Chapter VI

METHODOLOGY RELATED TO THE EVALUATION OF ATHEROSCLEROSIS

Magnet used for the experiment was cast alloy round and flat

plastic coated Delux magnet (North pole Blue and South pole Orange) in

pairs and in five different strength (1) 1500 (2) 2500 (3) 3000 (4) 3500

(5) 4500 Gauss, purchiased from Swami Homoeo and Magneto

Therapy Centre, Arjun Nagar, Sadarjunj Enclave, New Delhi, India.

The strength of the magnet was confirmed with Gauss meter

available in the School of Pure and Applied Physics, Mahatma

Gandhi University, Kottayam

Inbred Swiss a lb in~ rats of two months age weighing 125 5

12.5 g, purchased from ieterinary College Mannuthy, and Thrissur

were used for the study. T le rats were obtained from the stock inbred

colony, which was maintained by mating sisters and brothers. They

were housed in ventilated cages maintained on dry pellets of rat feed

(Gold Mohour Mouse chow, Hindustan Lever) and water adlibitum.

For magnetic field c!xposure, each animal was kept in a plastic

box of size 16 x 9 x 8 cm, with circular holes (6mm diameter) drilled at

equidistance to keep the box aerated. The box size was kept small so

as to keep the animal in the center of the pole pieces of the magnet

where the horizontal field was created. The animal was also restricted

to move inside the box. The experimental systems were positioned in

the north south direction of the Earth's magnetic field (the earth's

poles were determined b r magnetic compass). The animals were

exposed to magnetic field in the box with its head to the South and tail

to the North in order to avoid stress due to Geomagnetic North

orientation as reported by Rajeswary K R et al.' Each animal after the

exposure to magnetic field every day was transferred to the normal

cage with free supply of normal food and water. No food and water

was given during the expo:>ure period. The temperature inside the

experimental room was rnairtained around 25 5 5'C. Light conditions

were natural light plus fluor~,scent light during the day and complete

darkness during the night. A similar setup was created for the controls

but with out magnetic field.

6.1 Grouping of Animals; for Experimental Study

Rats weighing 125 :b 12.5 were divided in to five groups

consisting of 6 rats in each group. The experimental groups were

exposed to a magnetic field of 3000Gauss, 4hrslday for 45 days. The

same set up was created for c:ontrols but without magnetic field.

Group I- Normal control-maintained with normal laboratory feed and water adlibit dm.

Group-ll- Disease control--(Atherogenic diet)-Normal diet containing 49 Cholesterol, 5110rng sodium tourocholate and ground nut oil 1 1009 diet for 45 days.

Group-Ill- Atherogenic diet + simultaneous magnetic field exposure for 45 days.

Group-IV- Atherogenic rats exposed to magnetic field for 45 days

(Atherogenic diet for 45 days and magnetic field exposure for ano:her 45 days)

Group-V- Normal rats exposed to magnetic field for 45 days.

The average diet intake was found to be 12.5 5 2 gldaylkg body

weight. The body weight of 211 rats was noted before and after the

experiment. The experiment was completed between 45-90 days.

6.2 Preparation of Serum and Tissue Samples for the Biochemical Studies

After the experimental period, the rats were fasted overnight

and sacrificed by cervical jislocation. Blood was collected and tissues

were transferred to ice coltj containers and serum was separated from

blood cells by centrifugati,~n at about 2000 rpm for 30 min. Tissues

like Liver, Heart and Kidney were removed from rat's body, wiped

thoroughly using blotting paper to remove blood and other body fluids.

Then they were washed in normal saline, again wiped and desired

amounts of the dried tissu,?s were used for biochemical analysis. The

various tissue biochemic2l analysis include estimation of Lipids,

Lipoproteins, Antioxidants, activity of Antioxidant enzymes, activity of

Detoxifying enzymes, Lipicl peroxidation products, activity of clinically

important enzymes, enzynies of cholesterol metabolism, proteins and

calcium. Estimation of haemoglobin and RBC count in blood.

Biochemical analysis dore in serum include activity of clinically

important enzymes, lipid peroxidation products, Serum lron and lron

binding capacity.

6.3 Methods Used for the Biochemical Analysis

6.3.A Extraction of Serum and Tissue for Lipids

Both extractions wer,? done on the basis of method of Folch et at.'

Reagents

Alcohol - Ether mixture (3:l)

Chloroform - methaiol mixture (CHCI3 - CH30H) (2:l)

Procedure

About 1 g of tissue was ground in a mortar and pestle with 2 ml

alcohol ether mixture. The ?xtracts were transferred to a test tube and

made it up to 5 rnl. It was then placed at 60-70% in water bath for

2 hrs. Contents were occa!;ionally agitated using a glass rod. The

supernatant was then transferred to 25 ml standard flask through

folded filter paper fitted in a 'unnel. The residue was collected and the

process repeated three tirnes with alcohol-ether. Finally Small

amounts of CHC13-CH,OH was added to the residue, mixed and kept at

6 0 ' ~ for one hour. Repeated once more with CHCI, - CH30H. Pooled

the filtrate and made up to 25 ml with either of the solvents. This

aliquots were used for lipid e!;timations.

