MATERLQLS AND METHODS

T he present ~nvesvigations have been designed with a view to explore the

possible bioche~r~ical mechanisms involved in the formation of diabetic

ret~nopathy 'Rioche~iii~:al investigations were carried out of the samples of

following subJects, i e , ( 1 ) individuals without diabetes mellitus or any other

diseases (normal controls); (2) individual with diabetic mellitus, but no other

complication (diabetes without retinopathy); (3) individuals with background

diabetic retinopathy; and (4) those with pre-proliferative diabetic retinopathy.

Blood samples were obtained under fasting conditions from all these

subjects. The normal controls comprises of healthy volunteers from Mahatma

Gandhi University administrative staff and their family members. They did not

show any path~logic~i signs of liver, heart and eye diseases. For the diabetic

without retinopathy, blood samples were obtained from NIDDM subjects attending

Medical College Hospital, Konayam, Kerala. The details of the family history, age,

the duration of diabetics, mode of diabetic control (insulin/dmgs) etc. were noted.

The patients with heart, liver and kidney diseases were excluded. The blood

samples were obtained from retinopathic patients attending Medical College

Hospital, Kottayam and Little Flower Hospital, Angarnaly, Kerala. Retinopathy in

the patients were confirmed by fundal examination.

The subjects were undergone clinical examination under dilated pupil with

ophthalrnoscope and slit lamp. By using ophthalmoscopy and biomicroscopy yields

I~ttlc ~nfortnatlocr ~-e$nrc',iilg blood flow through the retinal nl- choronial \asculature.

Fluorescein angiography was also used for the classification of retinopathy.

Fluorescein angiography of the fundus demonstrate the abnormalities of the

vascular architecture of the fundus before retinal photocoagulation. A sodium

fluorescein solution was injected intravenously and its circulation through the

fundus recorded with a fundus camera. Normal retinal blood vessel walls are

impermeable to sodium fluorescein and retain the dye within the vessels. In

dtabetic retinopathy, leakage of dye through the wall of the new vessel occur due to

the break-down of the blood-retinal barrier. In addition, retinal ischaemia due to

capillary closure shows up as dark under perfused areas on the angiogram.

Venous retinal lesions shows the risk of progress of retinopathy and visual

loss. The first clinical signs of diabetic retinopathy are microaneurysms, which are

saccular outpouchings of retinal capillaries. Ruptured microaneurysms,

decornpensated capillaries and intraretinal microvascular abnormalities results in , . lntraretinal haemorrhages. The clinical appearance of these haemorrhages reflects

the retinal architecture of the retinal level at which the haemorrhage occurs.

Haemorrhages in the nerve fibre layer assume a more flame-shaped appearance,

coinciding with the structure of the nerve fibre layer that tuns parallel to the retinal

surface. Haemorrhages deeper in the retina, at which point the arrangement of

cells is more or less perpendicular to the surface of the retina, assume a pinpoint or

dot shape.

Intraretinal microvascular abnormalities (IRMAs) represent either new

vessel growth within the retina or more likely, pre-existing vessels with endothelial

cell proliferation that become shunts through areas of non-perfusion. IRMA may

be seen adjacent to cotton-wool spots. Multiple IRMAs mark a severe stage of

non-proliferat~ve retinopathy and frank neovascularisation is like to appear on the

surface of the retina or optic disc within a short time.

Venous cal~bre abnormalities are indicators of severe retinal hypoxia. These

abnormalities are venous dilation, venous beading, or loop formation. Proliferative

retinopathy is marked by proliferating endothelid cell tubules. The rate of growth

of these new vessels is variable. They grow either at or near the optic disc or

elsewhere in the retln:a.

3.1 Classification of Diabetic Retinopathy

In the present study, diabetic retinopathy were graded as:

I . NlDDM without retinopathy;

2. Background diabetic retinopathy (BDR) ;

3 . Pre-proliferative diabetic retinopathy (PPDR).

1) NIDDM without retinopathy

These are pure cases of diabetes, developed within a period of 3-1 1 years,

without any renal, hepatic or retinal problem.

2) Backgrountl diabetic retinopathy (BDR)

B D R is remarked by microaneurysms and dot haemorrhages. These

observed as tiny red dots, usually around the posterior part of the retina. This can

be confirmed by Fluorescein anyiography and microaneulysms are identified as

hyperfluorescent dots because of the localization of the dye within their lumina.

In some cases, there is leakage of dye from the endothelid linings of the retinal

vessel's. Dot haemorrhages fails to take up dye and block the fluorescence from

underlying vessels. When these lesions are present in relatively small numbers, the

condition is know1 as background retinopathy (Frank, 1995).

