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Material and Methods

32

MATERIAL AND METHODS

3.1 SOURCE OF DATA

The present data which is case controlled study was done during the period

from 1st Oct 2010 to 1st Jan 2012. All the participants were recruited from out-

patient departments of the Hospital of National Institute of Unani medicine,

Magadi Main Road, Kottege Palya. Bangalore. Located near industrial area of

Bangalore. The study was done on a total of 302 subjects of which 198 were

diabetic and 104 were controls, written consent was obtained from each

subjects after explaining the reasons, objective and methodology of the

research study. The copy of the consent form attached as annexure 1.

3.2 SPECIMEN COLLECTION AND PRESERVATION

The participants of the study were required to fast for at least 10 hours before

the sample collection; 5ml of the blood was collected in plain dry vacutainer

tube. The patients were asked to have breakfast and to come again after 1-1/2

hours for post prandial sample collection.

The vacutainer tube of the fasting sample was made to stand for 25-30 minutes

for separation of serum from the cells. Immediately after, the tubes were

centrifuged and serum was separated for the analysis.

From the fasting serum sample, glucose estimation, lipid profile, liver function

tests, kidney function tests, Gamma Glutamyltransferase, hs-CRP and

Lipoprotein (a) was analyzed.

Again the samples were collected for post prandial blood sugar for which only

2ml of the blood was drawn in vacutainer tube and serum separated and

analysis was carried out for the post prandial blood sugar levels,


33

3.3 Inclusion criteria

A comprehensive literature survey was done from various research journals to

design the study format and proforma. The copy of which is attached in

Annexure 2.

In building up of the proforma utmost care is taken to make it broad based so

that most of the desirable aspects could be included or incorporated in the

study. Information of the patient about sex, diagnosis and disease history,

lifestyle of the patients and family history was collected from each subject. A

criterion for inclusion of the subjects was taken as fasting blood sugar more than

110 mg/ at more than two occasions.

All the diagnosis was done by physicians of the hospital. Normal subjects were

selected from healthy random sampling from different age groups attending the

outpatient departments of the National Institute of Unani Medicine and the staffs

have also participated in the study.

a. Clinically investigated and diagnosed patients with Type-2 diabetes

mellitus.

b. Patients Investigated and Diagnosed for Hyper / Dyslipidemea.

c. Patients of either sex between ages 25 to 60 years.

3.4 Exclusion Criteria

a. Patients suffering from other serious illness / diseases.

b. Patients with Hypothyroidism

c. Patients with Pregnancy.

d. Patients on Hemodialysis.

e. Patients with complications of Diabetes.

f. Patients with Diabetic Nephropathy / Neuropathy.

g. Patients who are Alcoholics.

3.5 Laboratory Investigations The diagnostic tests of the patients was carried

out at the Biochemistry Laboratory. Hospital Block, National Institute of Unani


34

Medicine. (Govt of India.), Magadi Main Road. KottegePalya. Bangalore

560091

STUDY DESIGN

Subjective Parameters

• 1. Type-2 diabetes Mellitus

• 2. Hyperlipidimea / Dyslipidimea.

• 3. Obesity

• 4. History of the patient

Objective Parameters

� 1. Blood sugar (FBS, PPBS).

� 2. Serum Cholesterol.

� 3. Serum Triglyceride.

� 4. High Density Lipoprotein (HDL).

� 5. Low Density Lipoprotein (LDL).

� 6. LDL: HDL ratio.

� 7. Lipoprotein (a).

� 8. Gamma Glutamyltransferase

(GGT).

� 9. Hs-C Reactive Protein (hs-CRP).


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3.5.1 Estimation of Blood Glucose

The estimation of fasting plasma glucose (FBG) level was done by GOD-POD

method of Trinder (1969). (Aspen Diagnostics)

3.5.5.1.1 Principle

Glucose determination after enzymatic oxidation by glucose oxidase, The

quinoneimine is a colorimetric indicator , which is formed from 4 aminotipyrine

and phenol by hydrogen peroxide by the catalytic action of peroxidase.