6.3.8 Extraction of Serum ;'or Lipids

Reagents

Methanol (CH,OH)

. Chloroform (CHCI,)

Potassium chloride (KZL)

Procedure

2 ml serum sample was added drop by drop to 5 ml of

methanol taken in a stoppered tube. Then 10 rnl of chloroform was

added, so that the proportioi of CHCI, to methanol was 2: l (vlv).

After filtration the filtrate was collected and the residue was washed

with CHCI,: methanol (2: 1) 3 times. The filtrate of the washing were

also collected.The combined filtrate was treated with 0.7% aqueous

KC1 solution (20% of the total volume of the extract). The organic

layer was collected and wa;hed many times with 5 ml CHCI, :

methanol:KC1(3:48:47 V N ) solution. Each time the organic layer was

collected. The collected lipid containing solution is evaporated to

dryness and the residue was r~?dissolved in a known volume of CHCI,.

Aliquots of this solution were used for the estimation of lipids.

6.3.C Estimation of Lipitis

6.3.C (1) Estimation of Ct~olesterol

Cholesterol was est mated by the method of ~ a k . ~

Reagents

Ferric chloride - Acetic acid (FeCI, - CH,COOH) Reagent (stock)

dissolved 1 gm. FeCI, fiH,O in 100 rnl acetic acid. Used analytical

grade glacial acetic acid (aldehyde free).

FeC13-CH3COOH reagmt (working): Diluted 5 rnl stock to 100 rnl

with acetic acid.

Cholesterol (std) (1 mg,'ml): Dissolved 100 mg cholesterol in acetic

acid and made up to 100 ml in a standard flask with acetic acid

and stored in the cold.

Cholesterol (working) (0.04 rnglrnl): Diluted 4 ml to 100 rnl with

working FeC1,- CH,COOH reagent and stored in the cold.

. Concentrated Sulphuric acid (Conc H, SO,): AR

Procedure

To 0.1 ml of serum/extract was added 4.9 ml of working FeCI,

-CH,COOH reagent. Mixed well and allowed to stand for 15' and then

centrifuged. From this 2.5 rnl was taken. 2.5 ml of working standard

was taken as standard and 2.5 ml of FeCI,- CH,COOH was taken as

blank. 1.5 ml of con. H2S0, was added to all the three tubes. The

58

contents of each tube were rriixed well and allowed to stand for 30'. The

reddish purple colour developed was measured at 560 nrn.

6.3.C(2) Estimation of Total Lipids

Total lipids were estiriated by the method of Frings.'

Reagents

Con. H2S04 -AR

Vanillin - 0.6%

Phosphovanillin reagent - 250 rnl of 0.6 % vanillin in 800 ml

concentrated orthoph~lsphoric acid

Stock standard - 1 g0,; olive oil in ethanol

Working standard - 400 rng% in ethanol

Procedure

Three test tubes were labelled as test, blank and standard. 0.1 ml

serum1 extract, 0.1 ml water ~ n d 0.1 rnl working standard were taken

respectively in the three tubes. Added 2 rnl con. H,SO, to each tube.

Heated in a boiling water bath for 10'. Cooled, pepetted out 0.1 ml of

digested mixture from each tulle and transferred them to another 3 test

tubes. Added 0.1 ml con. t,SO, and 5 rnl phosphovanillin reagent.

Incubated for 15' at 37 '~ . Optical density was read at 540 nm.

6.3.C(3) Estimation of Phospholipids

Phospholipids were estimated by the method of Connerty

Reagent

Trichloroacetic Acid (TCA) - 5%

Digestion mixture: 500 ml distilled water, 25 ml con. H,SO, and 25 rnl 70% Perchloric Acid (HC104)

Sodium acetate: 5C% trihydrate (wlv) in water

Ammonium molybdate; 2.5% solution in H20

Metol (P-methyl a~ninophenol sulphate) - l g in 100 ml 3% NaHS03

Stock standard (Img phosphorous/ml): 4.394 g anhydrous

H,PO, in a litre of :.elution containing 2 ml con. H2S0, (0.4394

gllOO ml)

Working standard: Ililute the standard 1 in 250.

Procedure

Pipetted 0.2 ml serllm/ extract into a test tube and added 5 ml

5% TCA drop by drop mhile shaking. Centrifuged to give tightly

packed precipitate. Deczinted the supernatant and kept the tube

inverted on a filter paper wiped around the rim. Added 1 ml of the

digestion mixture and heated gently until the liquid becomes

colourless or almost so (3C-40 min). Allowed to cool, added carefully

1 ml distilled water and boiled for 15' to convert pyrophosphate to

orthophosphate. Added 1 n l 50% sodium acetate and made to 10ml

with distilled water. Then added lm l ammonium molybdate and lml

metol. Mixed well, kept for 15' and read at 700 nm. Blank was

prepared by mixing 025ml conH2S04, lm l 50% sodium acetate, 1 ml

ammonium molybdate, 1 rr~l metol and 8.75 ml distilled water. As a

standard, took 5 ml working jtandard 0.25 rnl con. H2S0, 1 ml acetate,

1 ml rnolybdate, lml metol and 3.75 rnl distilled water mixed well and

read against reagent blank.

