3. material and methods - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/25200/8/08_chapter...

Investigation of Insulin Like Protein From Spirulina

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3. Material and Methods

3.1 Collection of Microorganism

23 different Spirulina strains were procured from National Centre for Culture Collection

and Utilization of Blue Green Algae, IARI, New Delhi; Department of Botany, Universi

ty of Madras, Tamilnadu; Allahabad University, Allahabad National Facility for Marine

Cyanobacteria, Bharathidasan University, Triuchirapalli and Central Food

Technological Research Institute, Mysore.

3.2 Growth medium

Zarrouk´s medium was used for all fresh water strains (Zarrouk 1966) and ASN-III

medium was used for marine strain (Rippka 1988). Culture medium was autoclaved

(Yorco, India) maintaining 15lb/in2 or Kg/cm2 pressure for 15 minutes. Their

compositions are as follows__

A. Zarrouk´s medium

S.No. Chemicals g/liter

1. Sodium bicarbonate 16.8

2. Di potassium hydrogen phosphate 0.5

3. Ferrous sulphate 0.01

4. Sodium nitrate 1.5

5. Potassium sulphate 1.0

6. Sodium chloride 1.0

7. Magnesium sulphate 1.2

8. Calcium chloride 0.04


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B. ASN-III medium

S.No. Chemicals g/liter

1. Sodium nitrate 0.75

2. Di potassium hydrogen phosphate 0.75

3. Magnesium sulphate 3.5

4. Calcium chloride 0.5

5. Citric acid 0.003

6. Ferric ammonium citrate 0.003

7. EDTA 0.005

8. Sodium carbonate 0.02

9. Sodium chloride 25.0

10. Trace metal mix 1ml

Trace Metal Mix

S.No Chemicals g/litre

1. Boric acid 2.86

2. Manganese chloride 1.810

3. Zinc sulphate 0.222

4 Sodium molybdate 0.0390

5. Copper sulphate 0.0790

6. Cobaltous nitrate 0.0494

3.3 Sterilization

All the glasswares were thoroughly washed with HCl, liquid detergent (Teepol) and tap

water. Final rinsing was done with double distilled water. Dried conical flasks were


43

plugged with cotton and wrapped with aluminium foil and then steam sterilized in an

autoclave at 121˚C under 15Ib/cm2 pressure for 20 minutes.

3.4 Inoculation

Initial concentration of the cells was adjusted at optical density OD 560 = 0.1. For initial

inoculation 100 ml sterile medium was taken in 250 ml Erlenmeyer flasks and were

aseptically inoculated with 25 ml inoculums. For their bulk production inoculation was

done in 1L and 2L flasks.

3.5 Culture condition

The Spirulina strains were raised in BOD shaking incubator providing a light intensity

of 50 µ mol photons/m2/s light intensity 12:12 light/dark regime, 30±1ºC temperature.

Cultures were manually stirred twice daily. Spirulina biomass were harvested in their

late log phases of growth by filtration through nylon cloth. Harvested biomass were

thoroughly washed with distilled water to remove adhering salts and transferred into

sterilized Falcon tubes. These were properly corked and placed in refrigerator (- 20oC)

for one day. Stored harvested biomass was Lyophilised and then transferred in sterilized

Falcon tubes. Properly parafilm wrapped tubes were stored in refrigerator at - 20oC for

insulin like protein extraction.

3.6 Chemicals procurement

A highly purified antibody (cat no.GGHL-60P) against human insulin raised in guinea

pigs and a peroxidase conjugated guinea pig anti-IgG antibody (GTX82959) raised in

goats were purchased from GenX Bio, India, streptozotocin (STZ) from HIMEDIA


44

(RM1758), India. Rest of the reagents (Analytical grade) used were purchased from

Merck, India & Himedia, India.

