1 (76)

4. MATERIAL AND

METHODS

1 (77)

•

4.1 CLINICAL STUDIES

4.1.1 DEMOGRAPHIC STUDY :

All the patients with

mellitus a tt ending Shukla

Dec ember 1990 to March

demographic study. All the

hypertens i on and/or diabetes ­

General Hospital's OPD during

1993 were enrolled for the

patients were analysed to find

out the prevalence of hypertension in diabetes-mellitus I

occurrence of cardiac dysfunction among diabe tic and non­

diabetic hypertensive patients, and a 12 hours fas ting blood

samples were collected t o find out occur rence o f

hyperlipidaemia among diabetic and non- di abetic hypertens i ve

patients .

4 . 1 . 2 EFFECTIVENESS OF VARIOUS ANTIHYPERTENSIVES IN DIABETIC AND NON-DIABETIC HYPERTENSIVE PATIENTS :

4 . 1 . 2 . 1 Patient selection:

The study was a controlled open clinica l t r ial . It wa s

randomised, nonblind and based on a paral l e l g r oup design .

The protocol and proforma was approved by the local ethical

committee . The proforma and a pproval of the eth ical

c ommittee • 1S • g1ven • 1n Annexur e-1 and Anne xure-2 respectively .

From December 199 0 to May 1993, 48 7 patient s wi th either hypertension or diabetes visi ted the Shukla

hospital's , Bardoli. Out of 487 patients 198 patient s were selected for the study.

4.1.2 . 2 Inclusion criteria :

Patients of either sex with mild to modera t e

hypertens i on (defined as mean diastolic blood pressur e more

than 90 mm Hg but less than 115 mm Hg and systolic blood

pressure between 160 mm Hg and 190 mrn Hg ) were selected fo r

the study. Patients with non - inSUlin dependent diabetes f or

more than 2 years associated with or wi thout essen t ial

hypertension were also selected. Data was also collected for

67

1 (78)

a separate group of patiento who had severe hypertension but

unaware of it and did not receive any treatment. All the

patients belonged to the age group of 45 to 70 years . All

patients were within -15 to +25\ of the ideal body weight.

4.1.2.3 Exclusion criteria:

Patients were excluded from the study if their age ' .... as

above 70 years; had complicat e d hyperten s i o n o r re ce nt

myocardial infarction (1ess than 3 month s fr om the s tudy ) ,

bradycardia (heart rate le8S than 50 beat s /min), second or

third degree atrioventricular (AV) block , congestive hea rt

failure, recent cerebrovascular events (less than 3 months

previously), severe dyslipidaemia (familiar hyperlipidaemias

or total cholesterol more than 350 mg/dl), renal o r he pa t ic

failure, severe concomitant diseases, o r a hi sto ry of

hypersensitivity to any of the te s t drugs .

4. 1.2. 4 Treatment 8chedule :

The patients selected from hospita l' s OPO ' .... e re f ull y

explained about the procedures and writ ten in f ormed consent

(Annexure-3) wa s taken from them. Patien ts wh o met

eligibility criteria wer e admitted t o Shuk l a Ge n e r al

Hospital, Bardoli for one day, and underwen t a physical

examination and routine clinical examination f o r c hest x ­

ray, blood pressure, heart rate and ECG. Th e s el ected

patients received pl!3.cebo treatment for 2 - 3 weeks. Fo r the

follow up patients attended the OPO of Shukla Hospi tal .

Patients with diabetes-mellitus

usual diet and received the

5-20mg/day ) f or

were maintained on the ir

antidiabetic treatment

the control of diabetes. At (Glibenclamide

the end of the placebo

qualifying criteria,

washout period, if they Btill met

both essential hypertensive

the

and

diabetic hypertensive patients were randomised to receive

either Atenolol (50 mg/day), Nifedipine (10 mg/day),

Enalapril (5 mg/day), or Clonidine (50 meg/day) for 9 months

(Table:4.1) .

68

1 (79)

Table: 4. 1

GROUP OF PATIENTS WITH TREATMENT SCHEDUT"F:.

Sr. No.

1.

2.

3.

4.

Group

Non-insulin dependent diabetes mellitus (NIDDM)

UncQntrolled hypertensive pat1ents (UN -HTJ

Diabetic hypertensive patients (DM-HT)

Ess~ntial hypertensive patlents (EHl

Treatment

Glibenclamidc

-

1 . Enalapril 2. A,eno 01 3. N1fe<;lil?ine 4. Clon~dlne

1. Enala1ril 2. A,eno 0 1 3. Nlfe<;l pine 4. Clonldlne

/)onr:

5 - 20 1119/11 ,, '1

5 - 20 mq/d;y 50 - 100 Inrj ( j ,) y 10- 20 U1g /d ;y 50 - 100 W ; (J ddY

5-20 rn'J/d)y 50 - 10 0 1117 dd,/ 10 - 20 mg dlfl 50 - 1 0 0 HICg/tl I, i'

After four weeks of active treatment, pacicnuJ "J/ l(j/ P'

mean diastolic blood pressure was les s than 90 rnm 119 'l/I-(r'

instructed to continue taking the same dose. Pat i.cnLlJ ",h IH! "

diastolic blood pressure was more than 90 mm W J ""'r" instructed to increase their dose f o r two weckG. 1\.1 U.: ( I 'H ()

weeks, if diastolic blood pressure was still morc th.::..n 90 Hun

Hg, they were instructed again to increase t heir d OUD f o r

the remaining period of the study. The maximum in c rca n.; J I I

dose allowed was 20 mg/day for Enalapril, 100 mg/d<.ty 1 ( 1/

Atenolol, 20 mg/day for Nifedipine and 100 mc g/d ;. y I (H

Clonidine.

