Presented byRoll. -VU/PG/BIC-IVS No.-104Reg. No.-00110 of 2014-15

MSc. – BiochemistrySession- 2014-2016

Vidyasagar University

Project Guide

Dr. Sutapa MukherjeeAssistant Professor, Department of

zoologyVisva-Bharati, Santiniketan

WB

•Diabetes mellitus is the third leading cause of death worlwide.It is a chronic ,lifelong condition that affects our body´s ability to use the energy found in food.

•The body requires insulin to break down the glucose a carbohydrate that we take through food. Glucose fuels our cells in body. In case of diabetes mellitus the body cannot make insulin. So the cells can´t take the glucose.

•The glucose level increases in the blood and exceeds the normal range (80-120 mg/dl) and causes diabetes mellitus.

Introduction:

Regulation of blood glucose by insulin and glucagon

Mechanism of action of insulin:

Fig: Insulin mediated signal transduction

Glucose transport mechanism of insulin

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190

Hypoglycemic effects Hyperglycemic effects

Fasting(<100mg/dl)

p.p(<130mg/dl)

GlucagonGluconeogenesis↑Glycogenolysis↑EpinephrineGlycogenolysis↑ThyroxinGluconeogenesis↑GlucocorticoidGluconeogenesis↑Glucose Utilization↑Growth hormone and ACTHGlucose uptake↓Glucose utilization↓

Glucose uptake↑

Glycolysis↑

Glycogenolysis↑

HMP Shunt↑

Lipid Synthesis↑

Gluconeogenesis↑

Glycogenolysis↑

Renal Threshold

to Urine

Blood glucose (mg/dl)

Fig: role of hormones in blood glucose regulation

Symptoms of Diabetes Mellitus

Diagnosis of Diabetes:A1C Test

GTT

Glucosuria

•Neropathy

•Nephropathy

•Retinopathy

•Skin complications

•Heart problems

Effects of Diabetes:

Project Work Report

Catalase is a regulator of hydrogen peroxide metabolism that can , in

excess ,cause serious damage to lipids,RNA and DNA. Catalase converts H2O2

catalytically into water and oxygen and thus neutralizes it. In case of catalase

deficiency, beta cell of pancreas that contain large amount of

mitochondria,undergoes oxidative stress by producing excess ROS that leads to β

cell disfunctionand ultimately diabetes while investigating hyperglycemia-induced

functional changes,hydrogen peroxide production, superoxide, mitochondrial

membrane polarization, and gene expressio fingerprints of related enzymes in

endothelial cells suggest that hyperglycemia increased hydrogen peroxide

production and down-susceptible to oxidative stress leading to the development

of diabetic complications.

Diabetes and Catalase

Recent data suggests that at low conc. hydrogen peroxide acts as a cellular messanger

in insulin signaling, whreas at high conc. it is toxic ,particularly in pancreatic cells which

are catalse poor.

Life long increased hydrogen peroxide concentration, due to catalase gene mutations,

may be a risk factor for type 2 diabetes.This risk may be due to peroxide damage of

normally catalase-poor pancreatic β cells. In type 2 diabetic subjects without known

catalase gene mutations, blood catalase seems to be down regulated.One possible

explanation for this phenomenon may be that increased levels of hydrogen peroxide in

muscle cells due to decreased blood catalase may favour insulin signaling via

inactivation of the oxidation-sensitive tyrosine phosphates that could not

dephosphorylate insulin rec eptors.

EC 1.11.1.6

EC 1.11-Acting on peroxide as acceptor

EC 1.11.1 –peroxidases

Hydrogen-peroxide; hydrogen-peroxide oxidoreductase

Co-factors: Mn2+, heme

It catalyses the decomposition of hydrogen peroxide to water and oxygen

2H202 H20+O2 Dependance on pH:

Optimum pH for human is 7.

Catalase

Molecular mechanism of catalase:

Methods Used in work:

Lowrys Assay of total Protein Estimation

Catalase Enzymic Assay

Materials required:

Heart and liver tissue sample of normal(control )and treated(diabetic) mice

Heart samples:C6H2 (control) &T5H1(treated)

Liver samples:C5S2 (control)& T5L1(treated)

Catalase enzymic assay:

Catalase was assayed following the method of Cohenet. al [11].

Principle:

Catalase (EC 1.11.1.6) catalyses break down of H2O2 to water and oxygen. The

decomposition of H2O2 can be followed at 240 nm.

Reagents required:

•Potassium phosphate buffer, pH=7.0(50mm)

•Absolute ethanol

•Triton-x-100(10%,v/v)

•Hydrogen peroxides (H2O2, 12 mM) prepared in potassium phosphate

buffer, pH=7.0(50mM)

•Prior to assay, PMF was treated with ethanol in order to

inhibit compound formation.

•Briefly, PMF (0.25ml) and potassium phosphate

buffer(0.20ml) were taken in a microfuge in ice.

•To this absolute ethanol (5µl) was added and properly

mixed. This was kept in ice for 30 minutes.

•After incubation triton-x-100(50µl) was added.

•Freshly prepared H2O2 in buffer (2.9ml) was pipette into

glass tubes kept in water bath at 25 c. ͦ

Procedure:

•The solution was quickly transferred to quartz cuvette for

measurement.

•Decrease in absorbance was recorded at 240 nm in UV-VIS

spectrophotometer at every 15s interval till 2 min.

•The above treated PMF sample (0.1ml) appropriately diluted

to 80 as to contain 25-50µg protein was then added to the

tube.

Enzyme activity was calculated taking 43.6M-1cm-1 as molar

extinction co-efficient of H2O2, and expressed as nkatal/mg

protein (1katal=1mol/s).