β - D glucose +O2 GOD Gluconic acid +H2O2

H2O2 + 4-aminoantipyrine + phenol POD Red Dye + 4-H2O

3.5.5.1.2 Reagents composition

• Reagent: Phosphate buffer PH 7.5

• Phenol 5mmol/l

• 4-Aminoantipyrine 0.5mmol/l

• Glucose oxidase (GOD) ≥ 10kU/l

• Peroxidase (POD) ≥ 1 kU/l

• Standard 100mg/dl

3.5.5.1.3 System General parameters.

• Method End point

• Slope of reaction Increasing

• Wavelength 505 nm (49-530)


36

• Flow cell temp 37º C

• Sample volume 10 µl

• Reagent volume 1000 µl

• Incubation 10 minutes at 37º C

• Standard Conc. 100mg/dl

• Unit mg/dl

• Linearity upto 500mg/dl

• Zero setting Reagent Blank.

3.5.1.4. Test Procedure- Dispense into tubes Pipette into Tubes Blank Standard Test

Standard - 10 µl -

Sample - - 10 µl

Reagent 1000 µl 1000 µl 1000 µl

Incubate for 10 min at 37º C. Mix and read absorbance at 505nm against Blank.

3.5.1.5 Reference Range: Fasting 70-110mg/dl

Post Prandial Up to 140mg/dl


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3.5.2. ESTIMATION OF CHOLESTEROL The estimation of serum cholesterol was done by end point method of Allainet

al. (1974) (Siemens Kit).

3.5.2.1 Principle

Cholesterol Esters +H2O Chol Esterase Cholesterol +Fatty Acids

Cholesterol +O2 Chol Oxidase Cholest-4-en-3-one + H2O2

2H2O2 +Phenol+4-Aminoantipyrine Peroxidase Red quinine +4-H2O

3.5.2.2 Reagents composition

• Reagent 1 (Enzyme/Chromogen)

• Cholesterol Esterase ≥30IU/L

• Cholesterol Oxidase ≥250IU/L

• Peroxidase ≥1000IU/L

• 4-Aminoantipyrine 0.5 mmol/L

• Reagent 1A Buffer:

• Pipes buffer, pH6.95 50 mmol/L

• Phenol 24 mmol/L

• Sodium Cholate 0.5mmol/L

• Standard (Cholesterol 200mg/dl)


38

3.5.2.3 General parameters.









• Unit mg/dl

• Linearity up to 500mg/dl


3.5.2.4 Test Procedure Dispense into tubes

Pipette into Tubes Blank Standard Test

Reconstituted

reagent

1000 µl 1000 µl 1000 µl

Standard - 10 µl -

Sample - - 10 µl

Incubate for 5 min at 37º C. Mix and read absorbance at 500nm

3.5.2.5 Reference Range: Serum: Up to 200 mg/dl


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3.5.3. Triglyceride

The estimation of triglycerides was done by end point method of McGowan et

al., (1983)(Siemens)

3.5.3.1 Principle

Triglyceride +H2O Lipoprotein Lipase Glycerol + Fatty Acid

Glycerol +ATP Glycerol Kinase, Mg2+ Glycerol-3-Phosphate +ADP

Glycerol-3-Phosphate + O2 GPO DihydroxyacetonePhophate +H2O2

2H2O2 + 4-Aminoantipyrine + ADPS Peroxidase Red quinone

GPO = Glycerol-3-phosphate Oxidase

ADPS = N-Ethyl-N-(3-sulfopropyl)-m-anidisin

The intensity of the purple colored complex is measured.

3.5.3.2 Reagents

• Lipoprotein Lipase ≥ 1100U/L

• Glycerol Kinase ≥ 450U/L

• Glycerol-3-phosphate Oxidase ≥ 5000U/L

• Peroxidase ≥ 350U/L

• 4-Aminoantipyrine 0.7mmol/L

• ATP 0.3mmol/L

• Reagent buffer, pH 7.0 50 mmol/L

• ADPS 0.9mmol/L

• Magnesium salt 17.8 mmol/L

• Standard (Triglyceride 200mg/)


40

3.5.3.3 General system parameters.









• Unit mg/dl




Pipette into Tubes Blank Standard Test

Reconstituted reagent

1000 µl 1000 µl 1000 µl

Standard - 10 µl -

Sample - - 10 µl

Incubate for 5 min at 37º C. Mix and read absorbance at 546(520-570nm)

3.5.3.5 ReferenceRange: up to 150 mg/dl.


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3.5.4. ESTIMATION OF HDL-CHOLESTEROL

The estimation of serum HDL was done by precipitation method of Lopez-

Virellaet.al. (1977) (Siemens Kit)

3.5.4.1 Principle

Chylomicrons VL and L fractions in serum or plasma are separated from H by

precipitating with Phosphotungstic Acid and Magnesium Chloride. After

centrifugation, the Cholesterol in the H fraction, which remains in the

supernatant is assayed with enzymatic cholesterol method, using cholesterol

Esterase, Cholesterol Oxidase, Peroxidase and the chromogen 4-

Aminoantipyrine/Phenol.