4.1.2.5 Data Collection:

During 9 months of the active treatment, p,lLiJ ' ll l lJ

visited regularly the hospital atleast at a n intcrva J 0 1 (J I l l'

month and the clinical examination along wi th t l10 auv{://p'

effects of drugs were recorded by the inves tigato r. /\L t ILl'

end of the placebo wash out period, and after 1, ), 6 .:iJld ')

months of treatment, supine systolic and diaotolic bl ood

pressure were measured by sphygmomanometer on the Obm<' d' III

and whenever possible, by the same nurse or phYSician. IJ(1d ll

rate was assessed by palpation over a period o [ 1 rnJ nUl.0.

Before the initiatIon of treatment, and at the e nd o f 3 ~In d

9-months treatment period, the fasting blood sample/J wo ru

69

1 (80)

~o l l e c ted for the estimation of total cholesterol,

t riglycerides, high density lipoproteins (HDL), blood

gllicose, urea alld creati lline.

4.1.2.6 Life-style moni t oring:

No specific dietary prescription was provided to avoid

any diet fluctuation. Patients were required to continue

their usual diet habits throughout the study . Patients · .... ere

asked no to make changes i n physical exercise or smoking

hab i ts during the course of study. Drug compliance wa s

assessed by pill counts.

4.1.3 STATISTICAL ANALYSIS:

The results were analysed by t wo way analysis o f • f o llowed by Tucky's Var1ance test to find out the

Significant difference among the treatment. The val ue o f

p r obab ili ty less then 5\ ( P < 0.05 ) was considered as

statis tically Significant.

4.2 ANIMAL STUDIES

4 . 2.1 EFFECTS OF CH\<ONIC TREATMENT WITH CLONIDINE DIABETIC AND /OR HYPERTENSIVE RATS :

IN

4 . 2.1.1. Induction of Diabetes :

Healthy female albino rats of Wistar strain, we ighing 180-250gm were used in the experiment.

by a single tail vein injection of

(4Smg ,' kg) dissolved in O.lM citrate

Diabetes was induced

streptozot oc in (STZ )

buffer (pH 4. 5 ) . The

dIabetes ","'as confirmed by measuring urine glucose with the

help o f Di astixR U-1iles India ) after 48 hours o f STZ

In)ectl on. 5\ glucose solution was given 2 days befor e and 3

days after the STZ injection to prevent initial hypogl ycemic effect o f STZ.

•. 2.1.2 Induction of Hypertension :

Hypertension • 1n rats was induced by subcutaneous administration of deoxycorticosterone acetate {DOCA l in th e

dose of 5 mg / kg / day throughout the study period. Animals

receiving DOCA 'Here fed ' .... ith 2\ salt solution as drinking

.... ater . Anunals sho .... ed development of hypertension after 10

70

1 (81)

days of OOCA inject ion.

4.2.1.3 Treatment Protoco l :

The animals exhibiting glucosuria , 48 hours after the

'"-'i thdrawal of glucose feed ing i.e. 5 days after STZ

injection were considered a s diabetic . Control (Non·

diabetic) animals received only cit r ate buffer. Three days

after the administration of buffer or STZ, the animals ',..,ere

randomly divided in to two sub-groups

non-hypertensive.

• • hypertensi ve and

Clonid ine '"-'as administered daily (P.O.) in the dose of

25 meg / kg . The details of the groups of animals are given in

the Table, 4.2.

I.

II .

TABLE , 4.2

Name o f the Group Description

Cont r ol

Control treated

Non·diabetic , Non - hypertensive, Untreated

Non-diabetic, Non-hypertens ive, Clonidine treated

III. Diabetic control Diabetic~ Non -hypertens ive, Untreatea

IV. Diabetic treated

v. Hypertens ive control

VI. Hypertensive treated

Diabetic, Non-hypertensive , Clonidine treatea

Non-diabetic, Hypert ens ive, Untreated

Non-diabetic, Hypertensive , Clonidine treatea

Diabetic hypertensive Diabetic~ Hypertensive, con trol Untreatea

VII.

