CHAPTER II

ANALYTICAL PROCEDURES

1. Extraction of serum and tissues for lipid estimation

a) Extraction of serum

The serum was extracted according to the procedure

of Folch et al. 128 .--Inl ml of the serum sample was added dropwise to S

Inl volume of methanol in a stoppered tube. To this was

added S Inl volume of chloroform and mixed thoroughly. This

mixture was incubated for lS minutes at SSOC and after that

another S Inl volume of chloroform was added so that the

proportion of chloroform to methanol was 2:1(v/v). It was

filtered and the residue was washed with chloroform:

methanol (2:1) at least 3 times. The filtrates

combined.

were

To the filtrate, 0.7% KCl or 0.02% CaC1 2 (20% of

the total volume of the extract) was added in a stoppered

tube, mixed vigorously and allowed to stand for some time.

The aqueous upper phase was removed with a pasteur pipette

and the lower layer was washed three times, each time with

Sml of chloroform:methanol:KCl or CaC1 2 solution (2:48:47

33

v/v/v). The washed lower layer of chloroform was evaporated

to dryness and residue was redissolved in a known volume of

chloroform.

analysis.

From this, aliquots were used for lipid

b) Extraction of tissues for lipid estimation

The tissue was homogenised with washed, powdered

glass and extracted with chlorof9rm : methanol (2:1) and

processed as for serum. 0.25g of the tissue corresponds of

25 ml of the extract in the case of tissues other than

aorta. For aorta, the aortas from two rats (60mg) were.

pooled and the extract was made up to 10ml.

2. Estimation of cholesterol

Total cholesterol was estimated by the method of

Abell et al. 129 .

Reagents:- (a) 33% KOH. (b) Absolute ethanol. (c) Ethanolic

KOH - 6ml of 33% KOH in water was added to 94 ml of absolute

ethanol. (d) Petroleum ether (60-80oC). (e) Colour reagent ­

20 ml of acetic anhydride was cooled in ice. 1 ml of

concentrated sulfuric acid was added to this with shaking.

It was again cooled for 10 minutes and 10 ml of glacial

acetic acid was added

34

and allowed to attain room

temperature.

Procedure:- An aliquot from the lipid extract was pipetted

out into a glass stoppered centrifuge tube and evaporated to

dryness. 5m1 of ethano1ic KGH was added, stoppered and was

shaken well. It was then warmed in a water bath at 37-400 C,

for 55 minutes. After cooling to room temperature, 10 m1 of

petroleum ether (60-800C) was added and mixed. 5m1 of water

was added to this and shaken vigorously for one minute. It

was then centrifuged at low speed for 5 minutes. 4m1 of

petroleum ether layer was pipetted out into a test tube and

evaporated to dryness at 60 0 C. A standard was also treated

in the same manner. 6 ml of colour reagent was added to each

tube and kept at 250 C after thorough shaking. 6 m1 of

colour reagent was taken as blank. After 30-35 minutes,

the optical density was read at 620 nm.

3.Estimation of trig1ycerides

Handel

Trig1ycerides were estimated by the method of

d Z'l ,130 'h h d' f' .an 1 versm1t , W1t t e mo 1 1cat10n

Van

that

f10risi1 was used to remove phospholipids.

35

H2S04

0.5M

2.24g

600mlsalt) was dissolved in 200ml distilled water.

0.05M sodium metaperiodate (f) sodium arsenite

Chromotropic acid:- 2g of chromotropic acid (or

(e)

(g)

sodium

Reagents:- (a) Chloroform-AR. (b) Florisil (c) Ethanolic

KOH- 0.4%:- 2g of KOH was dissolved in 100ml of ethanol.

This was then diluted 5 times with ethanol. (d) 0.2N

of concentrated sulfuric acid was added slowly to 300 ml of

distilled water which was chilled in ice. This chilled and

diluted acid was then added to the chromotropic acid

solution (0.05 mg/ml).

