretically, to the extent that trans hydrogenation fromNADH to NADPH occurs, mixed function oxidase activity ofmicrosomes, which utilizes NADPH, will contribute to thedisposal of excess hydrogen generated by ethanol oxidation incytosol.Thus, in addition to promoting fatty acid elongation, ethanolmetabolism may result in production of two building blocksneeded for fatty acid synthesis, namely NADPH and acetylcoenzyme-A (Co-A) which is a means of disposing the excess ofHydrogen produced during ethanol oxidation in the liver.Hydrogen equivalents are transformed from cytosol intomitochondria by various shuttle mechanisms, namely malateshuttle and fatty acid elongation cycle.Normally, fatty acids are oxidized by beta oxidation andcitric acid cycle of the mitochondria, which serve as hydrogendonors for the mitochondrial electron transport chain. Thehydrogen equivalents generated by ethanol oxidation are shuttledinto the mitochondria; supplant the citric acid cycle as a source ofhydrogen there by depressing the citric acid cycle. Citric acid cycleactivity is affected by slowing down of the reactions that requireNAD+ and by decreasing hepatic oxaloacetate (OAA) (Williamsonetal 1969), the availability of which controls the activity of citrate 53. syntheses. Under these conditions mitochondria will utilizehydrogen equivalents from ethanol oxidation rather than from fattyacids oxidation there by decreasing fatty acid oxidation (Fritz1961).Administration of ethanol with cholesterol rich dietenhances hepatic cholesterol accumulation (Lefevre etal 1969).This is due to decreased cholesterol catabolism due to reduction inbile acid production by ethanol.Ethanol administration induces mild hyperlipemia (Baronaetal 1970) from enhanced lipoproteins production by esterificationof fatty acids. The hyperlipemia is probably potentiated byunderlying defective lipid metabolism, like diabetics, a lowlipoprotein lipase activity (Losowsky M. S. etal 1963) orassociated pancreatitis (Dimagno E. P. etal 1973) as risk ofdeveloping pancreatitis doubles with low intake. Increasedavailability of fatty acids in liver may also contribute to the hyperketonemia and ketonuria, which is seen in alcoholics.The over all effects of ethanol in all three main sub cellularsites of hepatocytes contribute to alterations in the lipidmetabolism.The increased availability of NADH also results in alterationof the hepatic steroid metabolism in favor of the reducedcompound (Cronholm etal 1970). Ethanol oxidation also producesmarked changes in the lipid membrane composition and affects the 54. fluidity, but it has not been clearly demonstrated (Uthus E. etal1976).1.4.2.4.Miscellaneous alterations associated with ethanol metabolism a. Vitamin abnormalitiesExcessive alcohol use commonly leads to vitamin deficiency(Leevy C. M. etal 1970). Liver not only converts vitamins intometabolically useful form (Cherrick G. R. etal 1965, Fennely J.etal 1967) but also a storage depot for vitamins, so injury to theliver alters vitamin metabolism. Vitamin can be released fromnecrotic liver, lost from the body and not adequately replaced(Frank O. etal 1964). Need for vitamins are increased, as there isliver regeneration (Leevy C. M. 1963, 1964) and increased nucleicacid synthesis. Fat soluble vitamins may not be adequatelyabsorbed by intestine because of increased fat loss in the stool,malnutrition associated with alcohol abuse, and inhibition ofabsorption of some vitamin (Hoyump A. M. etal 1975). The poordietary intake of alcoholics can also lead to vitamin deficiency. Inalcoholics low serum vitamin values are observed due to poordietary intake by alcoholics (Leevy C. M. 1965).Vitamin-A (retinal), which is absorbed by intestine and isconverted to retinal by alcohol dehydrogenase, is competitivelyinhibited by ethanol in liver (Thomson A. D. 1981). 