3) Pre-proliferative diabetic retinopathy (PPDR)

Among these, le:sions are greatly increasing numbers of blot haemorrhages

in several areas of the ,-etina. Arteriolar abnormalities shown as narrow arterioles

and surrounded by a white sheath, indicates non-perfused and dilated irregular

veins. Sometimes these veins are so irregular that they look like a string of

sausages or like hairpin loops, i.e., PPDR is characterised by retinal

microaneurysms, haemorrhages, edema, hard exudates, cotton wool spots, venous

beading, reduplication and loops and intraretinal microvascular abnormalities

(Frank, 1995).

3.2 Collection and Processing of Blood Sample

Venous blood samples obtained from subjects under overnight fasting

condition were anticoagulated with disodium ethylenediaminetetraacetic acid

(disodium EDTA) 1 rr~dml of blood. As far as possible, analysis was done on the

same day and remaining samples were preserved at 4OC till all the estimations were

completed as per Table 1

3.2.1 Stability of sample

Table 3.1 Stability of red blood cell enzymes and metabolic intermediates in blood stored in disodium EDTA ( 1 mglml)

I Glucose-6-phosphate dehydrogenase

6-Phosphogluconate dehydrogenase

Transketolase

Glutathione (reduced state)

Glutathione reductase

Glutathione peroxidase

Glutathione S-transferase

Catalase

Su~eroxide dir,mutase

3.2.2 Processing of blood samples

Reagents

1) Ice-cold 0.154 M NaC1.

2) p-Mercaptoethannl-EDTA stabilising solution.

Bringing 0.05 ml of P-mercaptoethanol and 10 ml of neutralised 10%

(0 117 M) EDTA to a volume of 1 litre w ~ t h water.

n) Preparation of packed red cell

T\\o ml of ' t f~c san~ples already collected were cent!-if~~ged at 1000 g fix

15 niin at 4" and :ieparated the plasma and stored for further analysis (diene

conjugate estimation). Buffy coat was removed completely. Erythrocytes were

resuspended in 10 ml of ice-cold 0.154 M sodium chloride solution and centrifuged

at 1000 x g for 15 inin. Removed the supernatant without disturbing the bottom

layer of erythrocyte:j. Resuspend once again in 10 ml of cold 0.154 M NaCl

solution. The suspension was centrifuged at approximately 1000 x g for 10 min

and removed the supernatant. The washing in cold sodium chloride solution was

repeated at least three times. The erythrocytes thus obtained were used for further

analysis.

I?) Preparation .o/ 1:20 haemobzatc

The erythrocytes obtained from the above process were resuspended in

cqual volume of cold sodium chloride. About 0.2 ml of this suspension was added

to 1.8 ml of P-mercaptoethanol-EDTA stabilising solution in a glass tube. Cap1

the tube and immersied in freezing mixture until it was completely frozen and was

then thawed by placing the tube into a beaker containing water at room

temperature. Repeated the freeze thawing until a crystal clear lysate was obtained.

When the haemolyz:ate was completely thawed, it was mixed and the tube was

transferred into ice-cold water and was maintained at O°C. The haemolyzate

prepared in this way was referred as 1 :20 haemolyzate which was used for enzyme

3.3 Storage Stability of Reagents

It 1s convenient to store all reagents in the frozen state. Although some

reagents are qulte stable even at room temperature, freezing prevents bacter~al

growth and mould format~on. There are some reagents wh~ch should always be

stored at 4°C

Reagents stable for several months at 4OC (if no mould forms)

T rls buffers 30% Hz02

Phosphate buffers Ethanol

MgClz Drabk~n's reagent

EDTA Glucose-6-P

NADP 6-Phosphoglucon~c acid (-20°C)

Reagents stable for two to four weeks at 4OC

p-mercaptoethanol-EDTA stabilizing solution (2 weeks)

Precipitating solution for GSH estimation (3 weeks).

EDTA-MnC12 solution for SOD estimation (1 month).

DTNB for GSH estimation (3 months).

Ribose-5-phosphate for TK estimation (3 months).

Reagents to be prepared daily

NADH t-Butyl hydroperoxide

NADPH Dilute Hz02

GSH

Most of the solutions are stored in refrigerated condition

Preparation of NADHINADPH (Beutler, 1975)

NADPFf and hADH are unstable not only in solution but also in the dry

state, at -20°C This is very critical for glutathione reductase (GR) estimation.