VIII . Dlabetic hypertensive Diabetic, Hypertensive , Clonidine treated

Du ring the SlX '",eeks study period, the f ollowing

phys i ol ogical parameters were recorded in all the groups of

anlmals

1 . Body .... 'eight

2, Water intake

3. Blood pressure

" . Pood lnt"ko

5 . Moreali t y

6. Heart rate.

71

1 (82)

•

All the ra ts were housed under identical conditions and

the standard diet and water was provided adlibi tum

throughout the study period. The

provided to animals during

Wh~at powder 1coarse! Malze powder coarse Grain powder coarse Vegetable oil Milk powder Iodized salt

the study

60% 20% 10%

5% 2 . 5% 2 . 5%

composition of

was as f ollows :

4.2.1.4. Measurement of Blood Pressure and Heart Rate:

diet

The blood pressure and heart-rate we r e recorded using

HARWARD BLOOD PRESSURE MONITOR (TAIL CUFF METHOD ) attached

with STUDENT'S OSC ILLOGRAPH . The tail was introduced into

cuff and initial gain s e t was es t abl ished by the use of

pulse sensor in order t o get minor de flection . The pressure

was ini t ially raised upto 200 mmHg a nd then it was s l owly

released by a screw attached in t he bulb . During this

decline in pressure, the point a t whi ch the magnitude of the

deflec t i on of pulse analyser i s increased was considered as systol i c blood pressure of rat. Heart rate was also r ecorded

by pul se analyser at particul ar chart speed .

Bl ood pressure and Heart r ate were measured befor e

t he start ing of clonidine therapy and at every lOth day upto

the completion of treatment .

4.2.1.5 Collection of Blood Samples and Their Ana lysis :

At the end of treatment with cl on idine, the blood

samples were collected from all the groups of animals from

retino-orbital plexus of the eye . About 4 -5 ml of blood

samples were collected in centrifuge t ubes and all owed to

clot for 30 min. at room temperature . Serum was separated by

centrifuging the tubes at 3000 rpm f or 30 mi nutes .

Supernatant. clear serum was separated and t r ansf e r red to

Ependorf tubes. The serum samples were s tored at -20 oC

unti l the analysis was done. Serum was ana l ys ed for

glucose, insul in, total choles terol, HD L - chol e s terol,

triglycerides, triiodothyronine (T3 ), thyroxin (T4 "

creatinine, blood urea nitrogen, alkaline phospha tase .

72

1 (83)

Glutamate oxaloacetate transaminase (GOT) and glutamate

pyruvate transaminase (GPT)

diagnostic assay kits.

using their respective

4.2.1.6 Recording of Cardiac Function :

After • sn weeks of the treatment animals were

sacrificed by stunning and bleeding to death. Heart was quickly diss ected out from the rat and placed in warm

aerated Chenoweth-Koelle buffer (37 oC) where extraneous

tissue was dissected free. The aortic stump was located and

tied to a 15 gauge stainless steel aortic perfusion canula.

Perfusion was initiated in the retrograde manner through the

aorta at 45 em H20 ( 3D mmHg ) aortic filling pressure. The

perfusion fluid was Chenoweth- Koelle buffer maintained at 37

± 10 C and bubbled wi th oxygen . A 16 gauge stainless steel

canula connected to atrial fi lling reservoir was then

inserted and tied to the pulmonary vein . Left ventr icular

developed pressure (LVDP) was measured by means of pressure

transduce r-polygraph system with a 3 em piece of

polyethylene tube attached to a 20 gauge needle. Th i s was

inserted through the apex of the heart into left ventricle .

Cardiac work was initiated by switching the perfusion system

from the retrograde mode to the working heart mode.

In . the working heart mode the perfusate entered the

left ventricle through the left atrium and was pumped out

through aortic pump '. Aortic outflow was subj ected to an

afterload of 45 cm polyethylene tubing. Left ventri cular pressure was recorded on polygraph.

The hearts were allowed to stabilise at 10 em H20

filling pressure for 10 mins . The cardiac function curve wa s

obtained by changing the height of the left atrial filling

reservoir from 2.5 to 20 em in 2 . 5 cm increment steps. At

each point, pressure development was allowed to stabilize

before it was rec orded. A complete functi on curve was

usually performed in about 30 min. The total time of

perfusion of each heart was approximately 45 min.

73

1 (84)

Compos i t i on o f Che noweth - Koell e Buffer wa s as f ollows .

Chemical n1l'I / Litre

Sodium chlo ride 119 . 80

Potassium chloride

Ca lcium chl oride

""agnesium chloride

Sodium bicarbonate

Glucose

Di s odium EDTA

5 . 63

2 .88

4.50

3.80

5.00

0 .03

Disti lled wa t er sufficient t o make 1. 0 Li t re.

After getting the cardiac fun c tion curve, the hearts

were taken out , bl otted with filter paper t o r emove ex cess

of water a nd weight of t he heart was no t e d down t o ca lcu l ate

the index of hypertrophy as wet heart weight t o body we ight

r at io .

' .2 .1.7 Histological Study :

Hi stologi cal study of heart and liver wa s carr ied ou t

to observe the effects o f chroni c clonidine treatment on

degenerative changes induced by diabet es and hypertens ion on

these organs .

Fi xa t i on of the tissues ;

Le f t ventricle o f the heart and the live r were

d lsse c ted fr e e and kept in Bouin's fixative f o r 1B t o 24

hours . Tissues were then washed twice with dist i ll ed water

and kept in 70 \ alcohol . A pinch o f lithium ca rbonate wa s

added and t i ssues were kept in overnight. Lithium carbonate

relT'.Qved a l l e xcessive s t ain and tissues were a gain kept in

fresh 70\ a lcohol. After that tissues were transferred i n t o

90 \ alcohol and kept in it overnight. Next mo rning al l

t issues ""ere transferred into 100\ alcohol and kep t f o r 3

hrs. Then tissues ''''ere transferred t o xylene and ke pt till

they become transparent.

