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Study of magnesium levels and oxidative stress in Type 2 DM patients with and without
METERIAL AND MEHTODS
This was hospital based study are conducted in the department of Biochemistry of Dr. V.M.G.M.C. Solapur; during the period of December 2013 to 2015.This study are approved by Institutional Ethics Committee. Selection of Subjects:- A total 90 sample were selected and divided into 3 groups. Groups Subject Number Control Age matched healthy subject. 30 Group 1 Type - 2 diabetic patients without any complication. 30 Group 2 Type - 2 diabetic patients with Hypertension. 30 Sample size calculation. P 1 = Prevalence of type 2 diabetes mellitus with hypertension. = 20.6 % P 2 = Prevalence of type 2 diabetes mellitus without hypertension. = 14 %. D= allowable error = 20%
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without
V = P 1 (1 – P 1 ) + P 2 (1 – P 2 )
V = 20.6 × (100 – 20.6) + 14 (100 - 14)
V= 1642 + 1204
In our study sample size calculation was done by following statistical formula n = Z 2 (1 - α/2) .V d^2 At 95% confidence interval Z 1 - α /2 = 1. n = 28. So according to calculation, minimum 29 sample should be taken in each group. In this study30 is minimum sample size in each group and since in study, subjects were divided in to three group, therefore total sample size came to be 90 with 30 subjects in each group. For group 1, we have selected well diagnosed type - 2 diabetes mellitus patient without hypertension. For group 2 we have selected well diagnosed with hypertension patients having history of type 2 diabetes. Hypertension patients were diagnosed on the basis of history, presenting symptoms like that B.P report changes.
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without Patients other than type - 2 diabetes, patients having hepatic disease or on lipid lowering drug or antioxidant vitamin supplements, or other drug known for affecting serum lipid peroxidation and antioxidant values. Collection of sample. Blood sample was collected from Dept of medicine .After 12 hour fast, 5 ml venous blood was collected in heparin bulb, centrifuged at 3000 r.p.m. plasma was separated and used for estimation of plasma magnesium ,plasma MDA ,plasma NO end products ( nitrite + nitrate ) and plasma TAS. Hemolysate was used to measure glycated HbA1c, Erythrocytic SOD. Statistical analysis. S.No Parameters Method of estimation. 1 Glycosylated haemoglobin Ion Exchange Resin Method.(43) 2 Plasma Magnesium D.W. Neill and R.A.Neely. (44) 3 Plasma Malondialdehyde S.K. Jain. ( 53 ) 4 Erythocytic Superoxide dismutase Kajari Das. (54) 5 Plasma Nitric oxide end products KortasNajwa, Wakid Nabil. (55) 6 Total antioxidant status I.F.F. Benzie.(56)
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without Results were expressed as mean ± SD. Unpaired student ‘ t ‘ test was applied to compare the results between the controls, Group 1 and Group 2. P value < 0.05 was considered statistically significant.
1. GLYCOSYLATED HEMOGLOBIN. (43) METHOD: - Ion Exchange Resin Method. Principle: - Glycosylated hemoglobin (GHb) has been defined operationally as the fast fraction hemoglobin HbA1 (Hb A1a A1b A1c) which elute first during column chromatography. The non – glycosylated hemoglobin, which consists of the bulk of hemoglobin, has been designated HbAo. A hemolysed preparation of whole blood is mixed continuously for 5 minutes with a weakly binding cation exchange resin .The labile fraction is eliminated during the Hemolysate preparation and during the binding .During this mixing, HbAo binds to the ion exchange resin leaving GHb free in the supernatant. After the mixing period, a filter separator is used to remove the resin from the supernatant. The percent glycosylated hemoglobin is determined by measuring absorbance’s of the glycosylated haemoglobin (GHb) fraction and the total hemoglobin (THb) fraction.
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without Dispense 0.5ml Lysing Reagent into tubes labeled as control (C) and test (T). Add 0.1 ml of the reconstituted control and well mixed blood sample into the appropriately labeled tubes. Mix until complete lysis is evident. Allow to stand for 5 minutes. B. Glycosylated hemoglobin (GHb) Separation. Removes cap from the ion – Exchange Resin tubes and label as Control and test. Add 0.1 ml of the hem lysatefrom Step A into the appropriately labeled Ion Exchange Resin tubes. Insert a resin Separator into each tube so that the rubber sleeve is approximately 1 cm above the liquid level of the resin suspension. Mix the tubes on a rocker, rotator or vortex mixer continuously for 5 minutes. Allow the resin to settle then push the resin separator into the tubes until the resin is firmly packed. Pour or aspirate each supernatant directly in to a cuvette and measure each absorbance against Distilled water. C. Total Hemoglobin (THb) fraction.