3.7 Instruments used

Autoclave (Yorco, India); UV-VIS spectrophotometer – Specord 200 (Analytikjena);

Incubator (Narang Scientific, India); Incubator shaker (JSR, Korea); Labconco

Lyophiliser (made in England); pH meter (Scientific Systems Control dynamics, India);

Balance (Sartorius Analytic, Germany); Homogenizer (Pro 200, Pro Scientific Inc.,

USA); Ice machine (Ice Flaker, Angelantoni, Italy); Centrifuge (Sigma Centrifuge 1-

15k, Germany); HPLC system (Shimadzu class –VP V 6.14 SP2); Syringe filter

(Millex®, Millipore corporation); Electrophoresis unit (BIO-RAD Laboratories, USA);

ELISA Reader (BIO-RAD Laboratories, USA); Semidry Western Blotting Device (BIO-

RAD Laboratories, USA); NMR (Bruker, Germany); Milli Q Water (SG, Ultra Clear

TWF UV plus Water system, Germany); Magnetic stirrer (Yorco, India); Gel shaker

(Rocker 100, Bangalore Genei); Molecular Imager® (BIO-RAD Laboratories, USA);

Spectropolarimeter (Jasco J-715).

3.8 Extraction of insulin-like protein (Khanna et al. 1976)

Five grams of fresh Spirulina biomass was used for extraction of insulin like protein

with a solution consisting of 2 ml distilled water, 10 ml 95 % ethanol and 0.72 %

concentrated sulphuric acid. After shaking for 20 min, 13 ml water and 40 ml 95 %

ethanol were added and the pH was adjusted to 1.7 with sulphuric acid. The suspension

was then filtered and the filtrate adjusted at pH 3.0 with ammonium hydroxide. To this

suspension 150 ml of 95 % ethanol and 200 ml diethyl ether were added. After standing


45

for 12 hours at 4oC the suspension was centrifuged at 3000 g for 10 min and the

sediment was washed with acetone and diethyl ether before dissolving in 25 % ethanol,

and pH was adjusted to 8.5. To this solution 100 µl of 1 M zinc chloride was added and

the preparation left to stand for 18 hours at 25oC to precipitate insulin like proteins. The

precipitate was collected by centrifugation after 18 hours.

3.9 Screening by ELISA (Gebara et al. 1995)

The presence of insulin like protein in Spirulina extracts were determined by qualitative

assay known as ELISA. The positive control insulin (bovine insulin, 1µg/100µl),

negative control (buffer without insulin) and the above extracts, all prepared in 0.05 M

carbonate/bicarbonate buffer, pH 9.6, were pipetted into the 96 wells of the plate (100 µl

of each solution) and left to stand for 16 hr at 4 ºC. The solution was then discarded and

the wells were washed for 1hour (changing the solution every 10 min) with Tween 20

dissolve in PBS (buffer A). After washing, 300 µl of a blocking buffer (1% BSA

dissolve in PBS) was added to the wells for 1hour. The plate was again washed for 1

hour as described above. 50 µl of the anti-human insulin antibody solution (1:5000

dilution) prepared with blocking buffer was added to the wells and left to stand at room

temperature for 45 min. The plate was then washed with buffer A for 30 min, with every

5 min changing. Then 50 µl of peroxidase-conjugated anti-IgG (1:2000) in blocking

buffer was added to the wells and incubated at room temperature for 45 min. The plate

was then washed four times with buffer A for 5 min each time. The substrate of

peroxidase was then added and the plates were left to stand for 10 min in the dark at

room temperature. The reaction was stopped by the addition of 50 µl of H2SO4 and the

absorbance was read with ELISA reader (Biorad) at 490 nm.


46

Reagents for ELISA

Carbonate buffer pH 9.6 100 ml

Sodium carbonate 159 mg

Sodium bicarbonate 293 mg

Distilled water 100 ml

Phosphate buffer saline (PBS), pH 7.4 1000 ml

Sodium chloride 8 g

Potassium dihydrogen phosphate 0.2 g

Disodium hydrogen orthophosphate 1.15 g

Potassium chloride 0.2 g

Distilled water 1000 ml

Blocking solution 100 ml

BSA 1 g

PBS 100 ml

Washing buffer 1000 ml

Tween 20 0.5 ml

Phosphate buffer saline (PBS) 1000 ml

Substrate buffer 10 ml

Citric phosphate buffer 5.5 pH (100 mM) 10 ml

H2O2 10 μl

O-Phenylene Diamine (OPD) 10 mg


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3.10 Characterization of insulin-like protein