Procedure:- 2g of florisil was taken in a glass stoppered

tube and 3ml of chloroform was added. An aliquot of the

extract was layered on top of florisil and mixed. More

chloroform was then added to this to a total volume of 10

mI. It was then stoppered, shaken intermittently for about

10 minutes and filtered through a filter paper. lml of the

filtrate was pipetted out into each of 3 tubes. The solvent

was evaporated at 60-70o C. Then 0.5 ml of ethanolic KOH was

added to two out of three tubes (saponified sample) and

0.5ml of ethanol was added to the 3rd (unsaponified sample).

0.5 ml of 0.2 N H2S04 was added to each tube and the tubes

were then placed in a gently boiling water bath for 15

minutes to remove alcohol. They were then cooled to room

36

temperature, 0.1 ml of sodium metaperiodate was added to

each tube and kept for 10 minutes. 0.1 ml of sodium arsenite

solution was then added. An yellow colour or iodine appeared

and vanished within few minutes. 5 ml of chromotropic acid

was added to each tube and mixed. The tubes were closed and

then heated in boiling water bath for 30 minutes. They were

then cooled and the absorbance was read at 570 nm.

4. Estimation of phospholipids

Phospholipids were estimated by the method of

zilversmit and Davis13l .

Reagents: (a) 5.0N H2S04 . (b) 2.5% ammonium molybdate. (c)

ANSA - 0.2g of l-amino-2-naphthol-4-sulfonic acid was mixed

with 1.2g of sodium bisulfite and 1.2g of sodium sulfite.

0.25g of this mixture was dissolved in 10 ml of water and

was used immediately.

Procedure:- An aliquot of the extract was pipetted out into

a Kjeldahl flask and evaporated to dryness. 1 ml of 5N H2S0 4

was added and digested in a digestion rack till it became

light brown. It was then cooled to room temperature. One or

two drops of 2N HN03 was added and it was digested till it

37

became colourless. The Kjeldahl flask ~as cooled, 1 ml of

water was added and heated in boiling water bath for about 5

minutes. 1 ml of 2.5% ammonium molybdate andO.l ml of ANSA

were added to this. The volume was then made upto 10 ml with

distilled water and the absorbance was measured at 660 nm

within 10 minutes.

5. Estimation of free fatty acids

Non esterified fatty acids were estimated by the

d . f' 132 I hmetho uSlng copper soap ormatl0n . n t e presence of

phosphate buffer the extract is shaken with a high density

copper reagent pH B.l. The copper soap remain in the upper

organic layer, the copper content determined colori-

metrically with diphenyl carbazide.

Reagents:- (a) Extraction solvent - chloroform: heptane

Methanol 5:5:1. (b) Phosphate buffer pH 6.4 33 roM/litre. Mix

2 volumes KH 2Po 4 (4.539g/litre) and 1 volume Na 2HP0 4

(5.93B9/litre) (c) Stock copper solution-500roM/litre.-12.07g

Cu(N0 3 )2· 3H 20

lM/litre-lOml

made upto 100

triethanolamine

mI. (d)

in 100 ml

Triethanolamine

water. (e) NaOH

1 M/litre. (f) Copper reagent-Mix 10 ml of copper solution,

10 ml triethanolamine and 6 ml NaOH and dilute to 100 mI.

38

add 33g NaCl and adjust pH to 8.1. (g) 1.5 diphenyl

carbazole solution - 4g/1itre. (h) Palmitic acid standard.

Procedure:- Take 0.1 ml lipid extract in a stoppered tube

and evaporate to dryness. Add 1 ml phosphate buffer standard

is also treated in the same manner. Shake vigorously for 90

sec and let stand for 15 min. Centrifuge at 4000 rpm for 5

min and transfer 3 m1 of the upper layer to a tube

containing 0.5 ml diphenylcarbazide solution and mix

carefully. Read after 15 min at 550 nm. Blank is 1 m1

phosphate buffer.