In order to get the correct concentration of NADH or NADPH, a solution

containing approximately 2 mglml was prepared. 850 p1 of water and 100 p1 of

I M tris-HCI-EDTA buffer at a pH 8 was taken in a cuvette, and its optical density

(A,,) was measured ai: 340 nm against a water blank. 50 p1 of the NADH or

NADPH solution w ; ~ added. and a second reading (A,) was taken. The

concentration of the pyndine nucleotide in the solution was then

where C is the conceritration (mM)

3.4 Instrumentation

Shirnadzu IJV-spectrophotometer 1601 was used for the measurement of

the enzyme concentration by uslng micro quartz cuvette (Pharmacla).

3.5 Calculation of results (Beutler, 1986)

A. Method of expressing quantities of red cells

The activity of red cell enzymes or levels of red cell intermediates were

expressed in terms of the quantity per gram or milligram of haemoglobin.

. B. Calculation of enzyme activities

In calculating the enzyme activity (E), in international units per gram of

haernoglobin,

1 OOA E = Hb

where A is the number of enzyme units per rnl and Hb is the concentration of

haemoglobin in grarns per 100 rnl in the haemolyzate.

where E - the: rnillimolar extinction coefficient of the indicator substance

(6.22 in the case of the NAD(P)iNAD(P)H system)

N - the number of molecules of indicator converted per molecule of

sut~strate consumed (N = I)

VI, - the volume of haemolyzate added to cuvette in ml

AOD - the change in optical density (absorbance) per minute

T h ~ s equation 1s mainly used for the estimation of G-6-PD, 6-PGD, GR, GSH-Px,

GST and catalase act:~vities.

3.6 Determination of Glucose-6-Phosphate Dehydrogenase (GI-PD, D-Glucose-6-Phosphate: NADP* 1-Oxidoreductase, EC 1.1.1.49) and 6-Phosphogluconate Dehydrogenase (6-PGD, 6-Phospho-D- Gluconate: NAD(P)' 2-Oxidoreductase, EC 1 .I .I .43) Activity

G-6-PD and 6-PGD activity of erythrocyte were assayed by the method of

Beutler (1986)

Principle

G-6-PD catalyses the oxidation of glucose-6-P to 6-phosphogluconolactone

which hydrolyzes spontaneously to 6-phosphogluconate (6-PGA):

Glucose-6-P + NADP 26'D 6-PGA + NADPH + H-

6-PGD catalyzes the oxidation of 6-PGA to ribulose-5-phosphate and COz:

6-PGA + NADP' -6- ribulose-5-P + C02 + NADPH + H'

The G-6-PD activity rneasured by the rate of reduction of NADP to NADPH when

the haemolyzate was incubated with glucose-6-P. Similarly 6-PGD was also

measured by incubating haemolyzate with 6-PGA in another test tube.

Assay

Cuvette

Tris-I-ICI, I M. EDTP,, 5 mM, pH 8 0

MgCI*. 0 I M

NADP, 2 mM

1 20 haemolyzate

Hz0

Udl m m 100 I00 100

100 100 100

100 100 100

20 20 20

680 580 580

Incubated at 37'C for 10 min

- 100 -

- - 100

Read the change in OD per minute at 340 nm

Comments

The difference between the reaction rate in cuvette 1 and 2 showed the

enzyme activity of G-6-PD. Using this assay N = 1, in the calculation of results to

conform with international usage.

The 6-PGA activity was calculated by subtracting the rate in cuvette 1 from

that in cuvette 3, using the equation no. I , where N = 1

3.7 Assay of Glutathione Reductase (GR, NADPH: Oxidised Glutathione Oxidoreductase, EC 1.6.4.2)

GR activity vvas assayed by the method of Beutler (1986)

Principle

GR catalyze:< the reduction of oxidized glutathione (GSSG) by NADPH or

NADH to reduced glutathione (GSH):

NADPH + H i + GSSG GR NADPf + 2 GSH

The act~vity of the enzyme was measured by the oxidation of NADPH

spectrophotometrically at 340 nm.

Assay

Blank (mi') Svstem (ul)

Tris-HCI, 1 M, EDI'A, 5 mM, pH 8.0 50 50

1.20 haemolyzate

HrO

Incubated at 37°C for 10 min

GSSG, 0 O i ? M (nr:ut ) 100

Incubated at 37°C for 10 min

NADPH, 2 mM 50 50

Read the change in OD per minute at 340 nm and calculated the enzyme activity as per equation no 1

3.8 Assay of Glutathione Peroxidase (GSH-Px, GSH: H202 ' Oxidoreductase, EC 1.11 -1.9)

GSH-Px activity was estimated by the method of Beutler (1986).

Principle

GSH-Px catalyzes the oxidation of GSH to GSSG by hydrogen peroxide.