3.10.1 SDS-PAGE (Laemmli. 1970)

SDS–PAGE was carried out on using a 15% (w/v) polyacrylamide gel and with 1.5 mm

thick spacer. Samples were extracted (1:10) with a solution of 4 % SDS, 12 % glycine,

50 mM Tris-HCl, 35 mM EDTA, pH 6.8, and 0.01 % bromophenol blue and manually

shaken at regular intervals (10 min) for 2 hours at 4ºC. The prepared protein extracts

(50µl) and commercial bovine insulin (10 µg, mixed in sample buffer in a ratio of 1:4)

was loaded in the wells of polyacrylamide gel. Electrophoresis was run for 1 h at 30 mA.

Protein bands were visualized by Coomasie Brilliant Blue R-250 (HI-MEDIA, India)

staining and followed by overnight de-staining solution. The gel was scanned by

Molecular Imager®.

Reagents for PolyAcrylamide Gel Electrophoresis

Separating gel buffer (1.5 M Tris- Cl pH 8.8)

Dissolved 181.8 g of Tris base in distilled water. Adjusted the pH to 8.8 with

concentrated HCl / 1N NaOH and made the volume to 1000 ml.

Stacking gel buffer (1 M Tris-Cl pH 6.8)

Dissolved 121.14 g of Tris base in distilled water. Adjusted the pH to 6.8 using HCl / 1N

NaOH and made the volume to 1000 ml.


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30 % Acrlyamide solution

Dissolved 29 g acrlyamide and 1 g Bis-acrlyamide in distilled water and volume was

made up to 100 ml. Filtered the solution through activated charcoal and degassed. Stored

at 4o C.

10 % Ammonium per Sulphate (APS)

Dissolved 0.1 g ammonium per sulphate in 1 ml distilled water just before use.

Stacking gel solution (5%) 5 ml

Water

30 % Acrylamide

1.0 M Tris buffer ( pH 6.8)

10 % Sodium dodecyle sulphate (SDS)

10 % Ammonium per sulphate (APS)

TEMED

3.6 ml

0.625 ml

0.625 ml

100 µl

50 µl

5 µl

Resolving gel solution (15%) 10 ml

Water

30 % Acrylamide

1.5 M Tris buffer ( pH 8.8)

10 % Sodium dodecyle sulphate (SDS)

10 % Ammonium per sulphate (APS)

TEMED

3.45 ml

3.75 ml

2.5 ml

200 µl

100 µl

10 µl


49

Running Buffer 1x 1000 ml

Tris buffer

Glycine

SDS

H2O

3.0 g

14.4 g

1.0 g

To 1000 final volume

After dissolution of all components volume is adjusted to 1000 ml with deionized water.

Staining solution 100 ml

Double distilled water

Methanol

Glacial Acetic acid

Coomassie brilliant blue

43 ml

50 ml

7.0 ml

0.25 g

Destaining solution 100 ml

Double distilled water

Methanol

Glacial Acetic acid

43 ml

50 ml

7.0 ml

3.10.2 Western Blotting (Towbin et al. 1979)

For immunological characterization of insulin like antigen of Spirulina sp.

Immunoprecipitation Assay (Western Blotting) was done. For Western blotting after

SDS-PAGE, the gel was washed with distilled water to remove excess SDS. The PVDF

(polyvinylidene fluoride) membrane cut to the size of gel was activated by soaking in

absolute methanol for two minute (Wear gloves at all times when handling the PVDF).