6.3.C(4) Estimation of Triglycerides

Triglycerides were e~ti~nated by the method of Van Handel "

Reagents

Chloroform AR

Florisil

Ethanolic potassium iydroxide (KOH): 2 g of KOH in 100 rnl

(KOH) ethanol. Dilute, 5 times with ethanol.

0.05M sodium metaperiodate

0.5 M sodium arsenitf

Chrorno tropic acid: Di:;solved 2 g of chrorno tropic acid (2.24 g of

sodium salt) in 200 rnl distilled water. Separately added 600 rnl of

con. H2S04 to 300 rnl c~f distilled water, cooled in ice. This chilled

and diluted acid was tt en added to chrorno tropic acid stored in

brown bottle.

Triglyceride (standard:: I 00 rngl 100 rnl distilled water

Procedure

29 of florisil were taken in a glass-stoppered tube and 3 ml

chloroform was added. Pn aliquot (0.2 ml serumlextract) was layered

on the top of florisil and mixed. CHCI, was then added to this to a

total of 10 ml. It was then stoppered and was shaken intermittently for

about 10 min. After filtrati~n, 1 ml was pipetted out into each of three

tubes. The solvent was evaporated at 60-70 OC. Then 0.5 ml of

ethanolic KOH was addea to each of the two tubes and 0.5 ml ethanol

was added to the third tut~e. The tubes were closed and kept at 660-

70% for 15 mln. To each tube, 0.5 ml 0.2 N H2S0, was added and

then placed in a gently bc,iling water bath for about 15 min to remove

alcohol. They were then cooled to room temperature; 0.1 ml sodium

metaperiodate was added to each tube and kept for 10'. 0.1 ml

sodium arsenite solution was added. A yellow colour of iodine

appeared and vanished mithin few minutes. To each tube, 5 ml

chromotropic acid was added and mixed. The tubes were closed and

heated in a boiling water bath for 30 min. They were then cooled and

absorbance was read at 5'0 nm.

6.3.D Lipoproteins

Lipoproteins were estimzted by the method of warnick.'

Reagents

Heparin solution: Diluted one vial of sodium heparin with NaCI(9 gll)

To get a final conceitration of 7500 USP units in each rnl

Manganese chloride(mnC1,): 2.02 molellitre -Dissolved 40.19 of MnC1,.4H20 in water to make to 100 ml.

Procedure

To lm l serum in a csntrifuge tube, added 0 1 heparin solution

and vortex mixed for 10'. Added 0.5 rnl MnCI, solution and remixed

similarly. Placed in an ice tlath for 30' and centrifuged at 4000 rpm.

The supernatant which con:ained HDL was analysed for cholesterol

by Zak's method.' The difference between total cholesterol and HDL

cholesterol gave LDL + VLDI. fraction.

6.3.E Ascorbic Acid and Cilutathione (GSHHAntioxidants)

6.3.€(1) Estimation of Ascc~rbic Acid

Ascorbic acid level in tissues was determined by using the

method of Roe.'

Reagents

Trichloro acetic acid (TCA) : 6%

Thiourea agent : 50% in alcohol

2,4, dinitrophenyl hydra.rine : 2% in 9NH,S04

Con. H,S04 : 85%

Ascorbic acid standard

Stock: 100mg ascorbic acid in 100 rnl of 6% TCA

Working standard: 1OOrrg ascorbic acid in I rnl 6% TCA.

Activated charcoal

63

Procedure

Immediately after sacrificing the animals, a weighed sample of

tissue was homogenized in 5 rnl ice cold 6% TCA in a pre-chilled

mortar. The extract was shaken well in a test tube, added activated

charcoal and allowed to stand for 15 min. The clear supernatant was

filtered through what man filter paper. To 4 ml of supernatant, added

a drop of Thiourea reagent (50% in alcohol) and 1 rnl of 2% 2.4

dinitrophenyl hydrazine in 9N H,SO, and incubated for 3h at 37' C in a

water bath. At the end o'the incubation, placed the test tubes in an

ice bath and added caref~lly 4 ml of 85% Con. H2S04. Kept for 30 min

in refrigerator. Centrifuged and OD was measured at 540nm in a

spectrophotorneter. The v: lues are expressed in mgI100g tissue.

6.3.E(2) Estimation of Glutathione (GSH)

Gluthione level in tissue was determined by using the method

of Patterson and Lazarrow.'

Reagents

Alloxan : O l M

Phosphate buffer : 0.5M (pH 7.5)

NaOH : 0.5N

NaOH : I N

GSH Standard : 3 Mg GSH in 5,25 ml metaphosphoric acid

Procedure

Weighed sample of tissue was homogenized in phosphate buffer.

The reaction mixture containiig 50 pI tissue extract. 50 pl alloxan, 50 pI

phosphate buffers and 50 p1 NaOH (05N) was incubated at 2 5 ' ~ for 6

minutes. The reaction was st2pped by the addition of 50 pl of I N NaOH.

Absorbance was noted at 305 nm in a quartz cuvette of l cm length path

in a spectrophotometer. A control tube was maintained with phosphate

buffer instead of extract. Tht! values are expressed in mgllOOg tissue.