6. Extraction of hepatic bile acids

Bile acids from the liver was extracted by the

procedure of Okishio et al. 133 . Homogenate of the tissue was

prepared in95% ethanol (v/v) containing 0.1% NH 40H (v/v,

specific gravity 0.88), refluxed for 30 minutes and

filtered. The residue was reextracted with the dame volume

of solvent and the filtrates from the two extracts combined.

The extract was concentrated in vacuum, made alkaline (pH

10) by the addition of NaOH. An equal volume of water was

added and extracted three times with petroleum ether (40­

60 oC) to remove neutral sterols. The alkaline aqueous

39

solution left after extraction of neutral sterols was

acidified to pH 1.0. The bile acids were extracted with

chloroform: methanol (1:1 v/v) three times. The chloroform

layer was washed with a little water and dried over

anhydrous Na 2S04 . After filtration and evaporation, the bile.

acids were dissolved in a known volume of chloroform and

aliquots were taken for the estimation of bile acids.

7. Assay of -hydroxy -methyl glutary1-CoA reductase (HMG-

CoA reductase, EC 1.1.1.34)

HMG-CoA reductase activity of the liver was

estimated as described by Rao and Ramakrishnan by

determining the ratio of HMG-CoA to Mevalonate134 .

Reagents:- (a) Saline arsenate - 19 of sodium arsenate/litre

of physiological saline. (b) Dilute perchloric acid-50

ml/litre. (c) Hydroxylamine hydrochloride reagent 138,

98g/litre .. (d) Hydroxylamine hydroxylamine hydrochloride

reagent and water were mixed freshly before use. (e)

Hydroxylamine hydrochloride reagent for HMG-CoA Equal

use.

volumes of hydroxylamine hydrochloride reagent and sodium

hydroxide solution (180g/litre) were mixed freshly before

(f) Ferric chloride reagent: 5.2g of trichloroacetic

40

acid (TCA) 10 9 of ferric chloride were dissolved in 50 ml

of 0.65N hydrochloric acid and diluted to 100 ml with the

latter.

Equal volumes of fresh 10% tissue homogenate and

dilute perchloric acid were mixed, kept for 5 minutes and

centrifuged at 2000rpm for 10 min. To 1 ml supernatant 0.5

ml of freshly prepared hydroxylamine reagent (alkaline

hydroxylamine reagent in the case of HMG-CoA) was added,

mixed and after 5 min, 1.5 ml of ferric chloride was added.

After shaking well, readings were taken after 10 min. at 540

nm against similarly treated saline arsenate blank. The

ratio of HMG-CoA to mevalonate is taken as an index of

activity of the enzyme which catalyses the conversion of p-

hydroxy p-methyl glutaryl-CoA to mevalonate. The lower

the ratio the higher the enzyme activity.

8. Fractionation of serum lipoproteins

The major lipoprotein classes in the serum (VLDL,

L L H) f . db' 1 fl . 135D, DL were ractlonate y sequentla otatlon .

Solutions required: (a) Density 1.006g/ml : 11.4g of NaCI

and 0.1 9 EDTA-Na2 are added to a 100 ml volumetric flask

500 ml of water and 1 ml I N NaoH are added and the solids

41

are dissolved by mixing. The flask is filled to volume and 3

ml additional water are added. (b) Density 1.182g/ml

24.98g NaBr are added to 100 ml of the above density (1.006

g/ml) solution. (c) Density 1.478g/ml: 78.32g NaBr are

added to 100 ml of the above density (1.006g/ml) solution.