50

The other blotting pads on the membrane were arranged in order of (-) blotting pad /

SDS-PAGE gel / PVDF membrane / blotting pad (+). This was soaked in transfer buffer

before transfer. Proteins on the gel were electrophoretically transferred to the membrane

at constant current 5.5 mA/cm2 for 30 minutes. The membrane was removed from the

western blotting device and was blocked with the blocking solution (1% BSA) for 2

hours at room temperature to block unoccupied sites with gentle rotating on a horizontal

rotator. The membrane was washed with washing buffer 3 times for 5 minutes each. The

membrane was immersed in 50 ml of primary antibody solution containing 1:5000

dilution of anti-human insulin antibody diluted with PBST and 0.1% BSA. This was

incubated at room temperature under gentle shaking for 90 minutes. The primary

antibody solution was discarded and the membrane was washed 3 times with PBST, 5

minutes each. The membrane was immersed in 20 ml of secondary antibody solution

containing 1:2000 dilution of Anti- Guinea pig IgG HRP (horseradish peroxidise)

conjugate diluted with PBST and incubated at room temperature under shaking

condition for 90 minutes. The membrane was washed with PBST for 5 times, 5 minutes

each and then 5 times with PBS. The reaction was developed by placing membrane in 20

ml of PBS containing 10 mg di-amino benzidine (DAB) and 60 µl of 30 % hydrogen

peroxide solution for few minutes under shaking until the bands were clearly visible.

The reaction was stopped by discarding the developing solution and washing the

membrane in water 2 times, 5 minutes each.


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Reagents for western blotting

Western Blot transfer buffer 1000 ml

Tris base (25mM) 3 g

Glycine (192 mM) 14 g

Absolute methanol 200 ml

SDS 1 g

Distilled water (make upto 1000 ml) 1000 ml

Developing reagent 20 ml

Di-amino Benzidine (DAB) 10 mg

Hydrogen peroxide (30% solution) 60 µl

PBS 20 ml

Western blotting is the electrophoretic transfer of separated proteins in a gel to the

surface of a thin support membrane matrix, where they are bound and immobilized with

specific antibodies. Semi-dry blotting is performed with plate electrodes in a horizontal

configuration, sandwiching a gel and membrane between sheets of buffer-soaked filter

paper that function as the ion reservoir. During electrophoretic transfer, negatively

charged molecules migrate out of the gel and move towards the positive electrode,

where they are deposited on the membrane. The plate electrodes, separated only by the

gel and filter paper stack, provide high field strength (V/cm) across the gel, allowing for

very efficient, rapid transfers.

3.10.3 RP-HPLC (Venancio et al. 2003)

Insulin-like protein were separated and purified by RP-HPLC (Shimadzu class –VP V

6.14 SP2 chromatograph with UV-VIS detector) using a C-18 Phenomenex column


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(250×4.6 mm, 5 μm) with 30–80% acetonitrile (A) in a 0.1% trifluoroacetic acid (TFA)

gradient (B). The fraction obtained by zinc chloride precipitation was dissolved in 500

μL of 0.1% TFA containing 0.1 M EDTA and applied to the column. The column was

operated at 1 mL min−1 and the proteins detected by absorbance at 216 nm. Bovine

insulin (50 μg of protein in 500 μL) was used as standard to compare retention times.

HPLC Reagents

Solvent (A) : Distilled water + TFA 0.1%

Solvent (B) : Acetonitrile

High Performance Liquid Chromatography (HPLC) is one mode of chromatography,

one of the most used analytical techniques. Chromatographic process can be defined as

separation technique involving mass-transfer between stationary and mobile phase.

HPLC utilises a liquid mobile phase to separate the components of a mixture. The

stationary phase can be a liquid or a solid phase. These components are first dissolved in

a solvent, and then forced to flow through a chromatographic column under a high

pressure. In the column, the mixture separates into its components. The amount of

resolution is important, and is dependent upon the extent of interaction between the

solute components and the stationary phase. The stationary phase is defined as the

immobile packing material in the column. The interaction of the solute with mobile and

stationary phases can be manipulated through different choices of both solvents and

stationary phases. As a result, HPLC acquires a high degree of versatility not found in

other chromatographic systems and it has the ability to easily separate a wide variety of

chemical mixtures.


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3.10.4 Circular Dichroism (Johnson. 1988)

CD spectroscopy is an adequate, convenient and well established physical technique

used for studying biomolecular conformation in solution under a wide range of

conditions. CD spectroscopy directly measures the differential absorption coefficients

between left and right handed circularly polarized light. The far UV CD bands of protein

(200–250 nm) derive primarily from the amide chromophore and they reflect the

secondary structure of protein (α-helix, β-sheets, β-turns).

