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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