55. Vitamin-D, deficiency is not a major problem amongalcoholics (Leevy C. M. etal 1965). However ethanol may alter tosome extent vitamin-D and related compounds.Vitamin-E deficiency is observed because of low dietaryintake and general malnutrition in alcoholics (Thomson A. D. etal1981).Vitamin-K stores are small, billary obstruction or severeparenchymal liver disease can produce bleeding abnormalities(Melntyre N. etal 1979).Among water-soluble vitamins deficiency of Thiamine,Nicotinic acid, Folic acid, Pyridoxine, Cyanacobalamine andAscorbic acid are encountered (Leevy C. M. etal 1970).Active transport of thiamine across the intestine is inhibitedby alcohol (Hoyumpa A. M. 1975). Moreover the carbohydraterequirement is more in alcoholics, so also thiamine (Melntyre N.etal 1979) requirement. Thiamine deficiency can produceneuropsychiatry disorder called Wernicke Korasakoff syndromein small number of alcoholics this can be genetically determined(Blass J. P. etal 1977). There is also increasing evidence thatthiamine deficiency may contribute to other forms of brain injury(Thomson A. D. etal 1981).The nicotinic acid is converted to coenzymes like NAD+ andNADP+. The deficiency symptoms pellagra of nicotinic acid found 56. in the alcoholics may be probably due to impaired absorption ofnicotinic acid.Folic acid deficiency is very common in alcoholics. This isdue to the malnutrition, which is associated with decreased folicacid absorption associated with alcoholics (Halsted C. H. etal1971). In addition, alcohol may directly block folate metabolism(Sullivan L. W. 1964).Pyridoxine deficiency affects the transformation of aminoacids as a result of which there is increase in both aminotransferases AST and ALT especially, greater increase in AST(Ludwig S. etal 1979).Cyanacobalamine absorption at ileum is inhibited by alcohol(Lindenbaum F. etal 1973, Powell L. W. 1982) but as suchCyanacobalamine deficiency is rarely a problem (Leevy C. M.1970). Because Cyanacobalamine is stored in liver, acute liver cellnecrosis of alcoholic hepatitis may actually produce a noticeableincrease in serum levels that parallels the severity of the liverinjury.Vit-C deficiency is not common in alcoholics but low levelshave been attributed (Beattle A. D. 1976) in alcoholics.b. Mineral abnormalitiesIt is observed that there is mild to moderate ironaccumulation in the liver of alcoholics which leads to cellular 57. damageresulting in cirrhosis, heartfailure etc. Inhaemochromatosis, there is accumulation of iron the mechanism ofwhich is unclear and controversial. This may be either due to theiron content of ethanol especially in red wine, or due to theinfluence of folate on iron absorption.Deficiency of zinc in alcoholics is due to reduced intake andincreased urine loss (Flink E. B. 1971) resulting in manysymptoms associated with zinc deficiency. Lead toxicity is due tothe high content of lead in some wines (Thomson A. D. 1981),which interferes with incorporation of iron into hemoglobin. Deltaaminolevulinic acid is increased and excreted in the urine (Flink E.B. 1971).Magnesium deficiency in alcoholics is quite common, asmagnesium activates many of the enzymes there by enhancingthiamine deficiency (Zieve L. 1969).Calcium level in alcoholic liver disease is very low as it isexcreted in the urine (Kalbfleisch J. M. etal 1963) and also due tolower Calcium absorption becauseofdecreased liverhydroxylation of vit-D.Phosphorus level is decreased in 50% of hospitalizedalcoholics [Jung R. T. etal 1978) resulting in hypophosphotaemia,which is due to cellular uptake and formation of phosphate esters(Krane S. M. etal 1980) poor food intake, diarrhea, vomiting andmagnesium deficiency. 58. Ethanol also affects the microsomal metabolism ofexogenous and endogenous steroid (Lieber C.S. 1968). The effectsinclude decreased blood testosterol levels (Gorden G. G. etal 1976)due in part to enhanced testosterone degradation and conversion toestrogens, as well as to decreased testicular synthesis of steroid.1.4.2.5. Alcohol induced lipid peroxidationLipid peroxidation is a complex process where bypolyunsaturated fatty acids (PUFA) in phospholipids of cellularmembranes under go reaction with oxygen to yield hydroperoxides (LOOH). The reaction occurs through a free radicalchain mechanism initiated by the abstraction of H+ atom fromPUFA by a reactive free radical, followed by a complex sequenceof propagative reactions. The LOOH and conjugated dienes thatare formed can decompose to form numerous other productsincluding alkanals, hydroxy alkanals, malonaldehydes and volatilehydrocarbons (Halliwell B. etal 1989). These diffuse from originalsite of attack and spread the damage to other parts of the cell.O2LHLH + R*RH + L* LOO* L* + LOOHLO*, LOO*, aldehydes (These are more radical species)LH is target PUFA LOO *is fatty acid peroxyl radicalL* is fatty acid radical R* is initiating oxidizing radicalLOOH is Lipid hydroperoxides. 