Far-UV CD was measured in Jasco Spectropolarimeter (model J-715) equipped with

peltier-type temperature controller (PTC-348) and interfaced with personal computer.

Protein concentration used was in the range of 0.2-0.3mg/ml and 0.1 cm path length.

Baseline correction was carried out with the phosphate buffer in which protein was

dissolved. Measurements were taken at 25oC. N2 was continuously flushed through the

machine at the rate of 2-2.5 lit/min and higher below 200 nm to minimize the noise

level. CD data was reduced to concentration independent parameter [q] (deg cm2 dmol-

1), the mean residue ellipticity using the relation:

[q]l =M0 ql/10lc

where ql is the observed ellipticity in millidegrees at wavelength, l, M0 is the mean

residue weight of the protein, c is the protein concentration in gm/cm3, and l is the path

length of the cell in centimeters.

Since most of the proteins contain β- sheet, the observed residue ellipticity ([θ]λp) of the

protein is written as the linear combination of all structures, i.e.

[θ]λp = ƒα[θ]λ

α + ƒβ[θ]λβ + ƒt [θ]λ

t + ƒr[θ]λr (1)


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Where ƒα, ƒβ, ƒt and ƒr are the fractions of α-helix, β-sheet, turn and random coil,

respectively, and [θ]λα

, [θ]λβ

, [θ]λt, and [θ]λ

r are the mean residues ellipticities of pure of

α-helix, β-sheet, turn and random coil at wavelength λ, respectively. Due to the

uncertainty of [θ]λt of proteins and inaccessibility of measurements beyond 200 nm for

all pH values, equation (1) was reduced to the following form,

[θ]λp = ƒα[θ]λ

α + ƒβ[θ]λβ + ƒr[θ]λ

R (2)

Where R represent everything which is not the part of α-helix and β-sheet. Using data

provided by Yang et al., (1986) for the reference spectra, we analysed each far –UV CD

curve for different pH values for the elements of secondary structures according to

equation (2).

CD spectroscopy plays an important role in the study of characterisation of secondary

structures of proteins. Circular dichroism (CD) is an instrumentation technology for

elucidating the secondary structure of the proteins and peptides. It also allows the

detection and quantitation of the chirality of molecular structures. CD is a variant of

absorption spectroscopy which measures the difference in absorption of left and right

polarized light in the ultraviolet (UV) band by a medium or sample. Although the

peptide bond is planar and hence symmetric, there is usually an asymmetric alpha-

carbon on either side; hence the peptide bond transitions interact to give a CD signal

which is very sensitive to secondary structure. In the far UV region (200- 250 nm) the

CD of a protein is primarily that of the amide chromophores along the backbone. With

the introduction of an optically active sample, a preferential absorption is seen during

one of the polarization periods and the intensity of the transmitted light now varies

during the modulation cycle. The variation is directly related to the circular dichroism


55

of the sample at that wavelength. Successive detection is performed at various

wavelengths leads to the generation of the full CD spectrum. CD uses very little sample

(200 μl of 0.5 mg/ml solution in standard cells), it is non-destructive and relative

changes due to influence of environment on sample (pH, denaturants, temperature etc.)

can be monitored very accurately.

3.10.5 MALDI-TOF (Sequencing)

Proteins, either in solution or trapped in a band from a gel, can be analyzed by MALDI-

TOF mass spectrometry. Isolated protein was run on SDS-PAGE; for analysis protein

band was cut and submitted in commercial lab (TCGA) for amino acid sequence. In in-

solution digest the protein sample was mixed with an aqueous buffer (NH4HCO3) and

trypsin (5%) was added. In-gel digestion is a little more complicated and involves a

number of different steps; first the gel spots have to be washed. This was done using