6.3.F Superoxide Dismutase (SOD), Catalase, Glutathione Peroxidase (GSH- px) - (Antioxidant Enzymes)

6.3.F(1) Estimation of Supc?roxide Dismutase

Superoxide dismutes in tissues was determined using the

method of Kakkar et al."

Reagents

Sodium pyrophosphate buffer

Tris-HCL buffer

Sucrose

Phenazine methosupll~hate

Nitro blue tetrazolium

NADH

: 0.052 M (pH 8.3)

: 0.0025 M (pH 7.4)

: 0.25 M

: 186 micro M

:300 micro M

:780MM

Glacial acetic acid

65

Procedure

100 mg tissue wa:; homogenized in 2ml 0.25M sucrose and

differentially centrifuged to get the cystosoal fraction. This fraction

was then dialysed against 0.0025M Tris HCI buffer (pH 7.4) overnight

before using for enzyme a!;say. Assay mixture contained 1.2 ml of the

sodium pyrophosphate buffer, 0.1 ml of phenazine methosulphate, 0.3

ml nitroblue tetrazolium, 3.2 ml NADH and 1.2 ml of the enzyme

source. Reaction mixture was shaken vigorously with 4.0 ml of

n-butanol. The mixture was allowed to stand for 10 min. and was

centrifuged. The upper b~tanol layer was taken out. Colour intensity of

the chromogen in butanol was measured at 560 nm, against n-butanol

blank. A system devoid of enzyme sewed as control. Protein

estimation was carried out on the same enzyme source by the method

of Lowry et al." The values are expressed as 50% inhibition of

bitroblue tetrazoliumlminl~rg protein.

6.3.F(2) Estimation of Catalase

Catalase level in tissues was determined using the method of

Cohen et al."

Reagents

Phosphate buffer : 0.067 M (pH 7.0)

Hz02 : 30m1110 PI buffer

Procedure

100 mg of tissue was homogenized in 2 rnl phosphate buffer

and centrifuged. To 30 pl cf the supernatant, added 3 ml of buffer and

0.75 ml of H202 Change in OD was measured at 240 nni at 0 sec,

30 sec, 60 sec respectivel). The control system devoid of tissue

extract was used as blank. Values are expressed in micromoles of

H202 consumed lminlmg protein.

6.3.F(3) Estimation of Glutathione Peroxidase (GSH-Px)

Glutathione peroxidase was estimated by the method of

Rotruck.''

Reagents

Tris buffer : 0.4M, pH-7.0

Sodium azide solutior : 10mM

Trichloro acetic acid(7 CA) : 10%

Ethylene diamine tetraacetic acid (EDTA) :O 4 mM

Hydrogen peroxide (H20,) : 0.2 mM

Glutathione solution (GSH) : 2mM

Procedure

Weighed sample of tissue was homogenized in known volume

of Tris buffer. To 0.2 ml of Tliss buffer, 0.2 ml EDTA, 0.1 ml sodium

azide, 0.5 ml tissue homoger ate was added and mixed well. To this

mixture. 0.2 ml of GSH follohed by 0.1 ml H,02 solution were added.

The contents were mixed well and incubated at 37'C for 10 min along

with a control containing all reagents except tissue homogenate. After

10 min, the reaction was arrested by the addition of 0.5 ml of 10 %

TCA. Tubes were centrifuged and supernatant was assayed for GSH

by the method of Beutler and Kelley.

6.3.F(4) GSH Estimation l)y the Method of Beutler and ~ e l l e y ' ~

Reagents

Phosphate buffer

Ell man's Reagent : 19.8 mg DTNBllOO ml of 0.1% sodium citrate

Standard glutathion? solution : I 0 mg GSHllOO ml H20.

Procedure

1 ml of the supernzitant from the above test tubes was taken,

0.5 ml of Ellman's reagent and 3 rnl of phosphate buffer were added.

The yellow colour developed was read at 412 nm with a blank

containing 3.5 ml of phosphate buffer. A series of standards were

also treated similarly. The amount of GSH is expressed in rngllOO g

tissue.

6.3.G Glutathione-S-transferase (GST), Glutathione Reductase (GR)-( Detoxifying Enzymes )

6.3.G(1) Estimation of Glutathione-S-Transferase(GST)

Glutathione-S-transferase in tissue was determined using the

methods of Beutler et a1.14

Reagents

Phosphate buffer

CDNB

GSH

: 0.5 M (pH 6.5)

: 25 mM in 95 % ethanol

: 20 mM

Procedure

Tissues were homogenized in phosphate buffer. The reaction

mixture containing 200 pl ph.,sphate buffer, 20 p1 CDNB, and 730 ml

distilled water were taken in the control tubes and 200 pl phosphate buffer.

20 p1 CDNB and 680 p1 distilleti water were taken in the test sample tubes.

Then the tubes were incubatel at 37% for 10 min. After the incubation,

added 50 p1 of GSH in both the set of tubes. After mixing well, added 50 p1

of tissue extract in the test sariple tubes. Absorbance was noted at 340

nm for 5 minutes in a quartz cuvette of 1 cm length path in a

spectrophotometer. Values are expressed in n moles of CDNB complexedl

minlmg protein.