I~ ic rotomy ,

Tissues ',;,Iere fixed in melted paraffin in wooden blocks ,

so that sectioning can be performed. Several sec tions o f 5

74

1 (85)

urn thi c~less were taken from each tissue and sections with

un iform s hape and s ize were selected f or histol ogy. Selected

sections were fixed on t he clear glass s lides with the he l p

of egg a lbumi n.

Sta i ning : Tissues V.' ere st a i ned with Haemo t oxyl l in a nd Eosin

stains. The staining procedure was as f ollows:

Slides ------ - --> Xyl ene 20 min

--- -- - >

Distilled ,",'ater 2-s

jmin

<--- -- - -30\

al cohol 2 - 5 min

< - -- --

,­Haem9 t oxyllin

s taln . 2 -4 m1.n

70%

acidic ---> Water - ----- ->

2-4 dips

alcohol <--- ---- --Eosin s t ain . 2 -3 m1.n

<-- ---2- 4

1mi n

100 \ alcohol 2 -3 dips

50% alcohol 2- 5 min

-- - - - >

<- - - - -

90 \ a lcohol 1- min

v 70 \ al cohol

2-5 min

Disti lled Water ----- ---> wate r

2- 4 dips 2 jin Ammonia

70 % alcohol 2 -3 min

v <--- 50% alc c;>hol

2 - 3 m~n

90\ alcohc;> l 2-3 d 1.ps

- - - - - - - - > 100% alcohol 2- 4 dips

--- -- > Xylene 48 hrs.

Per fectly stained s lides were mount ed with Diphenyl Xylene

(DPX ) and obse rved unde r li ght mi c r oscope . All the

hi st olog i cal s ec tions were examined by an individual who ... 'a s

unaware about t he section group being examined .

4 . 2 . 2 EFFECTS OF NIFEDIPINE TREATMENT IN DIABETIC RATS:

Ol.abetes .... 'as induced by a s ingle tail vein i njec tion of STZ

(45 mg / kg ) in fe male albino rats o f wi star strain as

descr ibed ea rlier i n sec tion 4 .2.1.1. The ra ts we r e div i ded

1.nto f our gr oups : Control , Control treated with nifedipine ,

Dl.abe t ics and Diabe ti c s treated with nifedipine . Nifedipi ne

suspenslon i n 1\ carboxy methyl cellulose (CMC) wa s gIven

oral ly i n t he dose of 35 mg / kg / day f or a period o f s ix .,.'eeks

"'i c. h f ood and .. 'ater given adlibitium .

The bl ood were collected from retina-o rbi t al pl exuses

before STZ in j e ct i on and four times during 6 ",'eek :;; o f

75

1 (86)

nifedipi ne treatment, at an interval of 10 days. Bl ood

samples 'Io.'e re all owed to clot and after half an hour serum

'Io.'a5 separated by centrifuging the tube at 3000 rpm. Serum

was aspirated and stored at -20°C until the biochemi cal

analysis. Serum glucos e and i nsulin levels were measured

initially and at an interval of 10 days upto six weeks study

period. While cholesterol, t r iglyceride, t r i - iodothyronine

{T3 1, thyroxin (T 4, 1 and thyrotropin (TSH1 levels wer e

analysed from all the groups at the end of six weeks o f

treatment period. All biochemical parameters were measured

by using a standard diagnostic kits.

At the end of six weeks the mean blood pressure and

heart rate were recorded using blood pressure monitor (Tail

cuff method ) attached with a student oscillagraph. The

1ndex of hypertrophy as wet heart weight to body weight • ratio were obtained as discribed earlier

4.2.1 6.

1n

4.3 ESTIMATION OF BIOCHEMICAL CONSTITUENTS

4 .3 .1 EST IMATION OF GLUCOSE (GOD/POD method )

Principle

the

In the single reagent system, glucose oxidase

glucose to gluconic acid and hydrogen peroxide. The

section

converts • • peroxloe

1n the presence of horseradish peroxidase f orms a colored

complex of hyd r oxybenzoate and 4-aminophenazone. The

1ntenS1t)' of the color formed is proportional to the glucose

le\'e .

Procedure

The tubes · .. ·ere arranged and the serum and the reagents were addea as follows :

-------------------------- - ---------------- - ------ --- --------Blank (B) Standard (S) Test (T)

--------------------------------- -------------------- ------- -Serum Reagent 2: (Glucose standard 100 mg% ) Solut10n 1 Distilled Water

- ---

1. 5 ml 1. 5 ml

--0.02 ml

1. 5 ml 1.5 ml

0 . 02 ml

1. 5 ml 1.5 ml

------------------------------------------------------- -------

76

1 (87)

The content of tubes was incubated at 37°C for 15

minutes, and colour developed was measured at 510 nm against

distilled water.