Phosphotungstate Serum/ Plasma HDL Fraction+ (LDL+VLDL+Chylomicrons) Mg2+ (Supernatant) (precipitate)

3.5.4.2 Reagents

• Reagent 1 (Enzyme/Chromogen) :

• Cholesterol Esterase ≥30IU/L

• Cholesterol Oxidase ≥250IU/L

• Peroxidase ≥1000IU/L

• 4-Aminoantipyrine 0.5mmol/L

• Reagent 1A Buffer:

• Pipes buffer, pH6.95 50mmol/L

• Phenol 24mmol/L

• Sodium Cholate 0.5mmol/L


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3.5.4.3 Precipitation step. Dispense into Centrifuge tubes

Test

Sample 200µl

Precipitating Reagent 2 200µl

Mix well, centrifuge separate the supernatant immediately and test for HDL-

Cholesterol.

3.5.4.4 General parameters.









• Unit mg/dl



43


Blank Standard Test

Reconstituted Reagent

1mL 1mL 1mL

Standard - 20 µl -

Supernatant - - 20 µl

Incubate for 5 min at 37º C Mix and read absorbance at 500nm

3.5.4.6 Reference Range: 30-70mg/dl

3.5.4.7 CALCULATION OF LDL-CHOLESTEROL

L CHOLESTEROL mg/ = TOTAL CHOL-Triglyceride –H CHOL

5

3.5.4.8 Reference Range: Up to 100mg/dl

3.5.5. ESTIMATION OF SGOT (AST Aspartate Aminotransferase):

The estimation of SGOT was done by IFCC methods of Bergmeyeret al., (a)

(1986) (Siemens kit)

3.5.5.1 Principle

L-Aspartate + α-KetoglutarateGOTOxaloacetate + L Glutamate

Oxaloacetate + NADH + H+ MDH L-Malate + NAD+

AST = Aspartate Aminotransferase

MDH = Malate Dehydrogenase


44

3.5.5.2 Reagents

• Reagent 1 (Enzyme)

• MDH ≥800U/L

• LDH ≥4000U/L

• NADH > 0.2.0mmol/L

• Α-Ketoglutarate> 13 mmol/L

• Reagent 1A (Buffer) :

• Tris buffer, pH 7.8

• L-Aspartate 264mmol/L


• Method Kinetic

• Slope of reaction Decreasing

• Wavelength 340 nm


• Delay Time 60 secs

• No of Readings 4

• Interval 60 secs



• Path length 1cm

• Factor 1746

• Unit IU/L

• Zero setting Distilled Water.


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• Linearity Up to 300IU/L

3.5.5.4Test Procedure Dispense into tubes

TEST

Reconstituted Reagent 1ml

Sample 100 µl

Mix and read immediately at 340nm

5.5.5.5 Reference Range: Up to 50 IU/L

3.5.6. ESTIMATION OF SGPT (ALT- Alanine Aminotransferase ):

The estimation of SGPT was determined by IFCC method of Berg Meyeret al.,

(b) (1986). (Siemens kit)

3.5.6.1 Principle

L-Alanine + α-KetoglutarateGPT Pyruvate + L Glutamate

Pyruvate + NADH + H+ LDH L-Lactate + NAD+

ALT= Alanine Aminotransferase

LDH= Lactate dehydrogenase

3.5.6.2 Reagents

• Reagent 1 (Enzyme)

• LDH ≥1200U/L

• NADH > 0.2.0mmol/L

• Α-Ketoglutarate> 16 mmol/L


46

• Reagent 1A (Buffer) :

• Tris buffer, pH 7.5 110mmol/L

• L-Alanine 550mmol/L


• Method Kinetic




• Delay Time 60 sec.