2 GSH + R-4-0--H "'''-& > GSSG + HzO + R-OH

where R-0-0-H IS a peroxide. t-Butyl hydroperoxide is the most suitable

substrate for assay of the enzyme. The rate of formation of GSSG is measured by

means

GSSG + N.4DPH + H ' -- 2 GSH + NADP'

Assay

Blank (ul) Svstem (ul)

Tris-HCI, 1 M, EDTA, 5 mM, pH 8.0 100 100

GSH, 0 I %I

GR, 10 Ulml

NADPH. 2 mM

1.20 haemolyzate

Hz0

Incubated at 37°C for 10 min

t-Butyl hydroperoxide, 7 mM - 10

ODImin was measured at 340 nm and calculated the enzyme activity as per equation no. I

3.9 Assay of Glutathione-S-Transferase (GST, EC 2.5.1 .I 8)

GST activity was measured by the method of Beutler ( 1 986)

Principle

The interaction of foreign compounds with GSH is catalyzed by GST and

the formation of GSH conjugates. Here, GST catalyze the interaction of CDNB

and GSH, resulting in glutathione conjugates

CDNB + GSH CDNB-S-glutathione

Assay Blank ( ~ 1 ) System (LA)

K2H PO~IKH~POJ, 0 . 5 M, pH 6.5 200 200

CDNB in 95% ethanol, 25 mM 20 20

H20 730 680

Incubated at 37OC for 10 min

GSH, 20 mM 50

Mixed well

1 :20 haemolyzate -

Read the in OD/ min at 340 nm.

Cu~nrnents

I n calculating results, N = 1 and E = 9 6, the extlnctlon coeffic~ent of the

CDNB conjugate, in Equatlon ( I )

3.10 Estimation of Catalase Activity (H202: H202 Oxidoreductase, EC 1.11.1.6)

Catalase activtty of erythrocyte was assayed by the method of Beutler

(1 986).

Principle

Catalase catalyzes the break-down of H202 as

The rate 3f decomposition of H202 by catalase is measured

spectrophotometr~cally at 230 nm, since H202 absorbs light at t h ~ s wavelength.

Ethanol was added to stabilize the haemolyzate by breaking down "complex II" of

catalase and H2O2.

Reagents and sample preparation

I . 1:2000 haemolyzate

1 :20 haemolyzate was further diluted to 1:2000 with ethanol. For this, 1 :20

haemolyzate prepared in P-rnercaptoethanol-EDTA stabilizing solution was diluted

further 1 : 100 and :2O pI of absolute or 95% ethanol was added per millilitre of

dilute haemolyzate to break-down any "complex II" which may be present.

2. H 2 0 2 , 10 m M

Measured CID of 0.9 ml of 1 :I0 dilution of 1 M phosphate buffer, pH 7.0 at

230 nm (OD)). Added 0.1 ml of a I :I00 dilution of 30% Hz02 solution to the

above and read Or) (ODZ). Since the extinction coefficient (E) of Hz02 at 230 nm

is 0.071, the H202 {concentration (c) of the 1: 100 diluted peroxide solution is

(OD2 - OD,) mM. Diluted 1 ml of the 1 : 100 dilution to (c) with water.

Assay

B l a n a k - Svstem (ul)

Tris-HCI, IM, EDTA 5 mM, pH 8.0 50 50

H202 10 mM - 900

Hz0 930 30

Incubated at 37" for 10 min

1 :2000 haemolyzate with ethanol 20 20

Measured change in OD at 230 nm

Comments

In calculating results, N = 1 and E = 0.071 in Equation (I) .

3.11 Determination of Superoxide Dismutase Activity (SOD, Superoxide: Superoxide oxidoreductase, EC I .I 5.1 .I )

SOD activiv estimated by the method of Paoletti and Mocali (1990).

Principle

The method consists of a purely chemical reactions which generate

superoxide from molecular oxygen in presence of EDTA, MnClz and

mercaptoethanol. NAD(P)H oxidation is linked to the availability of superoxide

anions in the medium. As soon as SOD is added to the assay mixture, it brings

about the inhibition of nucleotide oxidation. Thus the activity of the enzyme is

measured by the oxidation of NAD(P)H at 340 nm.

Sample preparation

Haemolyzate was prepared by simple lysis. 2.5 ml haemolyzate

(pre-warmed at 37OC) was treated with 1 ml of a mixture of ethanol-chloroform

(2:1, viv) and mixed thoroughly to obtain a thick precipitate. Added 2 ml of

distilled water and mixed again with the vortex. Incubated at 37OC for about

15 min with occasional stirring and then centrifuged to spin down the precipitate.

The supernatant was assayed after suitable dilutions.

Reagents

I Triethanolamir~e - diethanolamine (100 mM each) - HCI buffer (TDB).