Calculation

Serum Glucose in mg \ 0.0. (test) - 0.0. (blank) _ _ ______ _____ __ _____ x 100 - -----0 .0. (std.) - 0.0. (blank )

4 .3. 2 ESTIMATION OF INSULIN (Radioimmunoassay ) ,

Principle When serum containing insulin is added to a tube

containing a fixed amount of antibody and a fixed amount of

the radio - labeled insulin (I 12S -label l ed ) , insulin present • l.n serum and the radio labeled insulin competes f or t he

antibody. The amount of radiolabeled insulin bound t o the

antibody is inversely proportional to the amount of insulin in serum. A standard curve with known amounts of the test

substance can thus be constructed and the amount in t he

unknown samples can be calculated.

Procedure All reagents were allowed to reach room t empe rature

and mixed thoroughly before the use.

1. Tubes were arranged and labe l ed as total counts I non

specific binding (NSB) I standards I control s and

unknowns.

2. Insulin standards, controls and unknown (serum samples )

were added (100 ul) to the appropriate tubes while i n

NSB tubes 200 ul of the insulin standard (0 uIU/ ml ) wa s

added.

3 . 100 ul Insulin Antiserum was added to all tubes except

NSB and total count tubes.

4. 100 ul of insulin (I125) r eagent was added to each

tube.

S. All t .ubes were vortexed.

6. All tubes were i ncubated at 2°C-S OC for 16 hours.

7. 1 ml of precipitating reagent was added to all tubes

except total count tubes.

77

1 (88)

8. Again all tubes were vortexed and incubated at r oom

temperature for 10 to 15 minutes.

9 . All tubes except the total count tubes were centrifuged

for 15 to 20 minutes at 45 00 RPM.

10. All tubes except total count tubes were decanted by

simultaneous inversion with a sponge rack into a

radioactive waste receptacle. Tubes were allowed to

drain on absorbent material for 15-20 seconds and

blotted to remove droplets adhering to the rim before

returning them to the upright position.

11. All tubes were counted on a Garruna counter f o r one

minute.

Calculation

1. The blank count was substracted from all the other

counts t o give corrected counts.

2. %8 / 80 = Corrected count of sample or standard

- - - - - - - - - - - - - - - - - - - - - - - - - - - --------- )( Corrected count of zero standard

3. The standard curve was prepared as %8 / 80 on

and uU/ ml of insulin on the logarithmic scale

log graph paper.

4. The concentration of insulin • sample In was

the standard curve by extrapolation.

4 . 3.3 ESTIMATION OF TRI-IODOTHYRONINE (T3)

Principle

• •

100

the logit

of l og i t-

read from

It was estimated by RIA test system. The essential

reagents required f or a solid phase radioimmunoassay include

insoluble antibody, radio labeled antigen and negative

antigen. Upon mixing insolubilised antibody I radi olabeled

antigen and a serum containing the negative antigen , a

competition reaction results between the negative antigen

and the rad i o labeled antigen for a limited number of

insolubilised binding sites. The interaction is illustrated

by the following reaction.

125 I _Ag + Ag + Ab Ka AgAb + 125 I _AgAb ---->

<----K-a

78

1 (89)

Ab _ Monospecific insolubilised antibody (Constant quantity)

Ag = Negative antigen (Var iable quantity )

AgAb = Antigen -Antibody complex

Ag125 IAb = Radiolabeled antigen-antibody complex

Ka = Rate constant of association

K-a - Rate constant of dissociation

= Ka / K-a = Equilibrium constant

After equilibrium is attained, the antibody bound

fraction is separated from unbound antigen f ol lowing

centrifugation by aspirati on o f decantation.

simple

The

radioactivity in the antibody bound fraction is inversely

proportional to the negative antigen concentration.

Reagents (Supplied in the ki t)

Thyroxin human serum references :

0, 2.5, 5.0, 10.0 and 20.0 ul/dl

Non-specific reagent

Tracer reagent

Antibody reagent Human serum controls

Pr ocedur e

Before proceeding with the assay, all the reagents

brought to ambient temperature . Labeled test tubes f or each

serum references and test serum . 0.05 ml of the appropr iate

serum reference, cont'rol or specimen added into the assigned

tubes and 0.05 ml tracer reagent to all. Then properly mixed uniform slurry of antibody reagent di spensed 1.0 ml to each

tube . Vortex each tube for 2- 3 s econds, Then incubate if it R. T. for 30 minutes. Centrifuged all tubes at 3000 rpm for

10 minutes at 23 0 C ± 2oC. A clear separation of precipitate

and supernatant should be observed and if not, recentrifuge

at a higher gravitational force. Aspirated all tubes completE

counted for 30 seconds.

Calculations

t Bound -Sample count - NBB counts ----- ------- ------ ---- --- - x 100 Average total count

79

1 (90)

•

By utilizing several different serum references of

known a ntigen concentration, a standard cu r ve can be

generated from which the antigen concent ration of an unknown

can be ascerta i ned.

4. 3 .4 ESTIMATION OF SERUM THYROXIN (T4' :

Princ iple

It was estimated by solid phase RIA. The essent ial

requirement for a solid phase radioimmunoassay include

insoluble antibody, radiolabeled antigen and negat ive

antigen. It is same as described in T) assay.