• Interval 60 sec.



• Path length 1cm

• Factor 1746

• Unit IU/L


• Linearity Up to 250IU/L


47


TEST


Sample 100 µl


5.5.6.5 Reference Range: Up to 40 IU/L

3.5.7. ESTIMATION OF ALKALINE PHOSPHATASE

The estimation of ALKP was determined by PNPP methods of Z. Klin. Chem. U.

Klin).

3.5.7.1 Principle

Alkaline Phosphatase hydrolyses p-Nitrophenyl phosphate (PNPP) into p-

Nitrophenol and Phosphate. At the alkaline pH of the buffered medium.p-

Nitrophenol is yellow. The color developed by hydrolysis is measured at 405 nm

and is proportional to alkaline phosphatase activity.

p-Nitrophenyl phosphate + H2O ALKP p- Nitrophenol + Phosphate

3.5.7.2 Reagents

• Reagent 1 (Substrate):

• p-Nitrophenol Phosphate 10 mol/L

• Reagent 1A(Buffer) :

• Diethanolamine 1 mol/L

• Magnesium Chloride 0.5mmol/L


48

3.5.7.3 General system parameters

• Method Kinetic






• Interval 30 sec.



• Path length 1cm

• Factor 1826

• Unit IU/L


• Linearity Up to 700 IU/L


TEST


Sample 30 µl


3.5.7.5 Reference Range: 60-170 IU/L


49

3.5.8. ESTIMATION OF BLOOD UREA

The estimation of blood urea was done by kinetic method of Fawcett and

Scott,(1960)(Siemens).

3.5.8.1 Principle

Urea + 2H2O Urease 2NH4+ + CO3

-2

α-Ketoglutarate + NH4+ + NADH GDPH L- Glutamate + NAD++H2O

GLDH: Glutamate dehydrogenase

3.5.8.2 Reagents

• R 1 TRIS Buffer pH 7.8 120mmol/L

• α-Ketoglutarate 7mmol/L

• ADP 0.6mmol/L

• Urease ≥ 6kU/L

• GLDH ≥1kU/L

• R2: NADH 0.25mmol/L

• Standard: 50mg/dl

3.5.8.3 General System parameters

• Method Fixed Time




• Delta Time 60 sec.


50




• Standard Conc 50mg/dl

• Unit mg/dl


• Zero setting Reagent Blank


Pipette into Tubes Standard Test

Standard 10 µl -

Sample - 10 µl

Reagent 1000 µl 1000 µl

Mix well, Incubate for app 30 sec at 37º C. Then read absorbance A1.

After exactly 60sec further read A2 at 340nm.

3.5.7.5 Reference Range: 10-50mg/dl


51

3.5.8. ESTIMATION OF CREATININE:

The estimation of serum Creatinine was done by picrate method of Henry et.al.

(1974).

3.5.8.1 Principle

Creatinine in alkaline solution reacts with picrate to form a red-orange

compound. Under the specific condition of the assay, the rate of development of

the color is proportional to the concentration of creatinine in the sample when

measured at 500nm

Alkaline medium Creatinine + Picrate Orange color

3.5.8.2 Reagents

• Reagent 1 (Picrate):

• Picric Acid 34.9 mmol/L

• Sodium Hydroxide 45mmol/L

• Reagent 2 (Sodium Hydroxide)

• Sodium Hydroxide 0.26mol/L

• S. Creatinine 0.020g/L

3.5.8.3 General System parameters.

• Method Fixed Time






52

• Flow cell temp 25º C, 30ºC, 37ºC.



• Pathlength 1cm


• Unit mg/dl

• Linearity Up to 10mg/dl

• Zero setting Distilled Water


TEST

Working solution 1ml

Standard 100 µl

Mix and read immediately at 500nm.

3.5.7.5 Reference Range: Males 0.6-1.1mg/dl and Females 0.5-0.9mg/dl

3.5.8 ESTIMATION OF GAMMA-GLUTAMYLTRANSFERASE

(GGT/GGTP).

The estimation of GGT was done by Tris method of Bablock W. et al (1998).


53

3.5.8.1 Principle

L-Ɣ- Glutamyl-3-carboxy-4-nitronolide +Glycylglucine L-Ɣ-

GlutamyGlycylglucine + 5-amine-2-nitrobenzoate.