59. TBARS assay is the most popular and easiest method usedas an indicator of lipid peroxidation and free radical activity inbiological samples.The liver damage due to the reactive free radicals is througha variety of mechanisms, Ex. Lipid peroxidation, covalent binding,depletion of glutathione and protein thiols, derangements ofintracellular free calcium, homeostasis, DNA fragmentation withdifferent relevance in various condition (Freemann B. A. 1982).The liver injury due to acute or chronic abuse in ethanol intake (steatosis, plus necrosis, inflammation and fibrosis in lattercases) has been proved to be dependent to its oxidative metabolismat the cytosolic or microsomal (Lieber C. S. 1988). But despiteextensive investigation the molecular mechanism leading to thedamage is still need to be classified. Recent studies on the subjectsucceeded in directly demonstrating the involvement of freeradical species in ethanol metabolism and through their possiblerole in the pathogenesis of tissue changes.By using electron spin resonance, Albano etal were able todetect in rat liver microsomes incubated with ethanol, the hydroxyethyl free radical (CH3C*HOH) (Albano E. etal 1986, 1988).Further, these authors demonstrate that the formation of ethanolderived radical was mostly due to the activity of cyto-P450,dependant mono oxygenase system and minor amount of radicalcan be attributed to ethanol reaction with hydroxyl radicaloriginating (OH*) from iron catalyzed degradation of H2O2.Theformation of hydroxy ethyl radical is now proved to occur also in 60. vivo by its detection in the bile of deer mice which do not expressalcohol dehydrogenase (Knecht K. T. etal 1990) in the liver(Reinke L. A. etal 1987).The role of cyto-P-450 in catalyzing the free radicalactivation of ethanol was more stressed by Albano etal (Albano E.etal 1991) who showed that reconstituted membrane vesiclescontaining cyto-P450 reductase in presence of NADPH, spin trapand ethanol do not give rise to hydroxy ethyl radicals unless cyto-P450 is incorporated.An ethanol inducible form of cyto-P450 has beencharacterized in the liver and shown mainly responsible for theformation of ethanol free radical (Johansson I. 1988). There isevidence of presence of an analogous form of cyto-P 450 also inhuman liver (Ekstrom G. etal 1989). The hydroxyethyl radicaltogether with the reactive oxygen species whose endogenousproduction in the endoplasmic reticulum is strongly enhanced bythe induction of ethanol related cyto-P450 isoenymes can inprinciple trigger oxidative damage in chronic ethanol intoxication.As regards acute intoxication, it is likely that the excess ofacetaldehyde in the liver cytosol is oxidized by alternativepathways such as xanthine oxidase, aldehyde oxidase, with theproduction of super oxide radical (O2*) (Shaw S. 1987).The most investigated mechanism of free radical inducedliver injury during metabolism is lipid peroxidation and is linked toacetaldehyde oxidation. (Comporti M. etal 1967). Acetaldehydereacts quite readily with mercaptons and cysteine, and could 61. complex with acetaldehyde to form a hemiacetal. Binding ofacetaldehyde with cysteine or cysteine containing GSH or bothmay contribute to a depression of liver GSH (Shaw S. etal 1987)there by reducing the scavenging of toxic free radicals by thistripeptide. The depression of glutathione is predominant in themitochondrial compartment and may contribute to the strikingfunctional and structural damage produced by long term alcoholconsumption in that organ. The increased activity of microsomalNADPH oxidase after ethanol consumption may result in enhancedsuperoxideand