Reagent s (Supplied in t he k i t )

Thyroxin human serum references :

0, 2 . 5, 5.0, 10 . 0 and 20 . 0 ul / dl

Non-specific reagent

Tracer reagent

Antibody reagent

Human serum controls

Procedure

Before proceeding with the as say I al l the reagents

brought to ambient temperature. Labeled test tubes f or each

serum references and test serum. 0 . 05 ml of the appropria te

serum reference, control or specimen added into the assigned

tubes and 0.05 ml tracer reagent t o all. Then proper l y mixed

uniform slurry of an.tibody reagent dispensed 1. 0 ml t o each

tube. Vortex each tube f or 2-3 seconds. Then incubate if it

R.T. for 30 minutes. Centrifuged all tubes at 3000 rpm f or

10 minutes at 23°C ± 2°C . A clear separation of precipita te

and supernatant should be observed and if not , recentrifuge

at a higher gravitational f orce. Aspirated all tubes

completely and counted for 30 seconds.

Calculations

Aver age total count % Bound -

Sample count - NSB counts -- - - - - - - -------------- - - - - - - - - - x 100

By utilizing severa l different serum references of

known ant i gen concentration, a standard curve can be

80

•

1 (91)

generated from which the antigen concentration of an unknown

can be ascertained .

4.3.5 ESTIMATION OF SERUM THYROTROPIN (TSH) :

Principle The assay is based on a sandwich technique using two

monoclonal antibodies. It is performed in two steps.

Immunological Step

+<J <)

+ )--c j-c

2 3

Enzymatic Step

Incubation a 2 hours at 18-25 C with shaking

• Washing

--.:~)-c+ 0

Incubation 30 min at 18- 2S oC Readinq at 492 nm

__ ~~~~~~~~ ___ .~ Ye~lo~-orange

4 Stopping reagent colorlng

1 . Monoc lonal anti-TSH coated t ubes

2 . TSH present in the sample, standards and control

3 . Enzymatic con j ugate : Kors eadi s h per ox ida se ­

labeled monoclonal anti-TSH

4. Chromogen substrate : Ortho Ph e ny lene Diami ne (O.P.D.) / H202

The TSH concentration of each sampl e is determined us ing a calibration curve .

Reagents supplied in. the kit :

TSH reagents

Rl Anti-TSH tubes

•

R2-O to R2-E TSH standard (between 0 and 40 mIU/ l ) R3 Control (Human serum)

R4 Anti-TSH conjugate

Color ErA reagents

Color 0 Wash solution

Color 1 Chromogen

Color 2 Color 1 diluent

Color 3 Stopping reagent (18N H2SO4)

81

1 (92)

Procedure It is recommended to perform the assay in duplicate.

The dispensing and incubation times should be identical for

all tubes in the same series . Once started, the test should be completed without

• • 1nterrupt1on.

Immunological Step :

Dispense Anti-TSH tubes (R1 ) tubes

Reagent: Blank

Stanoaros RrO to R2 -E 200 m1 - - -Control (R3 ) 200 m1 - -Samples - - 200 m1

Conjugate (R4 ) 20 0 m1 200 m1 200 m1 -

Incubate for 2 hours at 18-2SoC with continuous shCtkiJl9 at

350 rpm.

Aspirate the liquid from each tube

Rapi dly dispense working wash solution (2 ml of diluLed

color 0) in series of no more than 4 8 hours. The s olution

should not remain in the tubes for longel- than one nunut e.

Aspirate immediately

Repeat the washing procedure

Aspirate thoroughly all traces of solution

Enzymatic Step :

r war 1ng

Solution (Color 1 to Color 4 )

Incubate 30 min at 1 8-2 SoC in the dark,

Stopping . Reagent Color 3

1 m1 1 ml

82

1 ml

un 11\1

1 ml

1 (93)

•

Shake on a vortex or equivalent

Read at 492 nm s t andard R2-O, A, S , C, 0 , E and samples

against the reagent blank.

Notes : The abs orbance of the reagent blank shou l d be <=0.2

The absorbance of s t andard R2-D should be >=1 . 0

If reading i s delayed ( 2 hours maximum ) , place t he tube s

immediately in t he dark and store at r oom temperature.

4 . 3.6 ESTIMATI ON OF TOTAL CHOLESTEROL (Wybenga and Pil e ggi Method)

Principle

Cho leste r o l rea ct s wit h hot solution o f Fel I ic'

perchlorate, Ethyl ace t ate and Sulphuri c acid (Cho!psU'I( 1

r eagent ) and gives a lave nde r colored complex , ''''hich \!"

measured calor imetrical l y.

Proc edure

The tubes were arranged and the reagent s and >- hf~ ;-;r'r Hfl

samples were added as foll ows :

.

Blank Standard 1'$0" c · -~ l (B) (S) (T)

Reagent 1 : Cnol esterol Reagent 5 . 0 mt 5 . 0 ml 5.0 Reagent 2 : Working Choles terol -- 0 . 05 ml

(2 00 mg %) Serum -- - - O.os

ml

ml

Tube s were then mixed wel l and kept i mmediately in lh'·

bo iling water bath exac t ly f or 9 0 seconds . They were coolr.d

immediately to r oom temperat ure under running tap wal£~r

The opt i cal d e nsity (0.0. ) o f sta ndard (S ) and test (T) '''d S

measured agai nst blank (B) on a spec tropho t ometer at 560 nm .