50% acetonitril for 2 x 15 min. Then the gels were treated with 100% acetonitril (the gel

pieces will shrink!) and rehydrated in the digestion buffer (NH4HCO3). The washing

step was repeated using 50% acetonitril and then the gel pieces were dried down in the

speedway. To reduce potential disulfide bridges the proteins in the spot were treated

with 10 mM DTT (in 0.1 M digestion buffer) and incubated for 45 min. at 56 0C. To

prevent reconstruction of the bridges the free cysteins were alkylated; the gel pieces

were incubated in 55 mM iodoacetamid (in 0.1 M digestion buffer) for 30 min. in a dark

place. The washing steps were then repeated. Loading of the sample/matrix mixture onto

the MALDI-target directly from a micro-column The protein was then cut into smaller

pieces (peptides) by a protease (e.g. 12.5 ng/µl trypsin in 50 mM NH4HCO3, 5 mM

CaCl2). This was done on ice. After 45 min. the enzyme was replaced by digestion


56

buffer and left for digestion at 37 0C overnight. If necessary, the peptides were extracted

from the gel using acetonitril and 5% formic acid. Although MALDI-TOF generally

tolerant towards contaminants, such as salts and detergents, these significantly lower the

sensibility of the spectra. When analyzing small amounts of biological analytes using

MALDI-TOF, optimizing sample preparation and removing contaminants from the

sample can improves the spectra a lot. The breakthrough of cleaning up contaminant

from samples came with the introduction of low price self-assembled nano-scale sample

preparation methods, e.g. Micro-column purification. Here, a small column of a

chromatographic material was packed inside a small GelLoader tip. By loading the

sample onto this column the peptides were bind to the chromatographic resin and the

contaminant washed away. By adding the matrix (dissolved in acetonitril) onto the

column, the peptides were elute together with the matrix. The sample/matrix mixture

was then eluted directly onto the target and is ready for analysis by MALDI-TOF.

3.11 Hypoglycemic activity (In-vivo studies)

3.11.1 Animals and housing conditions

Albino Wistar rats (both sexes) aged seven to eight weeks (150-200g) were housed in a

well-ventilated animal unit (25±2°C, 60-70% relative humidity, 12h light/dark cycle)

provided by the Central Animal House Facility, Jamia Hamdard, New Delhi, India. The

animals were fed standard chow and filtered tap water ad libitum. All the protocols were

performed in the accordance with the Institutional Animal Ethical Committee (IAEC) as

per the directions of the Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA).


57

3.11.2 Aqueous extraction

Ten grams of dried Spirulina was extracted in distilled water for 6 h at slow heat. Every

2 h it was filtered through 2 layers of muslin cloth and centrifuged at 5000 g for 15 min.

The supernatant was collected. This procedure was repeated twice and after 6 h the

supernatant was concentrated to make the final volume one-fifth of the original volume.

The extract concentration was determined by gravimetric method. The extract was then

stored at 4 °C for further experiment.

3.11.3 Ethanol extraction

Ten grams of dried Spirulina was extracted with 100 ml of 80 % ethanol kept on a rotary

shaker for 24 h. Thereafter, it was filtered and centrifuged at 5000 g for 15 min. The

supernatant was collected and the solvent was evaporated to make the final volume one-

fifth of the original volume. The dried extract was resuspended with 1 ml of phosphate

buffer and administered through orally (50 mg / kg body weight) to the experimental

rats. It was stored at 4 °C in airtight bottles for further studies.

3.11.4 Induction of diabetes mellitus

A freshly prepared solution of streptozotocin (45mg/kg) in 0.1M cold citrate buffer, pH

4.5 was injected intraperitoneally. After 72 h of STZ administration, blood was drawn

from the fasting STZ-treated animals. The rats with blood glucose above 250 mg/dL

were considered to be diabetic and used for the experiment. Control rats were given

citrate buffer (pH 4.5) only.


58

3.11.5 Experimental design

In the experiment, a total number of 36 rats (30 diabetic and 6 normal rats) were used.

The rats were divided into six groups of six rats each: group 1, normal untreated rats;

group 2, diabetic control rats; group 3, diabetic rats given crude Spirulina (50mg/kg

body weight); group 4, diabetic rats given aqueous extract of Spirulina (50mg/kg body

weight); group 5, diabetic rats given ethanolic extract of Spirulina (50mg/kg body

weight); group 6, diabetic rats given insulin-like protein from Spirulina (50 μg of

insulin-like protein dissolved in 100μl saline).