hydrogenperoxideproduction therebytheoretically favoring lipid peroxidation (Lieber C. S. etal1970).Following is the possible biochemical steps linking theobserved effects of ethanol on free radical production and on lipidperoxidation.Free radical mediated reactions potentially involved in ethanol induced liver damageEthanolAlcohol dehydrogenaseAcetaldehydeCyto-P450, 11E1Xanthine oxidaseAldehyde oxidase O2*, H2O2, OH*, CH3C*HOH Decreased GSH Lipid peroxidationProtein covalent binding 62. Further, it has been shown that long term ethanolconsumption is accompanied by increased formation of hydroxyradicals. Thus, ethanol provokes a significant, if not dramaticdecrease of intracellular GSH through series of mechanisms.(Albano E. etal 1991). Severe glutathione reduction favors lipidperoxidation and peroxidation may be prevented by theadministration of methionine, (Fernandez Cheeca J. C. 1987) aprecursor of cysteine and glutathione.So, hepatic ethanol over load is followed both by an increaseof reactive oxidant species, mainly free radicals, or by a decline ofantioxidant defense. The ethanol intoxication induces oxidativestress, that is an increased ratio between prooxidant and oxidantreaction.1.5. Metabolism of acetaldehydeEthanolmetabolism resultsin the productionofacetaldehyde, which is converted to acetate by aldehydedehydrogenase (RackerE.etal 1949) andthis occurspredominantly in the liver mitochondria. The acetate formed ishighly reactive compound and exerts some toxic effects of its own. Acetaldehyde dehydrogenaseAcetaldehyde AcetateAlthough acetaldehyde is rapidly metabolized significantamount of this reactive aldehyde has been shown to accumulate inthe liver and blood during ethanol oxidation (Braggins J. 1963, 63. Lindros K. O. 1982, Nuutinen H. etal 1983). This is associatedwith generation of reducing equivalents producing a rise incytosolic and mitochondrial NADH / NAD+ ratio (Lieber C. S.1984, Orrego H. etal and Sorrel M. F. etal 1979). Numerousstudies have implicated the role of either acetaldehyde or alteredredox state in many ethanol-induced alteration of hepatic functionsand structure. (Lieber C. S. etal 1984). The polymorphism of theseenzymes is interesting. Persons mainly Asians who harbor aninactive aldehyde dehydrogenase variant (Harada S. etal 1980,Yoshida A. etal 1984) have high blood acetaldehyde levels whenthey drink, with striking consequences in terms of ethanoltolerance and flushing. The beneficial effects include a relativeaversion to alcohol, with low consumption and related morbidity.Acetaldehyde has a carbonyl carbon, which is electrophilicin nature. This makes it susceptible to the attack by a variety ofnucleiophilic compounds (O. Donnel J. P. 1982). Sincemanynucleophilic groups are present in peptides, the likely targetmacromolecules of acetaldehyde in the liver would be proteins.Acetaldeyde can covalently bind to a variety of proteinssuch as albumin (Mohammed A. etal 1949) plasma proteins(Lumeng L. etal 1982) hepatic microsomal proteins andhaemoglobin under physiological pH of 7.4 and at 37c (Gaines U.C etal 1979, Nomura F. etal 1981). It can also form adducts withlipids and nucleic acid, but the reaction products are unstable,reversible and formed to lesser extent (Stevens V. J. etal 1981,Kenny W. C. 1982, Ristow H. etal 1978) to form mainly unstableschiffs base and latter stable compound (Medina V. A. etal 1985) 64. hence altering protein functions. This alteration in protein causesdisplacement of pyridoxal phosphate from its binding site, henceinterfering with the activity of some protein, inhibition of hepaticprotein secretion and induced liver function.Acetaldehyde reacts with protein to form unstable schiff sbase. The reaction is between carbonyl carbon of acetaldehydewith amino group of lysine of protein, which is later reduced byNADH to form stable secondary amines. This is supposed to be thechief causes of alcoholic cirrhosis. Any condition that wouldincrease acetaldehyde levels in the liver during ethanol oxidationwould also enhance the formation of acetaldehyde protein adducts.Chronic ethanol consumption elevates acetaldehyde