Procedure:- Aliquot of 4 ml of serum were measured into

0.5/2.5 cm (6ml) ultracentrifuge tubes. 2 ml of density

1.006 solution were layered over the surface. the tubes were

capped and centrifuged in a 40.3 rotor in the Beckman Model

L for 30 min. at 19,000 rpm without refrigeration. This was

spin I and fraction called chylomicrons and particles was

floated to the top of the tube. 4 ml of the bottom fraction

were transferred into clean tubes using a syringe and spinal

needle (18-19) gauge. These were again overlaid with 2 ml of

density 1.006 solution and were centrifuged 16 hrs at 40,000

rpm with a chamber temperature of 16-1So C. This was spin II

and the top I ml contained VLDL. The bottom 4 ml of spin II

were transferred to clean tubes. 2 ml of density 1.182

solutions were added, mixed and centrifuged 20 hrs as 40,000

rpm (16-1SoC). This was spin III and the top I ml contained

LDL fraction. The bottom 4 ml including the gelatinous

sediment of spin III were transferred to clean tubes with 2

ml of density 1.47S solution. The tubes were mixed and

42

centrifuged 40 hours under the foregoing conditions. This is

spin IV and its top fraction contained HDL.

Lipids were extracted from the top fractions of

Spins II, III and IV by Folch's

cholesterol129 was estimated.

128procedure and

9. In vivo incorporation of (1,2_14C) acetate into

lipids in the liver

The rats were deprived of food overnight for 16

hours were injected intraperitoneally with 0.5 ml solution

of (1,2_14

C) sodium acetate (5 uci/100 g body weight of rat)

at 09.00 hrs. After 3 hrs, the rats were killed by

decapitation. Liver was quickly removed to ice cold

container, then blotted and weighted.

The tissue was extracted with chloroform:methanol,

according to the procedure of Folch et al. 128 . Free

cholesterol, ester cholesterol, triglycerides and

phospholipids in the extract were separated by TLC over

silica gel (Silica gel G) using n-hexane : ether acetic

acid in the ratio of 80:20:1 (v/v/v) as solvent system. The

activity was counted in Packard's Priyas liquid

Scintillation Counter. The scintillant fluid was 6g 2,5-

diphenyl oxazole (pPO) and 0.2g 1,4-bis [2-(5-phenyl

43

oxazolyl)] benzene (POPOP) litre toluene.

10. Release of lipoproteins into the circulation

Release of lipoproteins into the circulation was

studied using triton WR 1339 by the procedure of Schurr et

al. 136

50 mg of triton WR 1339 per 100g body weight was

injected intraperitoneally in normal saline to overnight

fasted rats and 4 hours later, blood was collected in

heparinised tubes. control animals received the same volume

of normal saline instead of triton. Serum was separated and

total cholesterol estimated as described above. Percentage

increase in the cholesterol in the experimental animals

given triton as compared to saline treated control, is

measure of the release of lipoproteins into the circulation.

11. Lipoprotein lipase activity of heart and adipose tissue·

.(Ee 3.1.1.3)

Lipoprotein lipase activity of heart and adipose

tissue was estimated according to the procedure of Krauss et

al. 13?

Acetone dry power of the tissue was extracted with

44

0.025 M NH 40H-NH 4Cl buffer, pH 8.6 containing 1 unit of

heparin/ml and the extract was used as the enzyme source.

Protamin~ inhibited activity was taken as a measure of

lipoprotein lipase activity. The enzyme activity is

expressed as micromoles of glycerol liberated per hour per

gram protein.

12. Assay of plasma lecithin : cholesterol acyl transferase

(LCAT, EC 2.3.1.43)

Blood was collected in heparinised tubes

maintained at OOC and centrifuged immediately at oOC to

separate the plasma. An aliquot was immediately extracted

with acetone: ethanol (1:1) to extract the lipids. Another

aliquot of the plasma was incubated at 37 0 C for 3 hrs, at

the end of which it was extracted with acetone:ethanol.

Ester cholesterol and unesterified cholesterol were

separated using TLC as described before, eluted and

129estimated by Abell's method .

13. Estimation of different glycosaminoglycan (GAG)

fractions in the tissue

a. Preparation of dry defatted tissue

Defatting of the tissue (approximately 500 mg wet

4S

weight for other tissues and 60 mg in the case of aorta) was

carried out by successive extraction at 600 c with ethanol

ether (3:1, v/v) each for 2 hours. ~he defatted tissue was

then dried under vacuum to constant weight.

b. Papain digestion:

The procedure of Laurent as described by scott138

was used. A known amount of dry defatted tissue was digested

with Papain (crystalline papain, one third dry weight of the

tissue) in O.lM phosphate buffer (pH 6.S) containing O.OOSM

EDTA and O.OOSM cysteine hydrochloride for 48 hours at 6Soc.