Triethanolamine (14.9 p), diethanolamine (10.5 y) and approximately

13.8 ml of corlc. HCI were dissolved in ilitre of distilled water and adjusted

the pH to 7 4.

7 NADH or NA.IIPH (7.5 mM)

Dissolved 20 mg of NADH disodium salt in 4 ml of water.

3 . EDTA-MnCIz (100 mM/50 mM)

(a) Prepared 200 mM EDTA and adjusted the pH to 7 with 1 M NaOH.

(b) Prepared 100 mM MnC12.

(c) Mixed (a) and (b) stock solution in a ratio of 1:l (viv) and

adjusted the pH of the mixture to 7 by drop-wise addition of

10 M NaOH (- 0.14 m1125 ml of reagent)

4. Mercaptoethanol (10 mM) : Diluted 50 ~1 of conc. thiol (14.2 M) with

7 1 ml of walLer.

Assay

TDB

NADH

EDTA-MnCI?

Sample

Sample solvent

Control ( ~ 1 ) Sample (ul)

800 800

40 40

25 25

- 100

100 -

Mixed thoroughly and read at 340 nm against air for a stable base line recorded

ocer a 5 min period. Then added mercaptoethanol I00 111 to each tube. Mixed and

read at 340 nm. T'ne rate of nucleotide oxidation of control, calculated over an

8 min interval shoultl be in the range of 0.12-0.35.

Calculation

One unit of SOD activity is defined as the amount of enzyme required to

inhibit the rate of N.4DH oxidation of the control by 50%. So dilute the sample to

get 50% inhibition.

Sample rate % inhibition =. x 100

Control rate

3.12 Determination of Transketolase Activity (TK, Sedoheptulose-7- Phosphate: D-Glyceraldehyde-3-Phosphate Glycolaldehyde Transferase, EC 2.2.1.1)

TK activity of haemolyzate was estimated by the method of Brin (1974)

Principle

R-5-P and Xu-5-P are used as substrates for TK, the preceding reactions (1)

and (2) form Xu-5-P from R-5-P added to the assay. The two enzymes required for

ieactlon ( I ) and (2) are present excess in erythrocytes TK reaction 1s stopped by

deproteini7atron after a fixed time. the actiwty of the TK 1s determ~ned by anthrone

reaction

Reagents

Sodium chloride Hydrochloric acid, 1 N

Potasslum chloride Magnesium sulphate, MgS047HzO

Sulphuric acid, 66% (wlw) Trichloro acetic acid (TCA)

Potassium hydroxide, 5 N D-Glucose, anhydrous

Anthrone D-Ribose

Orcinol, cryste,lline Ferric chloride, FeC12.6Ii~O

Hydrochloric acid, AR 30% (w/w) Thiourea

Sodium sulphate, Na2S04. 1 OH20

Dipotassiurn hydrogen phosphate (K2HP04)

Ribose-5-phosphate monobarium salt (R-5-P-Ba)

Preparation of solutions

I Buffer (4 mhl ~ a ' , 115 mM K' . 20 mM POJ' , 5 mM M ~ " , pH 7.4).

Mixed 40 ml 0.9% NaCl solution, 1030 ml. 1.15% KC1 solution, 200 ml

1.75% K2HP04 solution (adjusted to pH 7.4 with 1 N HCI) and 10 ml

3.82% MgS04.7H20 solution. pH of the final solution was checked and

adjusted to 7 . 4

2 TCA (7.5%) -- Dissolved 7.5 g in distilled water and made upto I00 ml.

3. Hexose standard solution (0.55 mM) - Dissolved 25 mg D-glucose in 25 ml

dist~lled water Diluted 5 ml of this to 50 rnl with 7.5% TCA. This solution

contains I00 [ ~ g glucoseltnl.

4. Anthrone reagent (2 5 mM anthrone; 0.13 M thiourea) -- D~ssolved 0.5 g

anthrone and 10 g thiourea in ca. 200 ml 66% HzS04 by warming to

60-70°C Allowed to cool and diluted to 1000 ml with 66% HzS04.

Allowed to stand for at least 24 h in the cold before use.

5. Pentose standard solution (67 mM) - Dissolved 25 mg D-ribose in 25 ml

d~stilled water Diluted 1 ml of this to I00 rnl with 7.5% TCA. This

solution contains 10 pg riboselml.

6. Orcinol reagent ( I4 mM orcinol; 0.62 mM FeCI3) - Dissolved 4.0 g orcinol

and 335 mg FeC13.6H10 ~n distilled water, made up to 100 rnl and diluted to

2000 rnl with 30% HCI.