levelsby both increasing its formation as well as decreasing its oxidation(Nuutinen H. etal 1983). Presence of excessive reducingequivalents formed from ethanol oxidation favor the reduction ofschiffs base to form stable adducts. Conditions like fasting, intakeof certain drugs, particularly acute and chronic ethanolconsumption promote the formation of acetaldehyde proteinadducts (Pessayre D. etal 1979, Gillette J. R. etal 1981,Macclonald C. M. etal 1977, Fernaudez V. etal 1981). Evenacetaldehyde protein adduct formation is effectively reduced byreducing agent like vit-C.Biological nucleiophiles like cysteine, glutathione and lysinedecrease the binding of acetaldehyde to proteins presumably byvirtue of their ability to form addition products with acetaldehyde(Medina V. A. etal 1985). 65. HR-NH2HCH3CH2OH CH3C=OCH3C=NRCH3CH2-NR NAD+ NADH + H+Schiffs base Stable adduct H-carrier (reducing agent)1.5.1. Effect of acetaldehyde on lipid metabolismOne of the most conspicuous lesions induced by acute orchronic ingestion of ethanol in animals and man is, theaccumulation of triglycerides and cholesterol in the liver.Acetaldehyde, which is formed from ethanol oxidation,causes accumulation of triglycerides and cholesterol in the liver byforming schiffs base a stable adduct (as shown in) with protein inthe liver (Giudicelli Y. etal 1972) thus inhibiting the transport oftriglycerides and cholesterol out of liver. Thus, aldehyde stimulatesalcohol in many biochemical effects (Trult E. etal 1971) perhaps inmore toxic manner (Ttoltzman S. G. etal 1974).1.5.2. Effect of acetaldehyde on protein metabolismThere is diminution in both liver protein content and in theincorporation of leucine in to the liver protein showing that proteinsynthesis is affected in acetaldehyde treated in animals (PrasannaC. V. 1980). 66. 1.5.3. Effect of acetaldehyde on carbohydrate metabolismWith respect to carbohydrate metabolism, there is a decreasein TCA cycle as shown by diminished incorporation of glucose into respired CO2. It was also found that both hexokinase andpyruvate kinase activities of the liver and brain were diminished(Prassana C. V. etal 1980).Glycogen synthesis of the liver was increased, as seen bydecrease in the up take of glucose into the liver glycogen. Therewas diminished level of liver glycogen due to increasedglycogenolysis as cyclic AMP phosphosphodiesteras activity ofliver was found to be diminished.1.6. HyperlipidemiaBy definition hyperlipidemia is a condition with elevatedlevels of lipids in the blood. The hyperlipidemia may be due to anyof the following reasons.1. An over intake of dietary fat.2. An abnormal lipid metabolism in the body.3. Acute or prolonged alcohol ingestion.4. Exposure to hepato toxins. 67. 5. Protein malnutrition and malabsorption.As liver plays an important and extremely active role in thelipid metabolism along with adipose tissue and epithelial cell, theamount of lipid in the liver at any given time is the resultant ofseveral influences, some acting in conjunction with, some inopposition to others. Normal levels of lipid in the liver are theresult of maintenance of the proper balance between the factorscausing deposition of fat in the liver verses factor causing removalof fat from the liver. Any alterations in these processes as well asvulnerability of liver results in the accumulation of abnormalquantity of lipid in the liver resulting in so called fatty liver. Ifprolonged may lead to enlargement of liver and fibrotic changesultimately leading to cirrhosis of liver. The fat that accumulates inthe liver is derived mainly from following three sources,1. Dietary lipids, which reach blood stream as chylomicrons.2. Adipose tissue lipids, which are transported to liver as FFA.3. Lipids synthesized in the liver itself.The fatty acids from various sources are converted by liverinto low-density lipoproteins that may be returned to plasma, oraccumulate in liver because of a large number of disturbancesresults in hyperlipemia or hyperlipidemia.The heavy influx of fatty acids results in greatly increasedsynthesis of low-density lipoproteins by liver. Their liberation into 68. the plasma, in the face of decreased uptake by adipose tissuecontributes to the resultant hyperlipemia. The fat content of whichdecreases accordingly.