- 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