3.11.6 Biochemical analysis

At the end of 35 days, the animals were deprived of food overnight and anaesthezied and

sacrificed by cervical dislocation. Blood was collected in two different tubes, i.e. one

with anticoagulant, potassium oxalate for plasma, and another without anticoagulant for

serum separation. Serum was separated by centrifugation.

For blood glucose test, blood was collected from the tail of the rats. The blood sugar was

estimated using the ACCU-CHEK® Go, Glucometer (Roche, Germany). The glucose

estimation was made weekly throughout the study. Glycosylated hemoglobin (HbA1C)

was estimated by the method (Bannon 1982), LFT (SGOT, SGPT, ALP and Bilrubin

total) and KFT (Creatinine Serum, Uric acid Serum and Blood urea nitrogen) analysis

were estimated by using commercial kits.


59

3.11.6.1 Liver Functional Test

SGOT

SGOT (AST) catalyzes the transfer of amino group between L-Aspartate and a

Ketoglutarate to form Oxaloacetate and Glutamate. The Oxaloacetate formed reacts with

NADH in the presence of Malate Dehydrogenase to form NAD. The rate of oxidation of

NADH to NAD is measured as a decrease in absorbance which is proportional to the

SGOT activity in the sample.

SGOT L-Aspartate + a Ketoglutarate ———–––———> Oxaloacetate + L-Glutamate MDH Oxaloacetate + NADH + H+ ———–––———> Malate + NAD+

Reagent1:

L-Aspartate > 200 mmol/l

Malate Dehydrogenase > 2000 IU/L

Reagent2:

Alpha-Ketoglutarate >35 mmol/l

NADH 1.05 mmol/l


60

Procedure

Particulars Sample Volume

Reagent 1 800 µl

Reagent 2 200 µl

Mix and incubate at 37°C for 2 minutes then add Sample 100 µl

AST activity is determined by measuring the rate of oxidation of NADH at 340 nm.

SGPT

SGPT (ALT) catalyzes the transfer of amino group between L-Alanine and a

Ketoglutarate to form Pyruvate and Glutamate. The Pyruvate formed reacts with NADH

in the presence of Lactate Dehydrogenase to form NAD. The rate of oxidation of NADH

to NAD is measured as a decrease in absorbance which is proportional to the SGPT

activity in the sample.

SGPT L-Alanine + a Ketoglutarate ———–––———> Pyruvate + L-Glutamate LDH Pyruvate + NADH + H+ ———–––———> Lactate + NAD+

Reagent1:

L-Alanine > 200 mmol/l

Lactate Dehydrogenase > 2000 IU/L


61

Reagent2:

Alpha-Ketoglutarate >35 mmol/l

NADH 1.05 mmol/l

Procedure


Reagent 1 800 µl

Reagent 2 200 µl


ALT activity is determined by measuring the rate of oxidation of NADH at 340 nm.

ALP

This method utilizes 4-nitrophenylphosphate as the substrate. Under the optimised

conditions ALP present in the sample catalyses the following transphosphorylation

reaction.

ALP 4 - NPP + H2O ———–––———> 4-NPO + Phosphate Mg / Alkaline pH

At the pH of the reaction 4-nitrophenoxide has an intense yellow colour. The reagent

also contains a metal ion buffer system to ensure that optimal concentrations of Zinc and

Magnesium are maintained. The metal ion buffer can also chelate other potentially

inhibitory ions which may be present. The reaction is monitored by measuring the rate


62

of increase in absorbance at 405 nm which is proportional to the activity of ALP in the

serum. It is almost always measured in units/litre (U/L).

Reagent1:

p-Nitrophenylphosphate 15 mmol/L

Reagent2:

2-Amino-2-methyl-1- propanol

350 mmol/L

Procedure


Reagent 1 800 µl

Reagent 2 200 µl


Bilrubin Total

Bilibuin is coupled with diazotized sulphanilic acid in acidic medium to form the pink

colored azobilirubin.The intensity of the color produced is directly proportional to

bilirubin concentration present in the sample. Direct bilirubin or conjugated bilirubin is

water soluble and it directly reacts in acidic medium. However, for indirect bilirubin or

unconjugated bilirubin a surfactant is used for dissolving it in water and then the

dissolved material reacts similar to direct bilirubin.