(Owing to thenon-utilization ofcarbohydrates, adipose tissue is unable to take up low-densitylipoproteins, thus, aggravating the hyperlipemia.)1.6.1. Fatty liverFatty liver is an excessive accumulation of fat (lipid) in theliver parenchyma cells, as a result there is slight to moderateenlargement of liver. The fatty liver may result from number ofstresses or abnormality in lipid metabolism. The fat thataccumulates normally includes Triacyl glycerol, Cholesterol andPhospholipids, majority being neutral fat, Triacyl glycerol. Fattyliver can be separated into two categories based on whether the fatdroplets in hepatocytes are macro vesicular (large fat droplets) ormicro vesicular (small fat droplets). Macro vesicular fatty liver ismost common type and most frequently seen in the cases ofalcoholism, obesity and Diabetes Mellitus.In general the fat in the liver is not damaging perse, and willdisappear with improvement or elimination of predisposingcondition. Micro vesicular fatty liver is less common and isassociated with jaundice and hepatic failure. Fatty livers can bedivided into several types depending on the cause.The factors that tend to increase fat content are,a. Increased synthesis of FA in that organ by carbohydrates or 69. proteins.b. Increased influx of dietary lipid.c. Increased mobilization of fat from depots to liver.d. Decreased synthesis of VLDL.e. Deficiency of lipotropic factors.f. Exposure to hepato toxins.Thus, fatty liver may be caused due to,1. High lipid diet (HLD), or over feedingDue to intake of high lipid diet (HLD), fat appears in theplasma as chylomicrons, major fraction of this has to be clearedfrom liver, minor fraction by lipoprotein lipase of plasma andsome taken up by adipose and other tissues. In response to thisincrease, the liver synthesizes larger quantity of low density lipoproteins (LDL), which it liberates into plasma for transport intoadipose tissue, increasing the stores of fat in those organs with noor with increase in Triacyl glycerol and low density lipo proteins.Over feeding results in over synthesis of fatty acids, Triacylglycerol and low-density lipo proteins from carbohydrates. 70. 2. Deficiency of phospholipids Phospholipids are lipotropic factors and help in the normalthe mobilization of the fat from liver. Their deficiency anddecreased synthesis may lead to under mobilization of fat from theliver hence may result in the fatty liver.3. Decreased synthesis of lipoproteins Practically all the lipids of plasma are present as lipoproteincomplexes, which are synthesized in the liver and intestinalmucosa. They are involved in the transport of lipid from the liverto adipose tissue and vice versa. Thus possibly the decreasedproduction of lipoprotein may affect the fat transport, hence fatmay accumulate in the liver leading to fatty liver.4. Deficiency of Vitamins, flavoproteins and essential Fatty acids These help in the turn over of phospholipids. The Vitaminsspecially Pyridoxine (B6), Cyanacobalamine (B-12) as well asflavoproteins and essential fatty acids have been reported toproduce fatty liver in experimental animals by affecting their turnover. Their deficiency may lead to decreased production ofphsopholipid, hence may result in the accumulation of fat in theliver leading to fatty liver.Table 1.6.1 gives the list of different types of fatty liver. 71. Table-1.6.1 Classification of Fatty liver Effect on Effect ImmediateCurativeType Blood lipidsEffecton depotcauseagents (Postabsorptive) on lipids (lipotropic liverfactors)lipids1. None or increaseIncreaseIncreaseExcessive fat inCholine orOver dietprecursors orfeedingsubstitutes2. None or increaseIncreaseIncreaseExcessiveOver carbohydrates,Choline orsynthesiscysteine, B-precursors or Vitamines in diet substitutes Deficiency of Threonine3.Over Increase, normalIncrease, DecreaseCarbohydratemobilization pattern. Increase Normaldeprivation in unesterified pattern (dietary fatty acids hormonal)4. Decrease, IncreaseDeficiency ofUnderespeciallyin fat andDecreaseessential fatty Inositol, cholinemobilization in phsopholipid cholesteroacids,Pyridoxine, and precursors and cholesterol l,PanthothenicacidOr substitutes, decreaseCholine(direct or Lipoaeic acid in lecithin indirect, excess of cholesterol5.UnderIncrease in IncreaseIncrease, Deficiency of Choline andUtilizationphsopholipid andDecreasepanthothenicprecursors chlosterolacid, Hepato Decrease later if toxic agents. severe 72. 