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without Dispense 5.0ml of distilled water into tubes labelled as Control and test. Add 0.1 ml of the Hemolysate from step A into the appropriately labeled Ion Exchange Resin tubes. Insert a resin Separator into 1 cm above the liquid level of the resin suspension. Mix the tubes on a rocker, rotator or a vortex mixer continuously for 15 minutes. Allow the resin to settle, and then push the resin separator into the tubes until resin is firmly packed. Pour or aspirated each supernatant directly into a cuvette and measure each absorbance against Distilled water. C. Total Hemoglobin Fraction Dispense 5.0 ml of Distilled water into tubes labelled as Control and Test. Add to it 0.2ml of Hemolysate from Step A into the appropriately labelled tube .mix well. Read absorbance against Distilled water. Calculation: -
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without
2. 0.05 % Titan yellow. The dye powder 0.1 gm is dissolved in 200 ml D/W. 3. Stock standard Magnesium chloride (0.8348 gm.) A quantity 8.348 mg dissolved in D/W and made up to 1 Liter .This solution contain 1000 μg preml. 1. Working reagent :- Dilute 1 ml of stock standards to 200 ml with Distilled Water .to give a concentration of 5 μg magnesium chloride perml. 2. Calcium chloride: - (0.01338 gm.) A quantity 13.38 mg cacl 2 is dissolved in distilled water and made up to 100 ml .to given to final concentration of 0.05 mg Ca perml. 3. 4 N NaoH : - 40 gm. NaoH dissolved in to distilled water up 250 ml. 4. 10 % sodium Tungstate :- 10 gm is dissolved in to up 100 ml distilled water. 5. 0.67 N H 2 So 4 :-
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without 1.84 ml concentration H 2 So 4 is added in distilled water final volume adjusts up to 100 ml D/W. Preparation of protein free filtrate.- Taken 1 ml serum + 5ml D/W + 2 ml 10% sodium tungstate + 0.67 N 2ml H 2 So 4 in to test tube. Mix well the content and filter it, filtrate is called protein free filtrate.
- Preparation of protein free filtrate (PFF) :- 5 ml.
- Working standard : - 1.0 ml.
- Calcium chloride : - 1.0 ml.
- D/W : - 1.0 ml.
- Poly vinyl alcohol : - 1.0 ml.
- Titan yellow : - 1.0 ml.
- NaoH : - 2.0 ml. S.No Reagent T S B 1 Protein free filtrate 5.0ml -- -- 2 Working standards -- 1.0ml -- 3 Calcium chloride -- -- 1.0ml 4 Distilled Water 1.0ml 5.0ml 5.0ml 5 Polyvinyl Alcohol 1.0ml 1.0ml 1.0ml 6 Titan yellow 1.0ml 1.0ml 1.0ml
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without Reagent:
1. Phosphate buffer saline (PBS) PH = 7.4. Nacl = 8.4 gm. Na 2 HPO 4 = 2.302 gm. Na 2 H 2 PO 4 =0.194 gm. Double distilled water = up to 1 liter. 2. Butylated hydroxyl toluene ( BHT ) ( BHT ) = 88 mg = 0.088 gm. **Absolute alcohol = 10 ml.
- Trichloroacetic acid : -** TCA = 30 gm. Double distilled water = up to 100 ml. 4.. Ethylenediamnie tetracatic acid (EDTA) = 0.1 M (EDTA) = 2.92gm Up to 100 ml 5. Thiobarbituric acid ( TBA ) TBA = 1 gm. NaOH = 0.02 gm Up to 100 ml. Procedure:- In a clean centrifuge tube following addition take place. PBS (Phosphate buffer saline) = 0.8 ml. Plasma = 0.2 ml.