Fresh papain was added every 16 hours.

c) Fractionation and estimation of glycosaminoglycans:-

The papain digest after deproteinising with TCA

(final concentration of TCA 10%) was dialysed till free of

trichloro acetic acid (TCA). Total GAG was precipitated from

the solution by the addition of 4-S volumes of 9S% ethanol

(v/v) containing 1-2% potassium acetate (w/v). After

centrifuging, the precipitate was dissolved in a known

volume of water. An aliquot of the solution was used to

determine total GAG by estimating uronic acid by the

d · f' d d f' d' 139mo 1 1e proce ure 0 B1tter an MU1r •

46

d " " f " "d139• Est1mat1on 0 uron1C aC1

Reagents:- (a) 0.025 M sodium tetraborate

(b) 0.12% carbazole in methanol.

(c) Glucuronolactone standard, 40 ug/ml

inSOConc.H2 4

Procedure- 5.0 ml of sodium tetraborate was taken in a test

tube and cooled to 40 C by keeping it in an ice bath. 1.0 ml

of the sample was layered over this and stirred thoroughly

with a glass rod. It was allowed to attain room temperature

and then heated in a boiling water bath for 10 minutes.

After cooling to room temperature, 0.2 ml of carbazole was

added and heated again for 15 minutes in a boiling water

bath. It was then cooled and optical density was read at

530 nm.

14. Extraction of glycoproteins (GP) from the tissues

a. Preparation of dry defatted Tissues

Acetone dry powder of the tissue was prepared by

keeping the minced tissue in acetone at OOC for 72 hr. The

acetone was changed every 24 hr. The tissue was then

extracted with ether: acetone (3:1, v/v) at 370 C for 1 hr.,

47

followed by ether for 1 hr. The defatted tissue was then

dried under vacuum to constant weight.

b. Papain digestion

The dry defatted tissue was digested with papain

(crystalline papain, one third dry weight of the tissue) for

72 hr at 65 0 C in 0.2M acetate buffer (pH 7.0) containing 2

mg cysteine hydrochloride/mI. Fresh papain was added every

24 hr. The digest was then cooled to room temperature and 4­

5 volumes of ethanol was added at aOc and kept at this

temperature for 24 hr. It was centrifuged and the

supernatant was evaporated to dryness in the cold in vacuum.

The residue was dissolved in a known volume of water and

aliquot used for the analysis of carbohydrate components.

The procedure used is similar to that described by Wagh et

al. 140 except that ethanol was used instead of TCA to

deproteinise the digest, since TCA keeps the tissue glycogen

and GAG in solution.

15. Estimation of carbohydrate components of glycoproteins

a) Estimation of total hexose:

Total hexose was estimated by phenol-sulfuric acid

141method .

48

b) Estimation of fucose:

Fucose was estimated by the method of Dische and

Shett1es142 •

c) Estimation of sialic acid

Sialic acid was estimated by the thiobarbituric

acid method of warren143 •

d) Estimation of total protein in the dry tissue:

Total protein was estimated in the dry defatted

tissue by microkjeldhal digestion followed by nessleri­

. 144sat10n •

16. Estimation of protein

Protein was estimated in all enzyme extracts,

after TeA (trichloroacetic acid) precipitation by the method

145of Lowry et ale •

17. Estimation of lipid peroxides

Malondialdehyde was estimated by the

thiobarbituric acid assay method of Nichans and

49

Samuelsson146 • The tissue homogenate was prepared in 0.1 M

Tris-HCI buffer, pH 7.5. 1 ml of the homogenate was combined

with 2 ml of TCA-TBA-HCI reagent (15% w/v TCA and 0.375% w/v

thiobarbituric acid (TEA) in 0.25N HCI) and mixed

thoroughly. The solution was heated for 15 min. in a boiling

water bath. After cooling, the flocculent precipitate was

removed by centrifugation at 1000g for 10 min. The

absorbance of the sample was read at 535 nm against a blank

that contained no tissue homogenate. The extinction

5 -1 -1coefficient of malondialdehyde is 1.56xlO M cm •

Hydroperoxides was estimated by the iodometric

method of Mair and Hall147 . 1 ml of tissue homogenate (in

O.lM tris-HCI, pH 7.5) was mixed thoroughly with 5 ml of

chloroform : methanol (2:1) followed by centrifugation at

1000g for 5 min. to separate the phases. 3 ml of the lower

chloroform layer was recovered using a syringe and placed in

test tube and dried in a 45°C water bath under a stream of

nitrogen. While still under a stream of nitrogen, 1 ml of

acetic acid-chloroform, 3:2 (this reagent was depleted of 02

by bubbling with nitrogen at 4o C, then sealed and allowed to

come to room temperature) followed by 0.05 ml of Kl (6 g of

KI in 5 ml water in cold, which has previously been bubbled

with nitrogen for 15 min. This solution was made just prior

to use and shielded from the light) were quickly added, and

50

the test tube was stoppered and mixed. The tubes were placed

in the dark at room temperature for exactly 5 min. followed

by the addition of 3 ml of cadmium acetate (0.5g in 100 ml

water) The solution was mixed and centrifuged at 1000g for

10 min. The absorbance of the upper phase was read at 353 nm

against a blank containing the complete assay mixture minus

the tissue homogenate. Hydroperoxides have

.. ff" f 1 73 10 4 -1 -1extlnctlon coe lClent 0 • x M cm .

a molar

Conjugated dienes was estimated according to the

method of Recknagel and Ghoshal1 48 . Membrane lipids were

extracted and taken to dryness as described for the

iodometric assay for hydroperoxides. The lipid residue was·

dissolved in 1.5 ml of cyclohexane, and the absorbance at

233 nm was determined against a cyclohexane blank. The

conjugated dienes have a molar extinction coefficient of

2.52xl0 4 M- l cm-l .

18. Assay of Superoxide Dismutase (SOD)

SOD activity was determined by the method

described by Kakkar et al. 149 (SOD, EC.l.l5.l.l). The

tissues were homogenised in 0.25m sucrose and 10,000 rpm

under cold conditions to get the cytosol fraction. Before

estimating the activity of SOD an initial purification was

51

done by precipitating the Protein from the supernatant with

90% ammonium Sulphate and after dialysis against 0.0025M

Tris-HCl buffer (pH 7.4) the supernatant was used as the

enzyme source.

The assay mixture contained 1.2ml of sodium

pyrophosphate buffer (0.052M, pH 8.3), O.lml l86uM phenazine

methosulphate, 0.3ml 300uM nitroblue tetrazolium 0.2ml 780uM

NAOH, approximately diluted enzyme preparation and water in

a total volume of 3 ml. Reaction was started by the addition

of NAOH. After incubation at 30 0 C for 90 seconds, the

reaction was stopped by the addition of lml glacial acetic

acid. The reaction mixture was stirred vigorously and shaken

with 4ml of n-butanol. The mixture was allowed to stand for

10 minutes, centrifuged and butanol layer was taken out.

Colour intensity of the chromogen in the butanol was

measured at 560nm against butanol. A system devoid of enzyme

served as control.

One unit of the enzyme activity is defined as the

enzyme concentration required to inhibit the 0.0 at 560nm of

chromogen production by 50% in one minute under the assay

condition and expressed as specific activity in milliunits/

mg protein.