6.3.G(2) Estimation of Glutathione Reductase (GR)

Glutathione reductase was estimated by the method of David.

M. Goldberg.15

Reagents

Phosphate buffer : 0.12mol/l, pH-7.2

EDTA : 8.25mmolll

NADPH : 9.6 mmolll

GSSG : 65.3mmolll

Procedure

Weighed sample of tissue was homogenized in a known

volume of phosphate buffer. 2.6ml of buffer, Olml EDTA, and 0.lrnl

GSSG acts as reagent niixture to which was added 0.1 ml of the

homogenized sample and waited for 5 min. Then 0.05 ml of NADPH

was added, mixed thorous~hly and read at 340nm for 5 min. against

buffer blank.

6.3.H Lipid Peroxidatic~n Products-Malondialdehyde (MDA) and Conjugated Dienes(CD)

6.3.H(1) Estimation of Malondialdehyde (MDA)

Malondialdehyde bvas estimated by the method of Ohkawa."

Reagents

Sodium phosphate buffer : 50mM, pH 7.4

SDS : 8.1%

Glacial acetic acid : 20% pH3 adjusted with I N

NaOH

Thiobarbituric acid (TBA) : 0.8%

n-butanol and pyricline mixture : 15:l (vlv)

Procedure

Weighed sample of the tissue was homogenized in a known

volume of the buffer. 0.I8nl homogenate was taken and mixed with

0.2 ml SDS, 1.5 rnl g laci~~l acetic acid and 1.5 ml thiobarbituric acid.

The mixture was heated st 9 5 ' ~ for 1 hr on a water bath and cooled

70

under tap water. Then added 1 ml distilled water and 5 ml mixture of

n-butanol and pyridine. The mixture was shaken vigorously and

centrifuged at 2000 rpm fc~r 5 min. The upper organic layer was read

at 532 nm. The blank coitained all the reagents except the sample.

The result is expressed as moles of MDAImg protein.

6.3.H(2) Estimation of Conjugated dienes(CD)

Conjugated dienes were estimated by the method of Beuge J.A."

Reagents

Tris HCI buffer : 0.025 M. pH 7.5

Chloroform : Methanol : 2:1 (vlv)

Cyclohexane AR

Procedure

Weighed sample of tissue was homogenized in a known

volume of the buffer. An aliquot of the homogenate was shaken with

Chloroform: Methanol (2: l) and the lower layer was recovered. Then

evaporated this layer to dyness and re-dissolved in a known volume

of cyclohexane. Read the absorbance at 233 nm against cyclohexane

as blank. The amount of conjugated dienes is expressed in terms of

millimoles1100 g tissue.

.3.1 yGlutamyl Transpeptidase, (GGT), Lactate Dehydrogenasi? (LDH), Transamitlases [Aspartarte transaminase (AST) and Alanine Transalninase (ALT)], Phosphatases [Alkaline Phosphatase (ALP), Acid Phosphatase (ALP)] in Serum.

6 3 ( ) Estimation of ;-Glutamyl Transpeptidase (GGT)

GGT in serum was assayed by the method of S ~ a s z G ' ~

Reagents

Buffer (pH i .8) contained

Tris : 120 nmol

Mgcl, : 12 mmol

Glycyl Glyciie 90 mrnol

Substrate

Dissolved 1.28 g L- as y-glutarnyl 4-nitro anilide (anhydrous) in

0.15 molll HCI made up t~ 100 ml with acid. Constant stirring was

required to dissolve the SL bstrate. Stored at -20°C and it was stable

for several weeks (The reagent was stable for only a few days at room

temperature).

Procedure

100 PI serum and 1 rnl buffer were incubated at 3 7 ' ~ . Started

the reaction by adding 0.1 ml substrate, mixed and monitored the

reaction continuously at ,105 nm, so as to obtain the change in

absorbance per min. Valut?s are expressed in mglmin1100 rnl serum.

6.3.1(2) Estimation Serum Lactate Dehydrogenase

The serum lactate dehydrogenase was determined by the

method of Wrolewski eta\."

Reagents

. Phosphate buffer : 0.1 M (pH 7.4)

. Reduced NAD : 2.5 mglrnl of Phosphate Buffer

(Freshly prepared before use)

. Sodium pyruvate : 2.5 rnglml. Refrigerate

Procedure

Measured 2.7 ml of :he phosphate buffer in to a cuvette and

added 0.1 ml of serum and 1.1 rnl of reduced NAD. Allowed to stand

for 20 min at 37OC to reduc:e any keto acids already present in the

serum. Then added 0.1 rnl of sodium pyruvate. Read the extinction

for 5 min at intervals of 15-30 sec at 340 nm. Values are expressed in

IU 1100ml serum.

6.3.1(3) Estimation of S e r ~ m Transaminases

Serum levels of alanine and asparte transaminase were

determined by the method of Mohun and Cook.20

Reagents

Potassium phosphate buffer : 0.075 M (pH 7.5)

Buffered Substrates

73

Aspartate transaminiise: 300 mg, L-aspartic acid (Sigma) and

50 mg alpha Ketoglutaric acd (sigma) in 100 ml phosphate buffer and

the pH was adjusted to 7.5 with sodium hydroxide.

