Material & Methods - Shodhganga



The study was carried out in the Department of Biochemistry G.R Medical College, Gwalior. The study was done on 250 individuals, who included 100 uncontrolled type-2 diabetic, 50 controlled type-2 diabetic and 100 healthy individuals. All diabetic patients were on medication with oral hypoglycemic drugs. Age matched healthy control subjects were selected from known families. The written consent of patients was also taken before starting the study. A record of clinical history and previous investigations of patients disorders were compiled in a proforma (Proforma enclosed).  A proforma containing the relevant findings of clinical, biochemical and physiological investigations were recorded on preset questionnaire as base line record. All ethical measures were taken prior and during the study.

5 ml of blood sample was withdrawn from the anticubital vein following overnight fasting. The blood sample was collected in plain, fluoride and EDTA vacutainers. The blood sample was analyzed for biochemical and immunological investigations which include:

➢ Biochemical Parameters

1. Fasting Blood Sugar (FBS)

2. Glycosylated haemoglobin (HbA1c)

3. Total Cholesterol (TC)

4. Triglyceride (TG)

5. High density-lipoprotein cholesterol (HDL-c)

6. Low density lipoprotein cholesterol (LDL-c)

7. Very low density lipoprotein cholesterol (VLDL-c)

8. Urea

9. Creatinine

10. Insulin

11.    Insulin resistance by HOMA–IR calculation method

➢ Immunological Parameters

12.     Tumour Necrosis Factor-α

13.     Interleukin-6

Separation of serum

Blood sample was collected in plain vial and incubated at 37˚C for 30 minutes. After incubation, clot was removed and remaining sample was taken in centrifuge test tube. Samples were centrifuged at 3000 rpm for 10 to 20 minutes. Supernatant collected in clean and dry serum test tube for analysis of fasting blood glucose, lipid profile and insulin as well as inflammatory markers interleukin-6 and tumour necrosis factor-α.

PREPARATION OF HAEMOLYSATE

The blood samples collected in sodium citrate vial centrifuged at 3000 rpm for 15 minutes. Plasma was removed from Packed Cell Volume (PCV) Packed Cell Volume was washed 3 times with normal saline. Cells were lysed by adding 1ml of distald water. Mixture was refrigerated for 10 minutes and then vigorously shaken in vertex for 2 minute, and then 0.5ml chloroform was added as a preservative. Mixture was centrifuged at 3000 rpm for 20 minutes. The whole mixture was clearly separated into three layers, lower most layers was chloroform, middle layer was cell stroma (mucous) and upper most layer was clear haemolysate solution. This haemolysate solution was pipetted out and collected in microcentrifuge tube, which was finally utilized for the estimation glycosylated haemoglobin.

Biochemical Investigations

Biochemical parameters, fasting blood sugar and lipid profile, urea, creatinine were estimated by fully automated analyser in a commercially available kit (BioSystems S.A, Barcelona, Spain) according to manufacturer instructions. Glycosylated haemoglobin was estimated by Rai KB and Pattabiraman method TN (1984).

Immunological Investigations

Immunological markers interleukin-6 and tumour necrosis factor-α were estimated by a highly sensitive sandwitch-enzyme linked immunosorbent assay (ELISA) method in a commercially available kit (Immunotech, Backman Coulter, France). The assay was performed exactly as recommended by the manufacturer.

ESTIMATION OF SERUM GLUCOSE

METHOD: (Enzymatic- colorimetric- Trinder-End Point).

PRINCIPLE

Glucose oxidase (GOD) oxidizes glucose to gluconic acid & hydrogen peroxide. In presence of enzyme peroxides, released hydrogen peroxide is coupled with phenol & 4-Aminoantipyrine (4-AAP) to from colored quinoneimine dye. Absorbance of colored dye was measured at 505 nm & is directly proportional to glucose in the sample.

Reagent composition

Phosphate buffer, pH 7.4 13.8 mmol/L

Phenol 10 mmol/L

Glucose oxidase > 10000 mmol/L

Peroxidase >700 U/L

4-Aminoantipyrine 0.3 mmol/L

Sample: Serum free of haemolysis. Serum is stable 8 hrs.  At 25°C and up to 3 days at 2-8°C.