52

19. Assay of catalase (EC 1.11.1.6)

Catalase was assayed by the method of Machly and

150chance . The estimation was done spectrophotometrically

following the decrease in absorbance at 230nm. The reaction

mixture contained O.OlM phosphate buffer (pH 7.0), 2mM H20 2

and the enzyme extract prepared by homogenising the tissue

in 10 m M phosphate buffer (pH 7.0) and centrifuging at

5000rpm. Specific activity is expressed in terms of units/mg.

protein. Unit is defined as the velocity constant per

second.

20. Estimation of glutathione

Glutathione was estimated by the method of

Patterson and Lazarow151 .

Reagents:- ( 1 ) Metaphosphoric acid-5% ( 2 ) Alloxan

solution O.lM (3) Equivalent NaOH solution A 0.5N

Solution of NaOH was prepared and standardised so that 10ml

of the solution was equivalent to a mixture of lOml of 5%

metaphosphoric acid plus 10ml of O.lM alloxan. ( 4 )

Phosphate Buffer 0.5M, pH 7.5 (5) NaOH IN (6)

Glutathione standard:- 25mg glutathione was dissolved in

53

sufficient 5% metaphosphoric acid and made upto 500ml.

The reaction mixture contained tissue extract

(50ul) O.lM alloxan, (50 ul), 0.5M Phosphate buffer pH 7.5

(50ul) equivalent NaOH (50ul). After 6 minutes 50ul of O.lN

NaOH was added to stop the reaction. The contents were

transferred to the microquartz and read at 305nm.

21. Ceruloplasmin oxidase in serum

method

Ceruloplasmin oxidase in serum was determined by

152of Richard, Heneny, Jacobs and Milton Segalove .

Ceruloplasmin is a copper oxidase which can be determined

photometrically by rate of oxidation of P-Phenylenediamine

(PPD) by serum is catalyzed by serum ceruloplasmin and by

copper and iron present in serum as well as in water and

reagents used in the test. To determine oxidation due to

ceruloplasmin alone, it is nec~ssary either to correct for

or inhibit catalytic oxidation due to metals.

Reagents:- (1) Acetate buffer, O.lM, pH 6.0. Add 10ml O.lM

acetic acid (0.57ml glacial acetic acid plus water to 100ml)

to 200ml O.lM Sodium acetate (1.36g CH 3COONa.3H20/100m1), pH

should be 5.95-6.00. (2) Sodium azide, 0.1% in acetate

buffer. pH must be 5.95-6.00. Store in refrigerator.

54

(3) p-phenylenediamine. 2HC1, 0.25% in acetate buffer.

Recrystallize commercial salt as follows. Dissolve in water,

add Darco charcoal, warm in water bath at GOoc with

occasional mixing and filter. Add acetone to the filtrate

until turbidity appears, refrigerate several hours, filter

off PDD.2HCl and dry the crystals in the dark in vacuum

desiccator over anhydrous CaC1 2 . Store in brown bottle. To

prepare 0.25% reagent, dissolve 12.5 mg in 3.0ml acetate

buffer and, using narrow range pH paper, adjust pH to 6.0 by

adding IN NaOH dropwise from 0.2 ml serologic pipette

(approximately O.lml required). Add acetate buffer to final

volume of 5ml. This reagent can be used to about 2 hours

after preparation if kept in the dark. Procedure with

Beckman model UV spectrophotometer, equipped with

othermospacers, compartment temp. Kept at 37 C. To 'blank'

cuvette (1 cm light path) add lml azide reagent, lml buffer

and lml of PPD. To 'test' cuvette add 2ml buffer and lml of

PPD. Place cuvettes in compartment and allow 5 minutes for

temperature equilibration. Add O.lml serum to each tube from

a TC pipette. Read absorbance of 'test' against 'blank' at

530 nm at exactly 10 min. and again at 40min. after addition

of serum. The unit of ceruloplasmin activity is arbitrarily

defined as increase of 0.001 in absorbance in 30 min. under

our conditions. Calculation: ceruloplasmin units = (A 530 at

55

40 min A 530 at 10min.)x 1000. Procedure with Klett filter

photometer. The 'test' and 'blank' are set up in double

quantities in matched test tUbes. Tubes are incubated in

water bath at 37 0 C and covered by heavy black cloth to

exclude light except when tubes are removed at 10 and 40

minutes for reading with the Klett 54 filter. An artificial

standard for pontacyl violet 6R (Du pont) is employed. A

solution containing 15.9 mg of dye/liter of 0.5% acetic acid

is equivalent to 400 units (units same as absorbance units

defined above).