Ca l culatioDs

0 .0 (t e s t) -- -- -- - -- -- --- X 200 O.D (std . )

Serum c holester ol in mg / 100 ml =

4.3. 7 ESTIMATION OF HDL - CHOLESTEROL :

PriDciple

Chylomi c r ons, VLDL (Very Low Density Lipoproteins) and

83

1 (94)

, LOL fractions in serum or pl asma are separated from HDL by

precipitating with phosphotungstic acid - magnesium chloride .

After centrifugation, the cholesterol in the HDL fraction,

which remains in the supernatant is assayed with enzymati c

cholesterol method, using cholesterol esterase, cholestero l

oxidase, peroxidase and the chromogen, 4 amin oph e n­

zone / Phenol.

General System Parameters

Reaction Type • Endpoint •

Wavelength • 505 nm (505-530 nm) •

Fl owcell Temp . • 300e •

Incubation • 30 Min. • R.T./5 Min. 370e

Sample Vol. • 200 ul •

Precipitating 200 ul reagent Vol. • •

Supernatant Vol. • 20 ul •

Reagent Vol. • 1. 0 ml •

Standard Conc. • 50 mg/dl •

Zero Setting With • Reagent Blank •

4 . 3 .8 ESTIMATION OF TRIGLYCERIDE:

Principle

Triglycerides incubated with lipoprot e in l ipas e ar.c

hydrolyzed to free fatty acids and glycerol. Glycerol ki nase

catalyzes the conversion of glycerol and ATP to glycerol - 3

phosphate and ADP. The glycerol - 3- phosphate gets ox idized •

to dihydroxy acetone phosphate by glycerol ph os pha te

oxidase. Hydrogen peroxide (H20 2 ) formed in t his reaction

with the help of peroxidase, reacts with c hromogens 4

aminoantipyrine/3, 5, dichloro - 2 hydroxybenzenesulfoni c

acid to give a red coloured complex which is read a t 510 nm

(500 - 530 nm) .

Triglycerides LP Lipase

""'" '" ,> Glycerol + Free Fatty Ac ids

Glycerol ATP Glycerol Kinase

" ' '' ' ' ' "" " ~ > Glycerol 3-P + ADP

Glycerol-3-P Gly - 3- P Oxidase

+ °2 , ' 0 , 0 0 0 " 7 , , 0 I ,,, » > DHAP +

84

1 (95)

r-------- .-----------------------POD

H20 2+4-aminoantipyrine + DHBS _w __ > Red colou red compOu n(j

General System Parameters

Reaction Type • Endpoint •

Wavelength • 505 nm 1500 - 530) •

Flowcell Temp. 300 e Incubation • 15 Mi n. R. 1'. •

Sample Vol. • 10 ul •

Reagent Vol. • 1.0 m1 •

Standard Conc . • 200 mg/d1 •

Zero Setting Wi t h • Reagent Blank . ·

4.3.9 ESTIMATION OF LDL-CHOLESTEROL ,

After determining s erum cholesterol, triglycer i d e: illH l

HDL chol esterol, serum LOL l evels ... ,ere found wilil Lll(' hI-J P

of Fiedman's f ormul a .

LOL - Tot a l Chol este r ol -Triglyceride

HOI. ---- -- --- - ---5

4.3.10 ESTIMATION OF CREATININE (Alkaline Picrate method)

Principle

Creatinine forms a coloured complex wi th picra ~c i ll d l kdJ IW'

medium. The rate of forma t i on of the comple x i s m('~ d G un·d.

Procedure

Wavelength Sl Onm 1490 - 510 nm)

Spectrophotometer • 49 0 nm •

Cuvette • 1 em l i ght path •

Temperature • 25°C. •

Blank • Air •

• (j:('i r cr \1)"1 • , reagent mixtUre 5 2.0 MI 2. 0 ml Re'agent 1 0.2 m1 sample - 0.2 011 Mix ana start st~watCI1 a the same t1nm. ATr0Y--:fO CQC, read absorbance, 1 of standard and sample rcspccLjv0ly, and exactly 2 mln. later, read abso rba nce A? ot l~ ldn (tlrd and sample A2 - Al = A sample or A standard

----------------------------.-

85

1 (96)

Calculation • •• • The concentrat1on (c) of creat1n1ne 1n serum or plasma .

A sample c = 2.0 X ' "I.''''' (mg/100 ml)

A sc.andard

4.3 . 11 ESTIMATION OF UREA (DAM method) ,

Principle

Urea reacts with not acidic Diacetylmonoxime • In

presence of Thiosemicarbazide and produces a rose -purpl e

co l or complex, which is measured calorimetrically.

Reagents (Supplied in the kit)

Reagent 1 : Urea reagents

2 : Diacetylmonoxime (DAM)

3 : Working urea standard 30 mgt

Preparation of working solution

solution 1 : Dilute 1 ml of reagent 1 to 5 ml with

distilled water reagent 2 and reagent 3 are ready

for use.