Procedure: - Wavelength 505 nm, Temperature 37°C

Read against reagent blank

| |BLANK |CALIBRATION |TEST |

|REAGENT-R |300 µl |300 µl |300 µl |

|DISTILLED WATER |3 µl |- |- |

|CALIBRATER |- |3 µl |- |

|SAMPLE |- |- |3 µl |

Mix and after 11 minutes and 30 seconds of incubation read the absorbance (A)

Calculation

A sample / A calibrator X n n = calibrator factor

Conversion factor Mg/dl x 0.0555 = mmol/L

Mg/dl x 0.01= g/L

Reference Values 70- 105 mg/dl (3.89- 5.83 mmol/L).

ESTIMATION OF GLYCOSYLATED HAEMOGLOBIN (RAI KB and PATTABIRAMAN TN Method, 1984)

Reagents

1) 0.18 M HCL–acetone mixture

2) Acetone

3) Tris HCL buffer 0.05 M

4) 80% Phenol

5) Conc. H2SO4

6) Stock Glucose standard (100 mg %)

7) Working Glucose standard (10 mg/ ml)

Method

0.3 ml of haemolysate was added drop wise to 8 ml of 0.18 M HCI in acetone with mixing. After standing for 15 minutes, the contents were centrifuged in a table top centrifuge at 2000 g, for 10 minutes. The precipitated globin was washed twice with 8 ml of HCl–acetone mixture and finally with 8 ml acetone alone. The precipitate should be colourless. These washing steps also remove free glucose. The globin was dissolved in 1 ml of 0.05 M tris-HCl buffer, pH 7.0 and kept in a boiling water bath for 10 minutes to remove completely traces of acetone; to the cooled solution 0.05ml of 80% phenol was added followed by 3 ml of conc. H2SO4. The colour developed was

Measured at 480 nm after 30 minutes. The colour intensity was directly proportional to globin concentration in the range of 5- 30 μg of bound hexose. Fructose or glucose may be used as standard. The coefficient of variation of the method was 4.5%. HCl and acetone used should be of analytical grade, spurious colour with a reddish tinge may be obtained with substandard reagents, washing with HCL-acetone alone should be thorough; otherwise high values due to contaminating glucose will be obtained. It is absolutely necessary that traces of acetone should be removed by boiling, since acetone may also interfere in colour formation.

Calculation

Test Mol. Wt. Of globin 1

Reference values

0.25 to 0.28 M hexose / M Hb in normal healthy persons

0.50 to 0.60 M hexose / M Hb in diabetic patient.

ESTIMATION OF SERUM Total cholesterol

METHODE

Enzymatic- (Colorimetric Trinder End point).

Principle

Enzymatic colorimetric determination of total cholesterol according to the following reactions:

Reagent composition:

Pipes 50 mmol/L

Sodium cholate 5 mmol/L

Phenol 24 mmol/L

Cholesterol esterase > 180 U/L

Cholesterol oxidase > 200 U/L

Peroxidase > 1000 U/L

4-Aminoantipyrine 0.5 mmol/L, pH 7.0

STANDARD

Cholesterol 200 mg/dl

Reagent Preparation: Reagent provided is ready to use.

Stability of reagents:

To store 2-8°C and protected from light.

SAMPLES: Heparin or EDTA plasma from fasting patients.

STORAGE: Samples are stable 5to 7 days if stored at 4°C, 3months at-20°C and several years at -70°C.

PROCEDURE

This reagent can be used on most analysers; Semi automated analysers and manual Methods

The applications are available on request.

Wavelength : 505 nm

Temperature : 37°C

Read against reagent blank.

| |BLANK |STANDARD |SAMPLE |

|Reagent R |1000µL |1000µL |1000µL |

|Standard |-------- |10µL |-------- |

|Sample |-------- |------- |10µL |

Mix and incubate for 5 min. at 37°C. Read the absorbance of STD. And sample

against reagent blank.

CALCULATION

A Sample/A Standard × n n = standard concentration

REFERENCE VALUES: < 200 mg/dl

ESTIMATION OF SERUM TRIGLYCERIDES

METHOD: (Enzymatic-colorimetric end point method)

Principle: Triglycerides in the sample originate, by means of the coupled reactions described below, a coloured complex that can be measure by spectrophotometer.

