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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,
Material and Methods
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
Material and Methods
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).
Material and Methods
35
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)
Material and Methods
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
Material and Methods
37
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)
Material and Methods
38
3.5.2.3 General parameters.
• Method End point
• Slope of reaction Increasing
• Wavelength 500 nm (490-550)
• Flow cell temp 30º C
• Sample volume 10 µl
• Reagent volume 1000 µl
• Incubation 5 minutes at 37º C
• Standard Conc. 200mg/dl
• Unit mg/dl
• Linearity up to 500mg/dl
• Zero setting Reagent Blank.
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
Material and Methods
39
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/)
Material and Methods
40
3.5.3.3 General system parameters.
• Method End point
• Slope of reaction Increasing
• Wavelength 546 nm (520-570)
• Flow cell temp 30º C
• Sample volume 10 µl
• Reagent volume 1000 µl
• Incubation 5 minutes at 37º C
• Standard Conc. 200mg/dl
• Unit mg/dl
• Linearity up to 1000mg/dl
• Zero setting Reagent Blank.
3.5.3.4 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 546(520-570nm)
3.5.3.5 ReferenceRange: up to 150 mg/dl.
Material and Methods
41
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
Material and Methods
42
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.
• Method End point
• Slope of reaction Increasing
• Wavelength 500 nm (492-550)
• Flow cell temp 30º C
• Sample volume 20 µl
• Reagent volume 1000 µl
• Incubation 5 minutes at 37º C
• Standard Conc. 200mg/dl
• Unit mg/dl
• Zero setting Reagent Blank.
Material and Methods
43
3.5.4.5 Test Procedure Dispense into tubes
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
Material and Methods
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
3.5.5.3 General system parameters.
• Method Kinetic
• Slope of reaction Decreasing
• Wavelength 340 nm
• Flow cell temp 37º C
• Delay Time 60 secs
• No of Readings 4
• Interval 60 secs
• Sample volume 100 µl
• Reagent volume 1000 µl
• Path length 1cm
• Factor 1746
• Unit IU/L
• Zero setting Distilled Water.
Material and Methods
45
• 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
Material and Methods
46
• Reagent 1A (Buffer) :
• Tris buffer, pH 7.5 110mmol/L
• L-Alanine 550mmol/L
3.5.6.3 General system parameters.
• Method Kinetic
• Slope of reaction Decreasing
• Wavelength 340 nm
• Flow cell temp 37º C
• Delay Time 60 sec.
• No of Readings 4
• Interval 60 sec.
• Sample volume 100 µl
• Reagent volume 1000 µl
• Path length 1cm
• Factor 1746
• Unit IU/L
• Zero setting Distilled Water.
• Linearity Up to 250IU/L
Material and Methods
47
3.5.6.4 Test Procedure Dispense into tubes
TEST
Reconstituted Reagent 1ml
Sample 100 µl
Mix and read immediately at 340nm
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
Material and Methods
48
3.5.7.3 General system parameters
• Method Kinetic
• Slope of reaction Increasing
• Wavelength 405 nm
• Flow cell temp 25º C
• Delay Time 60 sec.
• No of Readings 4
• Interval 30 sec.
• Sample volume 100 µl
• Reagent volume 1000 µl
• Path length 1cm
• Factor 1826
• Unit IU/L
• Zero setting Distilled Water.
• Linearity Up to 700 IU/L
3.5.7.4 Test Procedure Dispense into tubes
TEST
Reconstituted Reagent 1ml
Sample 30 µl
Mix and read immediately at 405nm
3.5.7.5 Reference Range: 60-170 IU/L
Material and Methods
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
• Slope of reaction Decreasing
• Wavelength 340 nm (49-530)
• Delay Time 30 sec.
• Delta Time 60 sec.
Material and Methods
50
• Flow cell temp 37º C
• Sample volume 10 µl
• Reagent volume 1000 µl
• Standard Conc 50mg/dl
• Unit mg/dl
• Linearity up to 300mg/dl
• Zero setting Reagent Blank
3.5.7.4 Test Procedure Dispense into tubes
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
Material and Methods
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
• Slope of reaction Increasing
• Wavelength 500 nm (49-530)
• Delay Time 30 sec.
• No of Readings 2
Material and Methods
52
• Flow cell temp 25º C, 30ºC, 37ºC.
• Sample volume 100 µl
• Reagent volume 1000 µl
• Pathlength 1cm
• Standard Conc. 2mg/dl
• Unit mg/dl
• Linearity Up to 10mg/dl
• Zero setting Distilled Water
3.5.7.4 Test Procedure Dispense into tubes
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).
Material and Methods
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
Material and Methods
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
Material and Methods
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
Material and Methods
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.
Material and Methods
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)