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without TCA = 0.5 ml. Tube was then vertexes and allowed to store in ice 2 h and then centrifuged at 2000 rpm for 15 min.1ml separated supernatant was transferred to another Teflon lined stopper tube and following reagent were added. Mix well and boiled water with 15 min. Then cooled R.T. and absorbance were read at 532 nm to 600 nm. Calculation:- MDA nanomole /ml = 1.56 x 10 5 CM
- 1 M - 1 (1.56 x 10 5 CM
- 1 M
- 1 is extinction coefficient of MDA – TBA complex at 532 n**.
- Estimation of erythrocytes superoxide dismutase activity. (54) Method: - Kajari Das. Reagent Teflon lined tube Supernatant 1.ml. EDTA 0.075 ml.= ( 750 μl ) TBA 0.25 ml. (250 μ l)**
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without 0.069 gm hydroxylamine hydrochloride dissolved in D/W to make volume up to 100 ml.
4. Riboflavin – freshly prepared. 0.05 gm riboflavin dissolved in 100 ml D/W. 6. 50 μm EDTA (18.65 mg ) 0.01865 gm EDTA dissolved 250 ml distilled water. Sample preparation of Hemolysate:- Collected blood was centrifuged for 10 minutes at 3000 rpm. The plasma tube obtained was used for lipid peroxide estimation. Remaining packed RBC’s were washed thrice with normal saline to removes the buffy coat. Hemolysis was performed by pipetting out 1.ml of washed red blood suspension in ice cold distilled water. Erythrocytes ghosts were sediment in a high speed refrigerated centrifuge at 12000 rpm for 40 minutes .The cell contents was separated out carefully and used for superoxide dismutase estimation. 7. Griess reagent – Freshly prepared.
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without Mix equal volume of (0.172 gm) 1 % Sulphanilamide (4. gm ) 5 % phosphoric acid and (0.1 gm) 0.1 % N- Napthylethylnediamine. Procedures:- S.No Reagent Test Control 1 Phosphate buffer 1.110 ml 1. ml 2 Methionine 75 75 3 Hydroxylamine hydrochloride 75 75 4 EDTA 100 100 5 Sample 100 - 6 Buffer ---- 100 Tubes were incubated at 37 C for 5min. 7 Riboflavin 80 80 Tubes were exposed to two 20 W Philips Fluorescent lamps filtered parallel to each other in aluminum foil coated wooden box for 10 min. 8 Griess Reagent 1.ml 1.ml
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without 20 to 40 mg pieces in cadmium granules stored in 0.1 M H 2 SO 4. 2.Glycine NaoH buffer:- Dissolves 15 gm of glycine in de – ionized D/W adjust PH to 9. with 2 mol/ L NaOH solution and take up to 1L It is stable up to for one month at 0 to 8 C O. 3.Sulfanilamide :- Dissolve 5 gm of sulfanilamide in 500 ml of warm 3 mol/ L HCL Solution, then let cool. This stable for one year at room Temperature.
4. N- Napthylethylnediamine: - (50 mg) Dissolve 0.05 gm N- Napthyethylamine in 250 ml double distilled water. (Stable for 2 month at 0 to 8 C o ). 5. Standards:- Per pare working standard by diluting stock 0.1 mol/ L (0.69) NaNo 2 in 10 ml Na 2 B4O 7 one the day of use. (Stock standards for 9 month at Room temperature.) Working standared :- ( 10 μ mol /L).
- Copper sulphate= 5 mmol/L in glycine NaOH buffer. 7. Zinc sulphate = 75 mmol /L.
Study of magnesium levels and oxidative stress in Type 2 DM patients with and without
8.. NaOH = 55 mmol/L. Deproteinization.- In a clean and dry centrifuge tube following were added. Plasma 0.5ml. Zinc sulphate with mixing 2.0 ml. NaOH 2.5 ml. The final pH should be between 7 to 7.5 let stands for 10 min and centrifuge at 3000 RPM for 5 min Activation of cadmium granules:- Rinse the acid from the granules three times with deionized distilled water. Then granules were swirled in 5 mmol/ L CuSo 4 for 1 – 2 min the cuso 4 drained and granules were rinsed thrice with deionized. Drain and use the copper – coated granules within 10 min. Prolonged exposure of the granules to air diminishes their reduces ability. After use granules were rinsed with water and stored in 0.1 M H 2 So 4 solution. These can be regenerated by repeating the above producers.