Pipes 50 mmol/L

Magnesium chloride 14.8 mmol/L

Chlorophenol 2.7 mmol/L

ATP 3.15 mmol/L

Potassium ferrocyanide 10 µmol/L

Amino-4- antipyrine 0.31 mmol/L

Lipoprotein lipase 2000 U/L

Glycerol Kinase 500 U/L

Glycerol-3- phosphate Oxidase 4000 U/L

Peroxides 500 U/L

STANDARD

Glycerol (triglycerides equivalent) 200 mg/ dl

02 g/L

2.28 mmol/L

STABILITY OF REAGENTS

To store at 2-8°C and protected from light.

SAMPLES

Serum or plasma from fasting patients. Collects  the  samples in EDTA tubes and stoppers free of glycerol. Sample are stable 5 to7 days if stored at 2-8°C, 3 months at -15 to 20°C and several years at -70°C.

PROCEDURE:

This reagent can be used on most analysers, semi automated analysers and

Manual methods. The applications are available on request.

Wavelength : 505 nm

Temperature : 37°C

Read against reagent blank.

| |BLANK |STANDARD |SAMPLE |

|Reagent R |1000µL |1000µL |1000µL |

|Standard |-------- |10µL |------- |

|Sample |-------- |------- |10µ |

Mix and incubate for 10 min at 37°C. Read the absorbance’s of std. (A)

After a 425 seconds incubation.

CALCULATION

A Sample / A Standard× n n = Standard Concentration

REFERENCE VALUES: > 150 mg/dl.

ESTIMATION OF HDL-CHOLESTEROL

METHOD: PEG- CHOD- PAP, end point assay with lipid clearing factor (LCF).

Low density lipoprotein (LDL) Cholesterol, very low density lipoprotein (VLDL) Cholesterol and chylomicron fractions are precipitated by addition of polyethylene glycol 6000(PEG). After centrifugation, the high Density lipoprotein (HDL) fraction remains in the supernatant is determined with CHOD-PAP method.

REAGENT

1- Cholesterol reagent

2- Cholesterol standard

3- Precipitating reagent

4- HDL-Cholesterol standard

SAMPLE

9-12 hours fasting is recommended by national cholesterol education program, serum and plasma should be separated from cells within 3 hours of vein puncture. For plasma heparin or EDTA can be use as anticoagulants.

PROCEDURE

Step1: HDL cholesterol separation

|Pipette in to tube marked |Test |

|Serum / Plasma |200 µl |

|Reagent -3 |200 µl |

Mix well keeps at room temperature (15-30°C) for 10 minutes. Centrifuge for 15 minutes at 2000 rpm and separate clear supernatant. Use the supernatant for HDL Cholesterol estimation.

Step: 2 HDL cholesterol estimation

|Pipette in to tube marked |Blank |Standard |Test |

|Supernatant from step 1 |- |- |1000 µl |

|Reagent -3 |- |1000 µl |- |

|Reagent -1 |1000 µl |1000 µl |1000 µl |

Mix well keep incubate at 37°C for 10 minutes.

Calculation

HDL Cholesterol concentration (mg/dl) = Absorbance of Test/Absorbance                                                                                                                 of standard X 50X2

Reference Value: 40-60 mg/dl.

CALCULATION OF LDL-CHOLESTEROL & VLDL CHOLESTEROL BY FRIEDWALD’S FORMULA (1972):

LDL-c and VLDL-c can be calculated from the following formula:

LDL-c = TC – (HDL-c + VLDL-c)

VLDL-c = TG / 5

Reference Values

LDL-c = Up to 190 mg/dl (2.0 – 4.0 mmol/L)

VLDL-c = 14 -31.8 mg/dl (0.36 – 0.83 mmol/L)

ESTIMATION OF SERUM UREA

METHOD: Enzymatic- UV Kinetic

Principle

Enzymatic reaction according to following reactions

Urease

Urea + 2 H2O 2 NH4+ + CO3 -

GLDH

NH4+ + α – ketogluterate + NADH L- Glutamate +NAD+ H2O

GLDH- Glutamate dehydrogenase

Reagents Composition

Reagent 1: R1

Tris buffer, pH 7.60 (370C) 125mmol/ltr

ADP 1 mmol/ltr

α – ketogluterate 9 mmol/ltr

Urease ≥ 8100 U/L

GIDH ≥ 1350 U/L

Reagent 2: R2

NADH 1.5 mmol/L

PROCEDURE

This reagent can be used on most analyzers, semi-analyzers and manual method.