Calculation

Ceruloplasmin units

Klett reading at 40 min ­Klett reading at 10 min.

= ----------------------------x 400Klett reading of dye standard

against water

22. Activity of lipogenic enzymes

(a) Glucose-6-phosphate dehydrogenase

(Glucose-6-phosphate : NADP+ Oxidoreductase (EC1.l.l.49»

The enzyme was assayed by the method of Konberg

and Horecker153 • The chilled tissue was homogenised with 3

volumes of 0.04 M glycyl glycine buffer pH 7.5. The

homogenate was centrifuged at 2000 x g at 0 0 for 10 minutes.

56

The supernatant was used as the enzyme source.

Reagents:- (1) O.02M Glucose-6-phosphate, (2) 1.5 x 10-3M

NADP+, (3) O.lM MgCI 2 , (4) 0.04M glycyl glycine buffer,

pH 7.5.

To 1.0 ml of the substrate in a quartz cell, O.lml

+of NADP , 0.25ml buffer and O.2ml MgCl 2 were added. To this

was added 0.05ml of enzyme and the absorbance was read

immediately at 340nm and at I minute intervals.

A unit of enzyme is defined as that amount which

causes an initial change of O.D of 1.00/minute under the

above conditions of assay.

(b) Malic enzyme

(L-malate : NADP+ oxidoreductase (ECl.l.l.40l)

The activity of enzyme was determined by the

method of Ochoal54 . The chilled tissue was homogenised with

3 volumes of 0.25M glycyl glycine buffer pH7.4 at OOC and

the supernatant obtained by centrifugation at 2000xg at OOC

for 10 minutes which was used as source of the enzyme.

Reagents:- (1) 0.25M glycyl glycine buffer-pH 7.4, (2) O.05M

+MnC1 2 , (3) 0.000675M NADP , (4) 0.03M L-Malate, pH 7.4.

57

Procedure:- The reaction mixture, in a quartz cell (d=lcm)

consisted of O.3ml buffer (75 micromoles) 0.06ml of MnC1 2 (3

micromoles), 0.2ml of NADP+ (0.135 micromoles), 0.05ml of·

L~malate (1.5 micromoles), enzyme and water, to a final

volume of 3.0ml. The assay carried out at room temperature

(23-250 C). The reaction was started by reading of the

optical densities against a blank containing all components

except NADP+, at intervals of 15 seconds, for 1 to 2

minutes.

One unit is defined as the amount of enzyme

causing an increase in optical density of 0.01 per minute

under the conditions of the experiment.

23. Histopathological studies

The histopathological studies carried as per the

following method155 . The aorta was dissected entire and

fixed in 10% formalin. The entire aorta was rolled up like a

watch-spring and retained in position with a pin drive

through the coil. The coiled aorta was fixed on the 'chuck'

of the cryostat and frozen in position with gelatin. After

freezing the coiled aorta, the pin was removed. Sections

were cut at 8u-10u in the cryostat and preserved in 10%

formalin.

58

Staining:- (1) Wash sections in distilled water, (2) stain

0.7% Sudan black in propyl alcohol 10 min, ( 3 )

Differentiate in 85% propyl alcohol (about 5 min), (4) Rinse

in distilled water and (5) Mount in glycerol-jelly.

The fresh mounted sections were studied with

special attention to intracellular and extracellular lipid

deposition and alteration in the elastic tissue and collagen

of the intima and media.

Statistical analysis

The data given in the Tables are the average of

the values from the number of animals used given in

respective table +SEM. Statistical

calculated using students It' test156 .

significance was