4. material and methods -...
TRANSCRIPT
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4. MATERIAL AND
METHODS
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•
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
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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) .
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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
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~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
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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.
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•
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 .
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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.
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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
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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
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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
------------------------------------------------------- -------
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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.
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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
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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
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•
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
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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)
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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 .