The application is available on request

Wavelength: 340 nm

Temperature: 370C

Read against reagent blank

| |BLANK |CALIBRATION |TEST |

|Reagent :R1 |240 µL |240 µL |240 µL |

|Distilled water |3 µL |- |- |

|Calibrator |- |3 µL |- |

|Sample |- |- |3 µL |

Mix and wait 4 minutes and 43 sec. then add

|Reagent :R2 |60 µL |60 µL |60 µL |

Mix and read the variation of the absorbance (Δ A) between 24 sec and 106 sec

CALCULATIONS

Δ A sample

________________ X n n = calibrator Concentration

Δ A calibrator

Conversion factor Mg/dL X 0.1665 = mmol/L

Mg/dL X 0.01 = g/L

g /dL X 0.1665 = mol/L

REFERENCE VALUES

13 – 43 mg/dL

ESTIMATION OF SERUM CREATININE

METHOD: Jaffe Kinetic method.

PRINCIPLE

Protein is precipitated from serum using tungstic acid. Creatinine present is protein free filtrate reacts with alkaline picrate solution to form an orange coloured complex of creatinine picrate, the intensity of which is measured colorimetrically at 530 nm. A standard creatinine solution is treated similarly and the colour intensities are compared.

REAGENTS

1. Sodium tungstate, 10 ./.

2. Sulphuric acid, 2/3N

3. Saturated picric acid

4. NAOH, 10./.

5. Standard creatinine solution : concentration = 0.003 mg/ml

PROCEDURE

Prepration of protein free filtrate :

Distilled water 7ml

Blood 1ml

10 % Sodium Tungstate 1ml

2/3 NH2 SO4 1ml

Mix. Keep at room temperature for 5 minutes and filter.

| |BLANK (B) |STANDARD(S) |UNKNOWN(U) |

|Protein free filtrate | | |5.0ml |

|Creatinine standard | |5.0ml | |

|Distilled water |5.0ml | | |

|Alkaline picrate |2.5ml |2.5ml |2.5ml |

Mix and keep at room temperature for 10 minutes.

Calculations

ODU – ODB 100

________________ X Conc. of std/ml X Vol. of std X ___________________

ODS – ODB Vol. of blood

ODU – ODB 100

= ________________ X 0.003 X 5 X ____________

ODS – ODB 0.5

ODU – ODB

= ________________ X 3

ODS – ODB

= ………….. mg/dL

ODu: Optical densitiy of unknown sample.

ODb: Optical densitiy of Blank.

ODs: Optical densitiy of Sample.

NORMAL RANGE

The normal range for serum creatinine is 0.6 -1.2mg/dl.

ENZYME IMMUNOASSAY FOR THE DETEMINATION OF PLASMA INSULIN

(LDN LABOR DIAGNOSTIKA NORD, ELISA Kit, GmbH & Co. KG)

Principle of the Assay

The insulin ELISA kit is a solid phase enzyme – linked immunosorbent assay-linked immunosorbent assay (ELISA) based on the sandwich principle. The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site on the insulin molecule. An aliquot of patient sample containing endogenous insulin is incubated in the coated well with enzyme conjugate is washed off. During the second incubation step streptavidin peroxidise enzyme complex binds to the biotin –anti-insulin antibody. The amount of bound HRP complex is proportional to the concentration of insulin in the sample. Having added the substrate solution, the intensity of colour developed is proportional to the concentration of insulin in the patient sample.

Preparation of Reagents

Allow all reagents and required number of strips to reach room temperature prior to use.

Wash Solution

Dilute 30 ml of concentrated wash solution with 1170 ml deionised water to a final volume of 1200ml .The diluted wash solution is stable for 2weeks at room temperature.

SPECIMEN

Serum or plasma (only heparin – or citrate plasma) can be used in this assay. Do not use haemolytic, icteric or lipemic specimen.

Specimen collection

Serum

Collect blood by venipuncture(e.g. Sarsted Monovette # 02.1388.001), allow to clot, and separate serum by centrifugation at room temperature.