7 Ribose-5-phosphate (47 mM) - Dissolved 3.24 g R-5-P-Ba in 8.5 ml 1 N

HCI, diluted lo 45 ml with distilled water and slowly added 8 ml saturated

sodium sulphate solution. Allowed precipitate to settle in cold and

centrifuged off. Adjusted supernatant fluid to pH 7.4 with 5 N KOH.

Determined the ribose content of the above solution with the orcinol

reaction and then adjusted the concentration to 7 mg ribosefml(47 mM)

Determination of ribose content in R-5-P stock solution

Blank tml) StdLmJ T m r j ]

Pentosc standard (67 mM) - 1.0 -

R-5-P stock .. - 1 .O

Distilled rr-atcr 1.5 0.5 0.5

Orcinol reagent 4.5 4.5 4.5

Mixed thoroughly, placed in a boiling waterbath for 20 min and cooled in

cold water bath for 5 min. Read at 620 nm.

Preparation of sample

Weighed the packed erythrocytes, added the same volume of water and

m~xed. Capped the: tube and immersed it in freezing mixture until it was

completely frozen and was then thawed by placing the tube into a beaker

containing water at room temperature. Repeated the freeze thawing three times.

This haemolyzate can stored for 3 months in freezing condition.

Assay

I-laemolyzate

Buffer ( I )

R-5-P solution (7)

Blank 1 (mi)

0.5

0 6 5

Mixed and incubated for 60 min at 38OC

Mixed, centrifuged and used the supernatant for assay. Then four test tubes were

marked as follows.

Test Blank 1 Standard Blank 2 (mi) (mi) (mu (mu

Supernatant of abovt: test 1 0 - -

Supernatant of above blank 1 - 1 0 - -

Hexose standard solut~on (3) - - 1 0 -

TCA (2) - - - 1 0

Anthrone reagent (4 10 0 10 0 10 0 10 0

Added anthrone with. constant stirring. Heated all the tubes for 10 min in a boiling

water bath, then cooled for 5 min in a cold water bath. Allowed to stand for

20 min in the dark ar~d read at 620 nm against blank 2.

Subtracted the extinction of blank 1 from the test and used for calculation

&sampie C ~ t a n d a r d x 7.15 x 1000 Volume activity = -

AES,"d*d x 60 x 1.0 x 0.5

where AEs,,,,,~, - extinction of sample

AE ,,;,,, dJs,i - extinction of standard

CShlhd conc in standard solution (3 ) in pdml

7 15 - volume in ml after deproteinisation

60 - incubation period in min

1.0 - volume of supernatant in ml used for assay

0.5 - volume in ml of the original sample

3.13 Determination of Transaldolase Activity (TA, Sedoheptulose-7- Phosphate: D-Glyceraldehyde-3-Phosphate Dihydroxyacetone Transferase, EC 2.2.1.2)

TA activity was assayed by the method of Brand (1983).

Principle

T , i

a) Fructose-6-P t erythrose-4-P '--7 sedoheptulose-7P + GAP I'lhf

b) GAP '===i DAP GDH

c) DAP + N A D H + H '-===F glycerol-3-P + NAD'

Transaldolase activity is determined by the GAP formed per unit time from

fructose-6-P which 1s measured according to equations (b) and (c). Under the

conditions described below the rate of NADH oxidation is proportional to the TA

activity. The decrease of absorbance at 339 nm/min is measured.

Reagents and s o l ~ ~ t i o r ~ s

I Triethanolamine buffer (0. I molA; pH 7.6; EDTA, 10 mmolfl) - Dissolved

1.857 g triethar~olamine hydrochloride and 0.372 g EDTA-Na2H2.2H20 in

80 ml water, adjusted to pH 7.6 with NaOH, 1 molfl and diluted to I00 ml

with water.

2. Fructose 6-phosphate (0.15 mol/l).

3. Erythrose 4-phosphate (10 mmolA).

4. NADH (7.5 mmolfl in 1% NaHC03 solution).

5 . Glycerophospha.te dehydrogenase/triosephosphate isomerase (GDH,

30 kUA; TIM, 200 kUA) - GDH (t 170 Ulmg, 25°C) and TIM (2 10000

IJImg, 25OC) from rabbit muscle; 1 .S mg GDH + 0.2 my TIM suspended in

ammonium sulphate, 3 mol/i. Diluted the stock solution 1 + 9 with

ammonium sulphate solution, 3 molA Stored all solution at OA°C.

Assay

Pipetted success~vely into the cuvette (mi).

Triethanolamine buffer 2.50

Fructose-6-P sol~~t ion 0.05

Erythrose-4-P solution 0.05

NADH solution 0.05

GDHmIM suspension 0.05

Mixed and waited for 2 min.

Haemolyzate 0.05

Mixed and read the change in OD/min at 339 nm.

