The 5 th International & 10 th National Congress on Quality Improvement in Clinical Laboratories

Serum Superoxide dismutase activity in thalassemia patients and healthy subjects with p y j

new method

Elham Ghahramanlu, Abdollah Banihashem, Vahid salmasi,Shima Tavallaie,

M jid Gh M b hMajid Ghayour-Mobarhan

Blood Transfusion Research Center, High Institute for Research and Education in Transfusion

Medicine, Tehran, Iran

1

INTRODUCTION

Secondary iron overloadP i it ti f l h l bi h iPrecipitation of alpha-globin chain

Free iron , Drugs, Hypoxia

O2

SOD

H OH2O OHºFe 2+

H2O2

CatalaseGPX

2 OH

2

Cell damage

SUPEROXID DISMUTASE REACTION

3

INTRODUCTION

4

ACTIVE SITES OF SOD

5

ACTIVE SITES OF SOD

6

INTRODUCTION

7

AIM OF STUDY

h l i iThalassemic patients:

Increased of Reactive oxygen speciesyg p

Tissue injury

Zinc and copper defficiency

8

METHODS

Variables Males Females

n 79 61

Age (years) 14 13±4 59 15 05±4 303Age (years) 14.13±4.59 15.05±4.303

Blood samples were collected after 10h overnigh fasting, centrifuged and stored in a -20 freezer.

SOD activity was measured on 140 thalassemic patients and y p140 healthy subjects using microassay based on the inhibition of pyrogallol oxidation.

9

PROTOCOL

Cacodylic acid (Assay buffer) (C2H6AsNaO2.3H2O)

Cacodylic acid+ DW +Tris buffer PH=8.5

+pyrogallol+pyrogallol

Odd rows Even rows

Read the absorbance(405)( )After 60 min

Read the absorbance(405)

10

Read the absorbance(405)

METHODS

11

METHODS

BlankBlankA

BlankBlankB

S l 1S l 1C Sample 1Sample 1C

Sample 1Sample 1D

Sample 2Sample 2E

Sample 2Sample 2F Sample 2Sample 2F

Sample 3Sample 3G

12

Sample 3Sample 3H

CALCULATION

Mean Abs with pyrogallol 60' Mean Abs with pyrogallol 1'1 Mean Abs with pyrogallol 60 –Mean Abs with pyrogallol 1

Mean Abs without pyrogallol 60' –Mean Abs without pyrogallol 1'

1

2

- = /21 2- /21 2

13

RESULTS

Thalassemia group Control group P ValueThalassemia group Control group P - Value

N 140 140

SOD 1.027±0.019 0.98±0.012 0.046

Zinc 60 11±19 01 87 09±23 68 0 000Zinc 60.11±19.01 87.09±23.68 0.000

copper 90.45±29.8 105.59±32.51 0.000

PAB (HK) 78.76±25.58 70.16±19.93 0.004

14

15

METHODS

Superoxide Dismutase activity is measured as the inhibition of p ythe rate of reduction of Cytochrome c by the superoxide radical, observed at 550 nm:

Cytochrome c (oxidized) + O2- ·→ Cytochrome c (reduced) + O2

Th id di l i d d i ll b h iThe superoxide radical is produced enzymatically by the reaction with xanthine oxidase:

Xanthine + O2 + H2O → Uric acid + O2-. + H+

16

CURRENT METHODS

X/XOD/Cyt c3+ method McCord and Fridovich (1969)X/XOD/Cyt c3+ method McCord and Fridovich (1969)

and Crapo (1978).

X/XOD/NBT method follo ing the method of Bea champ andX/XOD/NBT method following the method of Beauchamp and

Fridovich (1971) as well as that of Oberly and Spitz (1984).

pyrogallol autoxidation method following the procedure of

Marklund (1974).

One unit of SOD is described as the amount of enzyme required to

cause 50% inhibition of pyrogallol autoxidation

17

py g

CURRENT METHODS

Winterbourn (1975), YI Sun, LarryW.

Is based on the ability of SOD to inhibit the reduction of NBT

by superoxide In this method :by superoxide. In this method :

Increased the concentration of xanthine oxidase

I d th C Cl t ti t 0 8 l/L tIncreased the CuCl2 concentration to 0.8 mmol/L to ensure

full termination of the reaction.

18

CURRENT METHODS

Spectrophotometric assay introduced by McCord & Fridovich

(1969).

In this assay high pH of 10 :y g p

The further increase in specificity,

High PH: a 4 fold increase in sensitivity comparedHigh PH: a 4-fold increase in sensitivity compared

with the classical SOD assay, carried out at pH 7.8

h l i f h º di l dThe complete trapping of the O2- radicals, accured at pH 10

19

SOD KITS

2O Furmazan dyeXantin + O2

2O2 Furmazan dye

Xantin oxidase

H2O2 2Oº-2 Tetrazolium salt

Xantin oxidase

Uric acidO 2

SOD

Tetrazolium salt

O2+H2O2

20

COMPARISON OF SOD METHODS

The X/XOD/Cyt c3 c method was applicable only to dialysed

crude tissue homogenates.

The X/XOD/NBT method, either with sodium carbonate

solution, pH 10.2, or potassium phosphate buffer, pH 7.8, was not

applicable to tissue.applicable to tissue.

In order to eliminate interference by cytochrome c oxidases and

id l t ti f id i hibit hperoxidases, low concentrations of cyanide as an inhibitor have

commonly been used (Salin et al., 1978; Geller & Winge, 1983).

21

CURRENT METHODS

Higher concentrations of KCN significantly inhibit Cu, Zn-SOD. g g y ,

Azzi et al. (1975) recommended the use of acetylated

ferricytochrome c in place of ferricytochrome cferricytochrome c in place of ferricytochrome c.

Besides ferricytochrome c, NBT is also used as a detector of O-2

d b h / O ( h d id i h 19 1)generated by the X/XOD system (Beauchamp and Fridovich, 1971).

Misra and Fridovich (1972) reported an assay for SOD based on

the ability of SOD to inhibit the autoxidation of epinephrine at

alkaline pH.

22

DISCUSSION AND CONCLUSION

The increased SOD activity in thalassemia :y

It is response to superoxide generated in greater amounts.

Our study results suggest that iron overload causesOur study results suggest that iron overload causes

peroxidative damage in β-thalassemia and antioxidant

systems try to lower tissue damage.

23

ADVANTAGESSimple, rapid, and sensitive method

Cu,ZnSOD and MnSOD, Fe SOD

Suitable for routine clinical use

Is not affected by the concentrations of ascorbic acid and

glutathione present in tissue homogenates

Sample = 20µ

In different tissuesIn different tissues

Identification of SOD on polyacrylamide gels after

l t h i

24

electrophoresisThe assay is best carried out in 200 λ, rather than 100 λ

ACKNOWLEDGMENT

We express appreciation to the staff of

Mashhad University of Medical Science Bu-Ali Research InstituteMashhad University of Medical Science Bu-Ali Research Institute

25dr sheikh Hospital