ESTIMATION OF ANTIOXIDANT ENZYME(CATALASE): A BIOMARKER

FOR TYPE 2 DIABETES

DEPARTMENT OF BIOTECHNOLOGY

INTERNAL GUIDE EXTERNAL GUIDECh.N.VANI DR. KAISER JAMIL

SHAYISTA NAAZ09J21A2316

AIM AND OBJECTIVESAim is to estimate the effect of antioxidant (catalase)

activity in Type2 Diabetes.

To identify and isolate human genomic DNA and amplify the antioxidant (catalase) gene using specific primers for catalase gene with polymerase chain reaction.

To use antioxidant (catalase) as a potential biomarker for type 2 diabetes.

INTRODUCTIONDIABETES:Type I diabetes:In type 1 diabetes, the pancreas

undergoes an autoimmune attack by the body itself, and is rendered incapable of making insulin.

Type II Diabetes:“Insulin resistance”When sugar cannot enter the cells

they start building up in the blood at high amounts in the blood stream is called “hyperglycemia”.

ROLE OF ANTIOXIDANT IN DIABETES:Anti-oxidants are involved to reduce diabetic complications

because they are involved to fight or destroy free radicals. These free radicals in hyperglycemia by glucose toxicity, glucose

auto oxidation and oxidative phosphorylation produces ROS and releases free radicals and these are involved in oxidative stress and leads to destroy the pancreas.

The beta cells are inhibited by their function and destroyed by free radicals.

The above process will prone to diabetic complications so the antioxidants role here is to inhibit activity of free radicals and avoiding diabetic long-term complications.

MECHANISM OF ACTION CATALASECatalase is a common enzyme found in nearly all living

organisms that are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen.

Catalase has one of the highest turnover numbers of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second.

catalase 2H2O2 2H2O + O2

Function of CAT geneThis gene encodes catalase, a key antioxidant enzyme in the

bodies defense against oxidative stress.Catalase is a heme enzyme that is present in the peroxisome of

nearly all aerobic cells.Cytogenetic Location : 11p13The CAT gene is located on the short (p) arm of chromosome 11 at

position 13. It contains 13 exons CAT Gene located 11p13

MATERIALS AND METHODBiochemical analysis of antioxidant (catalase) Estimation of antioxidant enzyme Molecular analysis of antioxidant (catalase)

Isolation of genomic DNA

Quantification of DNA

Amplification of DNA

ESTIMATION OF PROTEIN1 ml of lowry stock +10 μl of serum sample + incubation for 30

min

100 μl of F.C reagent + incubation for 30 min

Analyzing using UV-VIS spectrophotometer at 595 nm

ESTIMATION OF CATALASE790 μl of water+200 μl of reagent+ 10 μl of serum

Analyze using UV-VIS spectrophotometer at 240nm

Calculate the protein content

300 μl of blood sample + 800 μl of TKM1 and 1 drop of 100% Triton X 100 +incubate d for 5 min

Centrifuged at 10000 rpm for 5 minutes + then supernatant was discarded.

To the pellet 800 μl of TKM1 repeated for 2-3 times till white pale pellet is formed

To the pellet 800 μl of TKM1 repeated for 2-3 times till white pale pellet is formed

300 μl of TKM2 and 80 μl of 10% SDS + incubation for 30 min

80 μl of 6M NaCl + tapped for 5 min + centrifuged at 10000 rpm for 5min

GENOMIC DNA ISOLATION FROM BLOOD

Supernatant was transferred carefully to 680 μl of cold absolute ethanol + Centrifuged at 10,000 rpm for 5 minutes + discard

supernatant.

300 μl of 70% absolute ethanol was added to the DNA pellet + Centrifuged at 10,000 rpm for 5 minutes + Air dried the pellet.

To the dried pellet, 50 μl of TE buffer was added for hydration of DNA and preserve at freezing temperature.

Detection of DNA in the isolated samples using 1% of Agarose gel electrophoresis.

DNA is isolated successfully from both normal and diabetic patients blood samples.

QUNTIFICATION BY SPECTROPHOTOMETER

The isolated DNA is of pure quality and is not contaminated with proteins, RNA and other contaminants, since A260/A280 lies between 1.8 to 2.0. Which shows DNA is of pure form.∼

Polymerase chain reaction(PCR):The DNA obtained were used as templates for PCR using

specific primers.

Reagents Volume

Water 18.3µl

Buffer 2.5µl

dNTPs 1µl

Forward Primers 1µl (20 pmol/µl)

Reverse Primers 1µl (20 pmol/µl)

Taq Polymerase 0.2µl

DNA sample 1µl

The reagents as mentioned in the proportions were added to the nuclease free PCR tubes and placed them in a cycler and the following PCR conditions were maintained:

Stage 1: 94oC – 5 min Stage 2: 35 cycles Step 1: 95oC – 1 min Step 2: 60oC – 1 min Step 3: 72oC – 1 min Stage 3: Step 1: 72oC – 5 min Step 2: 15oC 

CONCLUSION

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