1.6.2. CirrhosisCirrhosis is a major cause of death in young and middleaged individuals in many West European Countries and USA(WHO).Cirrhosis is the destruction of normal tissue that leaves non-functioning scar tissue surrounding areas of functioning livertissue. Pathologically it is a defined entity that is generallypreceded by fatty liver, and is associated with spectrum ofcharacteristic clinical manifestations. Cirrhosis is derived fromGreek word Kirrhos meaning tawny as the projecting nodulesare of fawn or yellowish russet bordering on the greenish. Duringinitial stage, fat molecules infiltrate the cytoplasm of the cell,which later merge together so that most of the cytoplasm becomesladen with fat. By this time nucleus is pushed to the side of thecell, nucleus gets disintegrated and ultimately hepatic cell is lysed.As a healing process fibrous tissue is laid down causing fibrosis ofliver i.e. cirrhosis. Cirrhosis is classified as under, based onetiology or morphological features (Fauci Braunwald etal 1998).1.Alcoholic cirrhosis2.Cryptogenic and post viral or post necrotic cirrhosis.3.Biliary cirrhosis.4.Cardiac cirrhosis.5.Metabolic, inherited or drug mediated cirrhosis and,6. Miscellaneous type of cirrhosis. 73. 1.6.2.1. Alcoholic cirrhosisMajority of the cases, cirrhosis occurs in chronic alcoholicsand is usually preceded by a stage of fatty liver (Lieber C.S.1972).Alcoholic cirrhosis, which is also known as alcoholic hepatitis, isprinciple consequence resulting from chronic alcohol ingestion.Alcohol may cause three types of liver damage,1.Fat accumulation (fatty liver).2.Inflammation of liver (alcoholic hepatitis), and3.Scarring of liver (cirrhosis).Alcoholic cirrhosis is historically referred as Laennec,scirrhosis, which is the most common type in developed countries.It is an inflammatory lesion characterized by infiltration of liverwith leucocytes, liver cell necrosis and alcoholic hyaline is thoughtto be major precursor of cirrhosis. Deposition of collagen inperivenular spaces may be the earliest manifestations of theprocess that ultimately leads to cirrhosis. Loss of functioning ofhepatocellular mass may lead to jaundice, edema, coagulopathyand variety of metabolic abnormalities.Biochemical blood tests are usually normal in patients withalcoholic fatty liver except modest elevation of amino transferases(AST and ALT), occasional increase in ALP and bilirubin. Thedisproportional elevation in AST leading to an AST/ALT ratiogreater than 2 is associated with alcoholic hepatitis. This mayresult from proportionally greater inhibition of ALT synthesis byethanol, which may be partially inhibited by Vitamin B6. Serum 74. albumin level is depressed while serum globulin level is increased.Thishypoalbuminemia indicatesimpairment of proteinbiosynthesis while hyperglobulinemia is thought to result fromnon-specific stimulation of reticuloendothelium system.1.7. Lipotropsim and lipotropic factors Various processes help in the removal fat from liver. Theseinclude, 1.Mobilisation of fat from liver to depots. 2.Passage of cholesterol and phospholipids into blood. 3.Degradation of fatty acid in the liver. Agents, which have the apparent effect of facilitating theremoval of fat from the liver, are said to be Lipotropic, thephenomenon being called Lipotropism. Their effect is due totheir,1.Possible role in the synthesis of apolipoprotein, which helps in the transport of fat and fatty acid.2.They are directly or indirectly concerned with transmethylation reaction, thus involved in the synthesis of choline and choline related phsopholipid.3.They help in the passage of choline and phsopholipid into the blood. 