Alanine transaminasf:: 5 g DL- alanine (Sigma) and 2 mg alpha

Ketoglutaric acid (Sigma) n 100 ml phosphate and the pH was

adjusted to 7.5 with sodium hydroxide.

. Aniline-citrate reagent: Dissolved 50g citric acid (SRL) in 50 ml

distilled water and to this an equal volume of redistilled aniline

(Merck) was added.

. Dinitophenyl hydrazi~ie reagent: Dissolved 200 mg of 2,4,

dinitrophenyl hydrazir~e(Merk) in 85 ml of con.Hydrochloric acid

and made up to one litre with distilled water.

. Sodium hydroxide : 41'4

Procedure

Pipetted out 1 ml oi substrate solution into two tubes and

placed in a water bath at 37°C for few minutes. To one of the tubes

(test), added 0.2 ml of serum, shaken gently to mix. Exactly after one

hour, in the case of aspartate transaminase and half in the case of

alanine transaminase, with the tubes still in the bath, added two drops of

anilinecitrate reagent to both and added 0.2 ml of serum in the second

tube (control). Tubes were lef undistributed for 20m minutes. Then into

both the tubes, added lml of dinitrophenyl hydrazine reagent. Left the

tubes for another 20 minutes i i room temperature and then added 10ml

of 0.4 N sodium hydroxide ant1 read at 530nm after 10min a solution of

74

sodium pyruvate contair~ing 10mg pyruvate per ml was used as a

standard. Values are ex~ressed in IU/100 ml serum.

6.3.1(4) Estimation of !serum Phosphatases

Serum phosphatses were assayed following the method of

King and ~agatheesan'' using 4-amino antipyrene.

6.3.1.4(1) Alkaline phosphates

Reagents

. Disodium phenyl phosphate (Merck) : 0.01M

. Sodium carbonat'? and bicarbonate Buffer : (0.1 M) 3.18 g

anhydrous sodiurn carbonate and 1.68 g sodium bicarbonate

were dissolved in 500 ml distilled water.

. Buffered substrat- for use: Prepared by mixing equal volumes

of the above two .eagents

Sodium hydroxide :05N

Sodium bicarbonate : 0 5 M

4-amino antipyrer~e (sigma) ,0.6%

Potassium ferricy,mide :2.4%

Procedure

Measured 2ml of buffered substrate into each of the two test

tubes and was incubatelj at 37 OC for few minutes in a water bath.

Then 0.1 ml of serum was added to one of the test tubes (test) and

further incubated for 15rn1n At the end of incubation, removed the

tubes from the bath and aclded 0.8 ml of sodium hydroxide, 1.2 ml of

sodium hydroxide and 1.2 ml of sodium bicarbonate to both tubes.

Then, 0.1 ml of serum was added to the second tube (blank). To both

the tubes, added 1 ml amino antipyrene reagent and l m l potassium

ferricyanide solution. For 'he standard 1.1 ml of the buffer and lml of

phenol solution containing 0.01 mg of phenol and for the standard

blank 1.1 ml buffer and I rnl distilled water were taken, instead of

buffered substrate and serum. All other procedures were same as in

the case of test sample!;. The absorbance was read at 520 nm.

Values are expressed in King Amstrong units/100 ml serum.

6.3.1.4(2) Acid Phosphzhse

Reagents

Citric acid-sodiurr citrate buffer : 0. OlM(pH 4.9)

All other reagent$ were same as for alkaline phosphatase

Procedure

Procedure was same in the case of alkaline phosphatase

except that the citric acid-sodium citrate buffer was used for the

preparation of buffered substrate and incubation time in lhr. For

developing the colour w th aminoantipyrine, l m l sodium hydroxide and

Im l sodium bicarbonate were added. Values are expressed in King

Amstrong units/100ml serum

6.3.J. Hydroxymethyl Glutaryl Co.A ReductasHHMG Co.A Reductase), Glucose-Gphosphate Dehydrogenase(G4-P-D), Lactate Dehydrogenase(LDH), Lipoprotein Lipase and Triglyceride Lipase in Tissues

6.3.J.(1) Assay of HMG Co.A Reductase

HMG Co.A redud,ase was analysed by the method of Raa et al."

Reagents

Saline arsenate : l g of sod. arsenate I L saline

Dilute perchloric atid(Hc104) : 50 rnl HclO, made up to 1 litre

Hydroxylamine hydrochloride: 138.98 g / L

Hydroxylamine h!~drochloride : Equal volumes of hydroxylamine

reagent for mevelonate

hydrochloride and water were

mixed freshly before use.

Hydroxilamine hltdrochloride reagent for HMG Co.A : Equal

volumes of h,rdroxilamine hydrochloride reagent and

NaOH(180 gll) were mixed freshly before use .

Ferric chloride (FeC13).reagent : 5.2 g of TCA and 10 g FeCI,

in 100 m10.65 M Hydrochloric acid(HC1).