Calculation

Volume activity = 8731 x A,+lA, (UA)

Later i t was expressed in gHb.

3.14 Estimation of Reduced Glutathione Level (GSH)

Est~mation of reduced glutathione level was measured by the method of

Beutler ( 1986)

Principle

Virtually all of the non-protein sulfhydryl group of red cells is in the form of

GSH DTNB is a tlisulphide compound which is readily reduced by sulfhydryl

compounds, forming a highly coloured yellow anion (YA-). The OD of this yellow

substance is measured at 412 nm.

2 GSH + DTNB -+ GSSG + 2YA-

Reagents

1. Glacial metaphosphoric acid

2. Disodium EDTA

3. 5,s'-Dithiobis (2-nitrobenzoic acid) (DTNB)

4. Na2HP04

Preparation of reagents

1 . Na2HP04 solution (0.3 M)

2. Precipitating solution - Dissolved 1.67 g glacial metaphosphoric acid, 0.2 g

disodium EDTA and 30 g NaCl per 100 ml distilled water.

3 . DTNB solution - 20 mdlOO ml in 1% sodium citrate solution.

4. Sham filtrate - It was prepared by adding 3 ml precipitating solution to

2.0 ml water.

Preparation of sample

0.2 ml of blood was added to 2 ml distilled water and mixed rapidly. 2 ml

of the haelnolyzate thus formed was taken for GSH estimation and 0.2 rnl for Hb

estimation,

Assay

So the 2 ml haemolyzate added 3 ml of the preclpltatlng solut~on and mixed

thoroughly Then filtered the content through Whatman no 1 filter paper The

clear filtrate was used for GSH estlmatlon

Blank (UI) System (MI)

NazHP04 (0.3 M) 0.8 0.8

Filtrate - 0.2

Sham filtrate 0.2 -

Read OD1 at 4 12 nm

DTNB solution 0.1 0.1

Mixed Read OD2 at 412 nm

Calculation

The concentration of CiSH in micromoles per gram of Hb(C) is

C ~- - ( O D - O D ) i X - 11 X -~ 5 X -- 100 1000 13600 2a 2b Hb

where OD, - is the optical density measured at 412 nm before the addition of

D M B solution.

OD? - the optical density measured at 412 nm after the addition of

D'INB solution.

I I - final volume of assay system

2a - amount of sample used

5 - Volume of the diluted sample

2b - Amount of filtrate

Hb Conc in d l 0 0 ml lysate

3.15 Estimation of Haemoglobin

Haemoglobin estimation was carried out by cyanmethernoglobin method

(Beutler, 1975).

Principle

In the presence of potassium cyanide at alkaline pH, haemoglobin and its

derivatives were oxidised to methemoglobin. Methemoglobin so formed reacts

with potassium cyanide to form cyanmethemoglobin, a red coloured complex,

which was measured calorimetrically. The colour intensity was proportional to the

haemoglobin concentration of blood sample.

Reagents

1 . Potassium ferricyanide (K3Fe (CN),j)

2. Potassium cyanide (KCN)

7 - . Potassium d~hydrogen phosphate

4. Nonidet P-40 or Sterox SE

5 . Haemoglobin standard

Preparation of Drabkin's solution

Dissolved 200 mg of K3Fe (CN)h, 50 mg of KCN, 1.0 ml of 1 M KH2P04

and I ml of Nonitlet P-40 and made to one litre with distilled water and adjusted

between 7 and 7.4. This can be kept for several months in dark polythene bottle

between 4 and 20°C. Standards were diluted in Drabkin's solution with a range of

concentration fi.orn 5 mg% to 45 mgYo

Procedure

Poured 0.2 ml haemolyzate to 10 ml of Drabkin's reagent and mixed

t l~orou! ! l i !~ Kcp! at room temperature for 4 min Read the absorbance at 540 nm

against Drabkin's solution.

Calculation

'l'he concentration of haemoglobin in grams per 100 mi in any sample is

where ODJ40 - optical density at 540 nm.

V I ~ - the volume of Drabkin's solution used

Vllh - th,e volume of sample added to Drabkin's solution

F I I I ~ -- the calibration factor, which obtained by 11100A1, where A, is

haemoglobin concentration of 10 mg%, i.e., OD540 at which the

calibration curve intersects the haemoglobin concentration of

10 mg% designated Al. Then FHB := 1/100A1.

3.16 Estimation of Glycosylated Haemoglobin (HbAtc)

HbAlc was measured by the method of Chandal~a el al. (1980)

Principle

The interfering substances were removed by washing the red cells 4-6 times

with normal saline. Haernolyzate prepared by using carbon tetrachloride. Hexoses

bound to haernoglobin were quantitatively hydrolysed by heating the haemolyzate

at 100°C in presence of oxalic acid. Resultant chromogen was measured at

443 nm.