4.They are involved in the degradation of fatty acid in the liver. 75. Hence, choline, betaine, ethanol amine, methionine, casein,phospholipid, Vitamins like pyridoxine (B6), cyanacobalamine(B12), folic acid, folinic acid groups, panthothenic acid, carnitineand unsaturated fatty acids, etc are considered to be lipotropicfactors.Human organism is unable to synthesize methyl (-CH3)group and consequently must be obtained from preformed foods.Methionine is proved to be essential in the diet as its methyl groupcan be transformed to accepter molecules by transmethylation. Asa result many methylated derivatives like choline, carnitinebetaine, etc can be formed. Thus, the source of choline forphsopholipid synthesis is mainly of dietary origin. In liverphospholipid, mainly phosphotidyl choline in part may be formedfrom phsophotidyl ethalonamine by three successive methylationusing S-adenosyl methionine as a methyl donor by a minorpathway.-CH, -CH3,-CH3.Phsophotidyl ethanolaminePhsophotidyl cholineThis is of less significance as it drains off methionine, whichis an essential amino acid. Infact, choline can serve as theprecursor of the methyl groups for the synthesis of methionineprovided it is present in adequate amount in the diet. As choline isrequired for the synthesis of phospholipids, which is needed for theformation of lipoproteins specially LDL and HDL which arenecessary for the transport of lipids, hence choline along withmethionine, ethanolamine, betaine, function as lipotropic factors. 76. CholineBetaine aldehydeBetaine Homocysteine MethionineDimethyl glycene FH4 Favoproteins 5,10 Methylene FH4 Sarcosine FH4 5,10 Methylene FH4 Glycine Pyridoxine helps in the synthesis of phospholipids ingeneral, which are necessary for the transport of lipids hencepyridoxine acts as lipotropic factor. Coenzyme-A, the coenzyme form of panthothenic acid aswell as carnitine are involved in fatty acid oxidation, hence bothpanthothenic acid and carnitine act as lipotropic factor. Certain substances or agents, which act as antagonists tothese lipotropic substances are referred to as antilipotropicfactors. These include cholesterol, nicotinic acid or nicotinamide,saturated fat etc. 77. Cholesterol which may compete with phospholipid for polyunsaturated fatty acids hence bring down the formation ofphsopholipid there by act as antilipotropic factor.Nicotinic acid, nicotinamide and guanido acetic acid aremethylated in the body to form Nmethyl derivative (Nmethylnicotinamide, Nmethyl nicotinic acid and creatine) there bydepleting the supply of methyl groups for the synthesis of choline,hence acts as antilipotropic factor.Usually efforts to relieve fatty liver in alcoholics failbecause of continued alcohol abuse. Alcoholic fatty liver could notbe prevented by high protein diet, lipotropic factors or withclofibrate (Lieber C.S. 1972, 1965, Decarli etal 1967). 78. 1.8. Aims and objectives of the studyGarlic, a member of Allium species, is used in food andpharmaceuticals in India as well as in other parts of the world. It isclaimedthat garlichasantidiabetic, antioxidant,andantiatherogenic, anticancer and fibrinolytic effects.These beneficial effects of garlic may be due to itsorganosulphur compounds, which consist of either allyl orpropenyl grouping. The predominant sulphur compound in garlicis Diallyl Disulphide (DADS).Consumption of garlic and its extracts, as stated in earlierpart of this thesis, have certain biochemical toxic effects likeincrease in transaminases, increase in urea, creatinine as well asincrease in tissue thiobarbutaric acid reactive substances (TBARS).Hence it is necessary to have much care while using orconsuming garlic and its products.In accordance with the information given so far in thepresent thesis regarding the effects of garlic and its components,the present work is under taken to study the effects of garlicextracts as well as synthetic disulphide specifically pertaining to, 79. 1. Anti cirrhotic effects of garlic extracts (both aqueous i.e. AEG and Hexane methanol extract i.e. HEG) in chronic ethanol fed rats.2.Anti hyperlipidemic effects of garlic extracts (both aqueous i.e.AEG and Hexane meth