Procedure

Blank ( B) Test ( T) Standard (S)

Solution I 5.0 mI 5.0 mt 5.0 mI Sample - 0.02 ml -

Reagent 3 - 0.02 ml 0 . 02 ml

Mi x well

Reagent 2 0.5 ml 0.5 ml 0 .5 ml

Mix well and keep the tubes in a boiling wat er ba th

exactly for 10 minutes. Cool them immediately under running

water for 5 min ., mix by inversion and measure the col or

intensity within 10 mins us ing a green filter against blank. '

Calculation

Urea in mg/ 100 ml = O.D test

-- -- ------ - - x 30 O.D std.

86

1 (97)

•

4 . 3.12 ESTIMATION OF SERUM GLUTAMATE PYRUVATE TRANSAMINASE (SGPT) :

Principle and procedure

SGPT catalyses the following reaction.

Alpha keto glutarate + L-Alanine - - - > L-Gutamate + Pyruvate

Pyruvate so formed is coupled with 2,4-Dinitrophenyl

hydrazine (2, 4 - DNPH ) to give a corresponding hydrazone I

which gives brown colour in alkaline medium and this can be

measu r ed calorimet r ically. The standard curve with

differ ent concentrations of standard enzymetic activity is

created and from that curve enzymatic acti vi ty in serum

samples i s determined.

4 .3. 13 ESTIMATI ON OF SE RUM GLUTAMATE OXALOACETATE TRANSAMINASE (SGOT) :

Principle

GOT (AST) catalyzes the transfer of the amino

from L-aspartate to alpha ketoglutarate to

oxalocacetate and L - glutamate . Malate dehydrogenase

group

yield

(MDH) ,

then converts oxaloacetate and NADH to malate and NAD . The

conversion of NADH to NAD decreases the absorance at 340 nm,

the rate of which is proportional to the GOT (AST ) activity.

L-Asparatate + a -GOT (AST )

Ketoglutarate I", "" ,> Oxaloacetate + L - Glutamate

Oxaloacetate + + MDH + NADH + H "",> L - Malate + NAn

General System Parameters

Reaction Type

Wavelength

Flowcell Temp .

Delay Time

No. Of Readings

Interval

Sampl e Vol .

Reagent Vol.

Pa t hlength

Factor

Zero Setting With

• •

• •

• •

• •

• •

• •

• •

• •

• •

• •

Kinetic

340

60 Sec .

4

30 Sec .

100 ul

1.0 ml

1 em . 1749

Distilled Wate r.

87

1 (98)

•

4.3.14 ESTIMATION OF ALKALINE PHOSPHATASE:

Principle

Alkaline Phosphatase hydrolysis P-nitrophenyl phosphate

(PNPP) into P-nitrophenol and phosphate. At the alkaline PH

of the buffered medium, P-nitrophenol is yellow. The colour

developed by hydrolysis is measured at 405 nm and is

propor tional to the alkaline phosphatase activity.

P-nitrophenol phosPhate + H 0 (Colourless in acia/alkali)2

General System Parameters

Reaction Type

Wavelengt h

Flowcell Temp.

Delay Time

No. Of Readings

Interval

Sample Vol .

Reagent Vol .

Pathlength

Factor

Kinetic

: 405 nm

: 25 0C

: 60 Sec .

: 4

: 30 Sec.

: 30 ul

: 1.0 ml

: 1 Cm.

: 1826

ALKP - > r j , , P-nitrophe~ol + phosphate

(yellow In colour )

Zero Setting With ; Distilled Water.

88

-

1 (99)

No.

1.

2.

3 .

4 .

s .

6.

7.

8.

o ,.

10 .

11.

1:2.

13 .

14 .

Lis t o f Assay Kits

Name of Kit

• Glucos~ ( GOD I

~n serum POD Ne thod )

Insulin ( Radi o Immunoassay )

Tri -iodothy ronine (T3) ( RIA )

Thyr oxin (T 4) ( RIA )

Thyr ot r opin (TSH) ( ELISA 1

Cholesterol ( Wybenga and Pi leggi

Methoa )

HDL Cholesterol ( Phodphotungstate method )

Triglycerides (En zymatic )

Creatini ne

Blood Ur ea (DAM method )

Al kali ne Phosphatase

SGOT

SGPT

Glucos e in ur i ne ( Enzymat ic )

89

Sourc e

Span Diagnost ics uahana ( Su rat )

B.A .R.C. Bombay.

Ltd. ,

MonQbind Pvt . Lt d. , Cal~fornia , USA.

Monobind Pvt. Ltd . , California, USA.

Bi omeri eux , France .

Mi l es India Ltd . , Baroda .

t>lil es I nd i a Ltd. , Baroda.

Miles India Ltd . , Baroda .

Mil e s India Lt d. , Baroda.

Span Diagnostics Ltd. , uahana ( Sura t )

Mi l es India Ltd . , Baroda.

Miles India Lt d . , Baroda .

Miles India Lt d . , Baroda .

Mi les India Ltd . , Baroda .