3.5.8.2 Reagents

• TRIS 100mmol/l

• Glucylglycine 100mmol/l

• L-Ɣ- Glutamyl-3-carboxy-4-nitronolide 4mmol/l

REAGENTS STANDERDIZED TO IFCC Reaction type: KINETIC METHOD

3.5.8.3 ASSAY PROCEDURE.

Dispense into tubes

Pipette in Tubes Volumes

Working Reagent 1000 µl

Test 100 µl

Mix and read at 405nm.

Read absorbance initially after 60seconds and again after 1 & 2minutes.

3.5.8.4 Reference Range: 60-170 IU/L


54

3.5.9 LIPOPROTEIN (A) TEST

The estimation of Lp(a) was done by Gaubalz JW, et al. 1983

3.5.9.1 PRINCIPLE

Anti-humanLp(a) coated latex particles are agglutinated when mixed with

samples containing Lp(a). This agglutination causes an absorbance change

depending upon the Lp(a) contents of the patient samples, which can be

interpolated in a calibration curve.

3.5.9.2 Reagents

Lipoprotein (a) R1 Buffer Solution pH 8.3

Lipoprotein (a) R2 Lipoprotein latex

Lipoprotein (a) calibrator

3.5.9.3 ASSAY PROCEDURE (AGAPPE DIAGNOSTICS)

CALIBRATOR SAMPLE

Calibrator

Sample

R1 Buffer

R2 Latex

15 µl

-

800µl

200 µl

-

15 µl

800 µl

200 µl

Mix and read the absorbance against blank after 10sec and after

4minutes of the latex addition


55

3.5.9.4 Reference Range: Desirable ≤ 20mg/dl

Borderline risk: 20-30mg/dl

High risk: 31-50mg/dl

Very high risk: ≥ 50mg/dl

3.5.10 hs-CRP TEST (Turbilatex)

The estimation of hs-CRP was done by Thomas A et al. 2000.

3.5.10.1 Principle: This is a quantitative turbidometric test for the measurement

of low levels of C- Reactive Protein in human serum/plasma.

3.5.10.2 Reagents

Diluent Ultra1 Tris buffer 20mmol/L,pH 8.2, Sodium azide 0.95 g/L

Latex-ultra (R2). Latex particles coated with goat IgG anti- human CRP, pH 7.3

Sodium azide 0.95g/L

U-CRP CAL Liquid Calibrator,

3.5.10.3 ASSAY PROCEDURE

Pipette in Tubes Volumes

Working Reagent 1000 µl

Test/Sample 10 µl


56

Mix well and read the absorbance immediately (A1) and after 4 minutes (A2) of

the sample addition.

3.5.10.4 Reference range: Up to 3mg/dl

.

3.6 Blood pressure

Blood pressure was measured by the nursing staffs of the department with

accurate care and proper management of the patients in RELAXED STATE.

• SYSTOLIC above 130mm Hg

• DYSTOLIC above 90mm Hg

• Normal 120/80mm Hg.

3.7 Body Mass Index (BMI) = (Weight in Kg / Hieght in meters2)

• Up to 25 perfect Normal

• Overweight 25.0-29.9

• Obese 30 or more.


57

3.8 Statistical Analysis

SPSS (Statistical Package for Social Science) Software (SPSS Version 17.0:

SPSS Inc. Chicago, IL) was used for the statistical calculations of the study. The

prevalence was carried out using Microsoft Excel program. Results for

continuous variables were presented as mean ± SD.

The difference between the studied groups was also tested for their significance

using Students t-test.

The difference between the studied variables was analyzed using Pearson’s

correlation or Spearman’s0. rho correlation test. Differences and correlations

were considered as significant at p< 0.05. The differences and the correlations

were considered highly significant at p< 0.01.

3.8.1 Statistical Methods: Descriptive and inferential statistical analysis has

been carried out in the present study. ROC curve analysis is performed to find

the diagnostic role of GGT, Lp(a) and Hs-CRP

11. Significant figures

+ Suggestive significance (P value: 0.05<P<0.10)

* Moderately significant (P value: 0.01<P ≤ 0.05)

** Strongly significant (P value: P≤0.01)

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