Plasma

Whole blood should be collected in to centrifuge tubes containing anti coagulant and centrifuged immediately after collection.

Specimen storage

Specimen should be capped and may be stored for up to 5 days at room temp. 2-8°C prior to assaying. Specimen held for a longer time should be frozen only once -20°C prior to assay.

Specimen dilution

If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can be diluted 10- fold or 100 hold with zero standard and reassayed as described in assay procedure. For the calculation of the concentrations this dilution factor has to be taken in to account .

Example

A) Dilution 1:10: 10 µl serum + 90 µl standard Zero standard (mix thoroughly)

b) Dilution 1:100 10 µl dilution a) 1:10 + 90 µl zero (mix thoroughly).

Assay Procedure

1. Secure the desired number of microtiter wells in the holder.

2. Dispense 25µl of each standard controls and samples with new disposable tips into appropriate wells.

3. Dispense 25 µl Enzyme conjugate into each well.

4. Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

5. Incubate for 30 minutes at room temperature without covering the plate.

6. Briskly shake out the contents of the wells.

7. Rinse the wells 3 times with diluted wash solution (400 µl per well). Strike the wells sharply on absorbent paper to remove residual droplets.

8. Add 50 µl of enzyme complex to each well.

9. Incubate for 30 minutes at room temperature.

10. Briskly shake out the contents of the wells. Rinse the wells 3 times with diluted wash solution (400 µl per well). Strike the wells sharply on absorbent paper to remove residual droplets

11. Add 50 µl of substrate solution to each well.

12. Incubate for 15 minutes at room temperature.

13. Stop the enzymatic reaction by adding 50 µl of stop solution to each well.

14. Read the OD at 450± nm with a microtiter plate reader within 10 min after adding the stop solution.

Calculation of Results

1. Calculate the average absorbance values for each set of standards,

controls and patient samples.

2. Construct a standard curve by plotting the mean absorbance obtained from each standard against its concentration with absorbance value on the vertical (y) axis and concentration on the horizontal (x) axis.

3. using the mean absorbance value for each sample determine the corresponding concentration from the standard curve.

4. The concentration of the samples can be read directly from this standard curve. Samples with concentrations higher than that of the highest standard have to be further diluted. For the calculation of the concentrations this dilution factor has to be taken in to account. Below is listed a typical example of a standard curve with the Insulin ELISA.

|STANDARD |Optical Units (450 nm) |

|Standard 0 (0 µIU/ml) |0.03 |

|Standard 1 (6.25µIU/ml) |0.07 |

|Standard 2 (12.5µIU/ml) |0.14 |

|Standard 3 (25 µIU/ml) |0.35 |

|Standard 4 (50 µIU/ml) |0.88 |

|Standard 5 (100µIU/ml) |2.05 |

Expected values

It is strongly recommended that each laboratory should determine its own normal and abnormal values. In a study conducted with apparently  normal healthy adults, using the insulin ELISA the following values are observed : 2 to 25 µIU/ml.

Insulin Resistance

Insulin resistance values were derived using the homeostasis model assessment (HOMA) method, employing the equation below.

IR = (Fasting plasma insulin micro units/L) × (Plasma fasting glucose mmol/L) / 22.5

IMMUNOASSAY FOR THE IN VITRO DETERMINATION OF IL-6 IN SERUM

Immunotech ELISA Kit, 96 wells (cat. # IM1120)

Principle of the Assay

Samples and calibrators are incubated in the microtiter plate coated with the first monoclonal antibody anti-IL-6, in presence of the second anti-IL-6 monoclonal antibody linked to acetyl cholinesterase (ACE). After incubation, the wells are washed and the bound enzymatic activity is detected by addition of a chromomeric substrate. The intensity of the coloration is proportional to the IL-6 concentration in the sample or calibrator.

Reagent provided

Plate: 12 x 8 wells (ready-to-use)

Unused strips may be stored at 2-8°C in the self-lock bag provided until expiration date of the kit.

Calibrator: one vial (lyophilized)

The vial contains bovine serum albumin. Reconstitute the calibrator with the volume of distilled water stated on the vial label. After reconstitution use immediately the calibrator or store at ................
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