Reagents

1 . Normal saline (0.85 g/dl NaCI)

2. Carbon tetrachloride (CC14)

3. 0.3 N oxalic acid

4. 40 ddl , Trichloroacetic acid (TCA)

5 Drabkin's reagent

6 . 0.7 g/dl thiobarbituric acid (TBA)

Procedure

Centrifuged the blood specimen.

Aspirated plasma and buffy coat.

Washed the packed cells by using normal saline for six times.

Added 'A part of distilled water to the packed cells and '/i part of CCI4.

Shaked vigorc~usly and centrifuged for 20 min at 3000 r.p.m.

Aspirated the haemolyzate and determined its haemoglobin concentration.

Adjusted haetnoglobin concentration to 10 g/dl by using normal saline (use

formula CIVI - C2V2).

Added 2 ml of haemolyzate to 1 ml of oxalic acid reagent and mixed well.

Kept in a boiling water bath for one hour. Covered the tubes using cotton

or marbles to prevent loss of water by evaporation.

i ) Cooled the tubes to room temperature and added I ml of TCA reagent.

Mixed thoro~~ghly

j) Centrifuged at 3000 r.p.ln.

k ) Added 2 ml of supernatant to 0.5 ml TBA and mixed well. Kept at 37'C for

40 min.

I ) Readings were taken against blank (2 ml distilled water and 0.5 ml of TBA)

at 443 nm.

OD of test GHb% = - x l

0 079

3.17 Measurement of Lipid Peroxidation

Membrane lipid peroxidation was determined by thiobarbituric acid (TBA)

reactivity by the modified method of Stocks and Dormandy (1 971).

Principle

Malondialdehyde (MDA), an end-product of fatty acid peroxidation can

react with TBA to form a coloured complex having maximum absorbance at

523 nm. EDTA is added to chelate the metals, viz., iron or copper, from the

extract, which othenvise may initiate lipid peroxidation during boiling and results

in falsely elevated TBA reactivity.

Reagents

I . Phosphate-buffered saline (pH 7.4)

Added 8.1 g 1VaC1 + 2.302 g Na2HP04 + 0.194 g NaH2P04 in 1000 ml

distilled water.

2. Trichloro acetlc acid (30%).

3 EDTA (0.1 M)

4 Thiobarbituric acid ( 1 % TRA in 0.05 M NaOH)

0 2 ml of packed erythrocyte was suspended in 0.8 ml phosphate-buffered

saline. To this, 0.5 ml of 30% TCA was added. Tubes were vortexed and allowed

to stand in ice for at least two hours. Tubes were centrifuged at 2000 r . p m for

15 mln. One ml each of the supernatant was transferred into another tube. To this

add 0.075 ml of 0. l M EDTA and 0.25 ml of TBA. Tubes were mixed and kept in

a bo~ling water bath for 15 min. Cooled at room temperature and read the

absorbance at 532 nrn

Calculation

MDA values in nanomoles per millilitre packed cells were determined using

the extinction coefficient of MDA-TBA complex at 532 nm - 1.56 x 10' per cm

per molar solut~on. Later it was expressed in g Hb.

3.18 Diene Conjugate Measurement

Diene conlugates in plasma were measured by the method of Lunec et al.

(1981), where diene: conjugates measured in optical density units and it has been

shown that the predominant DC component of human lipid is octadeca-9,

I I-dienoic acid.

Reagents

1 . Chloroformlmethanol 2: l (vlv)

2. Deionized water.

Procedure

0 5 ml of plasma was m~xed w ~ t h 4 ml chloroform/methanol solut~on for

30 seconds and then centrlfuged for 5 mln at 1500 x g Deionized water (1 ml)

was added to 2 5 ml of the lower phase, vortex m~xed and centrlfuged for 5 mln at

1500 x g Thlr, procedure separated a lower (chloroform) and upper

(waterlmethanol) phase Dlene conjugate concentration was measured in the

chloroform phase at 240 nm

Calculation

Result expressed as optical density unit per ml (OD unitlml).

3.19 Statistical Methods

Results are stated as mean + standard devlation In order to compare

d~fferent groups, unpa~red 't'-test was used (Rao, 1996)

X I '-- X Z Test criterion (I) = -------

SE[XI - X!]

Standard error of ( x I u 7 ) was measured by

where xi = meim of first sample ~~. x2 mem of second sample

SE standard error

S D ] ~ = variance of the first sample

S D ~ ~ = variance of the second sample

n~ - tir:;t sample size

n2 = second sample size

Degree of freedom (ti0 = nl + n2 - 2.