The principle behind Lowry’s method of determining protein concentration

lies in the reactivity of the peptide nitrogen with the copper ions under alkaline

condition and subsequent reduction of the

folinciocalteauphosphomolybdicphosphotungstick acid to heteropolymolybdenum

blue by the copper catalyzed oxidation of aromatic acids. The Lowry method is

sensitive to pH changes and therefore pH of the assay solution should be

maintained at 10-10.5.

The Lowry method is sensitive to low concentration of protein. Dunn

[1992] suggests conc. ranging from 0.10-2 mg of protein per ml while Price [1996]

suggests conc. of 0.005-0.10 mg of protein per ml .The major disadvantage of

Lowry method is the narrow pH range which is accurate. However we will use very

small volume of sample, which will have little or no effect on pH of the reaction

mixture.

Lowrys assay of total protein estimation

Principle:

A variety of compounds will interfere with Lowry procedure.

These include some amino acid derivatives, certain buffers, drug

lipids,sugars,salts,nucleic acid and sulphyhydril reagents. The

ammonium ions,zwitter ionic buffer and thiol compound may also

interfere with the Lowry reaction. These substances should be

removed before running the Lowry assay

Reaction mechanism

Reagents required:

A.2% Na2CO3 in 0.1 ml NaOH

B. 1% Sodium potassium tartarate in H2O

C.0.5% CuSO4.5H20 in H20

D.Reagent I: 48 ml of 1A, 1 ml of B, 1 ml of C

E: Reagent II: 1 part Folin Phenol [2N]:1 part water

Bovine Serum Albumin standard: 1 mg/ml

Procedure:

0.2 ml of BSA working standard in 5 test tubes and make up to 1 ml using

distilled water.

•The test tube with 1 ml distilled water serves as blank.

•4.5 ml of reagent I was added and incubate for 10 minutes.

•After incubation 0.5 ml of reagent II was added and incubated for 30 minutes.

•The absorbance at 750 nm was measured and the standard graph was plotted.

•The amount of protein present in the given sample was estimated from the standard

graph.

The protein content of each sample is estimated before performing the catalase assayThen the catalase assay was carried out of each sample.The catalase activity was measured at 240 nm at 15s interval using UV-VIS Spectrophotometer

time time

Observation:

Sample heart:

Absorbance at 240 nm at 15s interval

Sample- Liver

Liver sample(treated)

time time

Absorbance at 240 nm at 15s interval

C5S2 (control) T5L1(treated)

Liver sample(control) Liver sample(treated)

timetime

timetime

Comparative study of Catalase activity in tissues :

The Enzyme activity was expressed in nkatal/mg

It is observed from the bar diagram that the catalase activity is increased in heart than the liver.while among the individual tissues the activity is increased in case of diabetic i.e. treated tissue sample.

Summarized bar diagram of catalase activity in tissues

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in

diabetes. Catalase is biomarker of oxidative stress in diabetes mellitus. It is

believed that oxidative stress plays important role in the development of vascular

complications in diabetes particularly type 2 diabetes.As it can be observed that

there was only slight increase in catalase activity in the liver of diabetic animals,

the difference was well marked in the case of heart sample. This shows that the

enzyme activity is increased. Due to less number of samples, statistical significance

could not be ascertained. However, it is evident from the result that the enzyme is

upregulated and this might be a very important adaptive response to oxidative

stress in diabetes. It is known that higher amount of reactive oxygen species can

stimulate the antioxidant enzyme catalase.

Discussion:

Hydrogen peroxide is one of the component of oxidative stress and is

enzymatically processed by catalase and catalase deficiency in blood

is known as acatalasemia.Increased activity of catalase in heart tissue

indicates increased oxidative stress in heart . This is one of the reason

for several cardiovascular complications in diabetes. Thus it can be

concluded from the work on the study of the effect of diabetes on the

activity of an antioxidant enzyme catalase that catalase serves as a

biomarker of oxidative stress in diabetes mellitus which shows higher

amount of oxidative stress in heart which causes several

cardiovascular disease in heart.

Conclusion:

•Biochemistry-U.Satyanarayan,U.Chakrapani .Publisher &Co-publisher:Elsivier and Books and Allied pvt.ltd.4th edition P-672,675,676,679-682,ISBN-978-81-312-3601-7

•Cohen G, Dembiec D ,Marcus J (1970) measurement of catalse activity in tissue extracts.Anal.Biochem.34:30

•Diagnosis and classification of Diabetes Mellitus-Diabetes care.2010Jan;33(suppl l):S62-S69.PMCID :PMC 2797383-American Diabetes Association.

•Giacco F and Brownlee M (2010) Oxidative Stress and Diabetic complications.Cir.Res.107:1058-1070

•Halliwell B, Gutteridge JMC.Free Radicals in Biology and Medicine, 3rd ed, Oxford University press, New York,pp.617-783(1991).

•http://www.wikipedia.org

•http://www.diabetes.co.uk

References:

•Lowry OH,Roserberg NJ ,Farr AL, Randall RJ (1951) Protein measurement with the Folin-phenol reagent. J.Biol.Chem.193:265-275

Maritim AC, Sanders RA and Watkins 3rd (2003) Diabetes, Oxidative Stress and antioxidant review.J.Biochem.Mol.Toxicol.17:27-38

•principles of physiology and Anatomy-Gerard.J.Tortora,Bryan Derrickson- 12th edition. John & Willey, P-683,ISBN-978-0-470-08471-7

•The current state of diabetes mellitus in India Australas Med J. 2014; 7(1): 45–48.Published online 2014 Jan 31. PMCID: PMC 3920109. Seema Abhijeet Kaveeswar and Jon Cornwall

Thank you