Procedure

Fresh 10% horrogenate of tissue in saline arsenate was

prepared and mixed witt equal volume of dil HCIO,. It was kept for 5'

and centrifuged at 200C rpm for 10'. To 1 ml filtrate 0.5 ml freshly

prepared hydroxylamine reagent (alkaline hydroxylamine reagent in

the case of HMG C0.A) was added. Then 1.5 ml of FeCI, reagent was

added. After shaking and keeping for 10 min, the readings were taken

at 540 nm against similarly treated saline arsenate blank. The ratio of

HMG Co.A to mavelonate was taken as an index of enzyme activity

which catalyses the conversion of HMG Co.A to mavelonate. The

lower the ratio the higher the enzyme activity.

6.3.J(2) Assay of Glucose-6- Phosphate Dehydrogenase(G-6-PD)

G-6-PD was Assayed by the Method of 6eutleP3

Reagents

Glucose-6-phosphatt?: 0.02M

NADP : 0.02mM

MgCi, : 0. lM

Tris HCI-EDTA : 1M Tris,5mM EDTA. H 8.0

Procedure

Arranged two Cuvettes and marked as 1 and 2. To both the

cuvettes added 100 pl Tris HCI-EDTA. 100 p1 Mgcl*, 100 111 NADP,

20 p1 sample and 680 p1 clistilled water. Incubated at 37% for 10'.

Added 100 pl 0.02 M Glu~;ose -6-Phosphate.Read at 340nm at 1'

intervals immediately after the addition of the substrates. The

difference in reading of cuvettes 1 and 2 gave the G-6-PD activity. A

unit of enzyme of defined as that amount which causes an initial

change in optical density of 1.00 per minute at room temperature.

Specific activity is expressei as units per milligram of proteh2'

78

6.3.J(3) Lactate Dehydrogenase (LDH)

The lactate dehydrogenase was determined by the method of

Wrolewski eta]."

Refer 6.3.1(2)

6.3.J(4) Estimation o'f Lipoprotein Lipase and Triglyceride Lipases

Procedure

Estimation of lipoprotein lipase and triglyceride lipase enzyme

assay was carried out by the method of Krauss et a ~ . ' ~

Reagents

Ammonium chloride-ammonium hydroxide buffer : 0.2M pH 8.6

Protamine

Methods

The chilled tissues, free of blood was homogenized with 3

volumes of ice cold ammonium chloride-ammonium hydroxide buffer

(0.2M pH 8.6). The hc~mogenate was centrifuged at 0 OC at 20009 for

10 minutes and the supernatant was used as the enzyme sources.

Protamine inhioited activity is taken as the lipoprotein lipase

activity and the protarnine resistant activity as the triglyceride lipase.

The enzyme activity i:; expressed in terms of micromoles of glycerol

liberated per hour per gram protein.

Protein in the t?nzyme extract was determined by the method

of Lowry et al."

6.3.K Serum lron and l n ~ n Binding Capacity

6.3.K(1) Estimation of Serum lron

Serum lron was an.3lysed by Dipyridyl method a am say)^"

Reagents

2. 2' - dipyridyl - 0.1 % in 3% Acetic acid (VN)

Sodium sulphite - 01M

Chloroform

Standard ferrous Sulphate (FeS0,.7H20). 100pg ironlml

. Working standard - Diluted 3 ml of the stock solution to

100 ml(3 pg iron lml)

Procedure

Mixed equal volume of serum, 0.1 M sodium sulphite and dipyridyl

reagent in a glass stoppert:d tube which was centrifuged. Heated in a

boiling water for five minu:e. Cooled and added 1 ml of chloroform,

stoppered well and shook vigorously for thirty seconds. Removed the

stopper and centrifuged for five minutes at 3000 rpm. If the supernatant

fluid is not completely clear, repeated the shaking and centrifuging. Read

at 520 millimicrons or using a green filter. For the standard, did with the

working standard in the same? way.

6.3.K(2) Estimation of Serum lron Binding Capacity

lron binding capacity was analysed by Ramsay's Dipyridyl

Method (1 957.1 9 ~ 8 ) ' ~

80

Reagents

Ferric chloride solution - 5 pg iron per ml in 0.005N HCI

Magnesium carbonate "Light" for absorption.

Sodium sulphite-(0.2M) - 0.2M

2.2' dipyridyl - 0.2% in 3% acetic acid(v1v)

Chloroform.

Standard ferrous sulphate(FeS047H20)

Stock standard - FeSO, 100p ironlml

Working standard - Diluted 3ml of stock solution to

100ml (3pg ironlml)

Procedure

Added 4 rnl of the ferric chloride solution to 2ml serum. After

standing for five minutes add 430rng of magnesium carbonate (100 rng for

each ml of fenic chloride). Shook frequently and vigorously for thirty to

sixty minutes. Centrifuged and 4 ml of the supernatant fluid was used for

iron determination. Added lrnl each of the 0.2M sodium sulphite and 0.2%

dipyridyl and proceeded as dtscribed previously for determining serum

iron. The result gives the total lr>n binding capacity.

6.3.L. Estimation of Hemoglobin And Red Blood Cell Count (RBC Count)

6.3.L(1) Estimation of Herloglobin

Est~rnation of Hemoglob~n (Cyanmethaemoglobin method) "

81

Reagents

Drabkin's solution : Ferricyanide-Cyanide Reagent

Cyanmethaemoglobin : 60rngllOOrnl

Procedure

Added 0.02 rnl of blood lo 4 or 5 rnl of the Drabkin's reagent.

Allowed to stand at least 4 mi11 and read against blank at 540nm.

Read the standard in the same way. Haernoglobin is expressed as

grams of haemoglobin I100 ml blood.

6.3.L(2) Total RBC count

Total RBC counted by hemocytorneter neubauer counting

chamber

Requirements:

Hernocytorneter, RBC pipette, sterilized blade, microscope,

cover glass, cotton, spirit, RBC d luting fluid etc.

Procedure

The tail tip of rat was sterilized by rubbing with cotton soaked

in spirit. Cut the extreme tip of the tail with a surgical blade. The tip of

the tail was pressed and blood oozed out. The drop was wiped out

with the help of cotton. Then the blood was aspirated into the RBC

pipette exactly up to the 0.5 ml marks. Immediately RBC diluting fluid

is loaded up to the 101 mark. The pipette is rotated between the

thumb and forefinger. This gave a dilution of 1:200.

Counting chamber and cover glass were cleaned thoroughly.

Place of the cover glass 11 position over the ruled area using gentle

pressure. Mixed the suspznsion thoroughly by rotating the pipette for

about a minute holding il is horizontal position, and finally shaken

sidewise, expelled the fluic from the steam of the pipette, and filled the

chamber. Allowed two to three minutes for the red corpuscles to

settle.

Counted the numbttr of RBC'S in 80 small square (4 square at

the four corners and one a: the center of central area)

6.3.M Estimation of Proteins

Protein content of the serum and tissues were assayed by the

method of Lowry et al."

Reagents

Sodium hydroxide 5olution : 0.1 N

Sodium Carbonate solution : 2% in 0.1 N sodium Hydroxide

Copper Sulphate solution : 0.5%

Sodium potassium -artrate solution : 1%

Alkaline copper reagent : A mixture of 50ml solution

carbonate solution and 0.5 ml

of each of copper sulphate

solution and sodium potassium

tartrate solution

Folin's phenol reagent : 1 :I dilution with distilled water

Standard protein solution : 100mg% in 0.1 NaOH

Procedure

Pipetted out 0.2 rnl sf serum or extract to the test tube and

added 1.8 ml of sodium hydroxide solution and 5 ml of alkaline copper

reagent. Shaken well and kept the mixture for 10 rnin, 0.5 rnl of

Folin's phenol reagent was added and mixed well. The mixture was

kept for another 30minutes. The optical density was measured at

675nm, in a spectrophotometer. The system devoid of sample was

used as the blank

6.3.N. Estimation of Tissue Calcium

Tissue calcium were estimated by the method of Baginski et a ~ . ~ '

Reagents

Cresolphthalein comple:ton : Added l m l conc. HCI to 40mg CPC in 50ml beaker. Swirled until dissolved. Added few drops of water wash the solution in to a litre volumetric flask with 50ml dimethyl sulphoxide. Added 2.5 8-hydroxy quinoline and wash down the neck of the flask with a further 50ml dimethylsulphoxide. Mixed thoroughly until dissolved and add water to the mark.

Diethyl amine buffer solution: Dissolve 500rng potassium

cyanide in water, add 40ml

diethyl amine and dilute to a

litre with water.

Ethyl glycol bis(2-aminoethyCether)tetraaceticacid(EGTA) : 5glL

84

Stock standard calc um solution : 12.5 m mollL

Working calcium solution : 2.5 mol/L containing0.8m mol/L magnesium.

Stock standard Magnesium solution : 40 m mol/L

Procedure

Pipetted 20 pl tissue homogenate 20 p1 standard in to two

cuvettes. To each added lml cresolphthalein complexon mixture

(CPC) reagent and mixed To a further cuvette added lm l reagent for

blank. To each then added 1 ml diethyl amine reagent and mixed

thoroughly preferably with a plastic paddle stirrer. Read the test and

standard against the blan< at 575 nm. The values are expressed in

pglg tissue weight.

Estimations were done with UV spectrophotorneter (UV-16601-

Shimadzu).

Statistical Analyses

All statistical analpis was done using computer software

created by Russel E. F~red (Crop & Soil Sciences Department,

Michigan state University) named M-STAT (version 1.2). 3'

References

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3. Zak. 0, Am. J. Clin. patk, 27, 583, 1957.

4. Frings. C. S, Anedonmt. J. Clin. pathway, 53,89.1970.

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6. Van. H. E, and Zilversmit. D. 6, J. Lab. Clin. Med., 50,152,1957

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9. Patterson. J. Wand Lazarrow. A, Methods of Biochemical Analysis. Vol. 2, (Ed, Glick), Interscience, New York, pp. 259-278, 1955.

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12. Rotruck. J. J, Pope. A. L, Gantha. H. E and Swanson. A. B, Science, 179,588-590, 1973.

13. Beutler. E (ed), Red cell metabolism, Churchill Livingston pub. New York, pp. 51-53, 1986.

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15. Goldberg. D. M and Spooner. R. J, Berg Meyer, 2, 258-265

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Freed. R. E, Crop and Soil Science Department, Michigan University, Version 1.2.