63

Reagent1:

Sulphanilic acid

4 mmol/l

Surfactant

0.6 mmol/l

Reagent2:

Sodium nitrite

144 mmol/L

Procedure


Reagent 1 500 µl

Reagent 2 10 µl


Read the absorbance at 546/670 nm.

3.11.6.2 Kidney Functional Test

Creatinine

In alkaline solution, creatinine forms a yellow-orange complex with picrate. The color

intensity is directly proportional to the creatinine concentration and can be measured

photometrically. Assays using rate-blanking minimize interference by bilirubin. Serum

and plasma samples contain proteins which react non specifically in the Jaffe method.

Serum and plasma results must be reduced by 0.3 mg/dl (26 µmol/l) to obtain accurate

values.


64

NaOH creatinine + picric acid ———–––———> creatinine - picric - acid - complex

Reagent1:

Sodium hydroxide

0.2 mmol/L

Reagent2:

Picric acid 25 mmol/L

Procedure


Reagent 1 500 µl

Reagent 2 100 µl

Sample 200 µl

Mix and incubate at 37°C for 60 second

Read the absorbance at 492/500 nm.

Uric acid Serum

Uric acid is oxidized by phosphotungstic acid in alkaline medium containing the

tetrasodium salt of ethylenedinitrilotetraacetic acid (EDTA-Na4), sodium tungstate, and

hydrazine sulfate.

Reagent1:

Sodium tungstate 10% w/v


65

Reagent2:

Sulphuric acid 1/12 N

Reagent3:

EDTA 5% w/v

Reagent4:

Hydrazine sulfate 0.025% w/v

Reagent5:

EDTA-Tungstate 5% EDTA and 60% Sodium tungstate

Reagent6:

Phospho-Tungstate 40 g Sodium tungstate and 32 ml of 85% orthophosphoric acid

Reagent7:

Saline 0.9% NaCl

Procedure


Reagent 2 1.6 ml

Serum 200 µl

Reagent 1 200 µl

Mix vigorously allow to stand for 10 min and centrifuge


66

Supernatant 0.5 ml

Reagent 4 1 ml

Reagent5 4 ml

Reagent6 1 ml

Mix and incubate at 37°C for 30 minutes

Read the absorbance at 700 nm

Blood urea nitrogen

Urea in an acidic medium condenses with Diacetyl monoxime at 100°C to form a red

coloured complex. Intensity of the colour formed is directly proportional to the amount

of urea present in the sample.

100°C

Urea + Diacetyl monoxime ———–––———> Red Coloured Complex

Reagent1:

Urea

0.2 mmol/L

Reagent2:

Acid

0.2 mmol/L

Reagent3:

DAM

0.2 mmol/L


67

Procedure


Reagent 1 1 ml

Reagent 2 1 ml

Reagent 3 1 ml

Sample 100 µl

Mix and incubate at 100°C for 10 minutes

Read the absorbance at 520 nm.

3.12 Growth measurement (as protein)

The growth of selected Spirulina strain(s) was determined as protein (Lowry et al.

1951). The algal biomass (1mg) was taken in a test tube. 1mL 1N NaOH was added to it.

The test tube was placed in a boiling water bath for 10 minutes. The blank / sample

solution were taken and added 5mL of Reagent-A (prepared by adding 1mL freshly

prepared 1% Na-K tartarate solution containing 0.5% CuSO4 into 50mL 2% Na2CO3

solution) and incubated at room temperature for 10 minutes. Then added 0.5 mL

Reagent-B (Folin reagent) and again incubated at room temperature for 15 minutes. The

absorbance of the supernatant was observed at λ650 nm. Protein content was evaluated

from the concentration of BSA solution known from standard curve.

3.13 Culture condition manipulation

S. platensis (CFTRI, Mysore) was grown in different concentrations of NaNO3, K2HPO4,

NaHCO3 and Calcium in Zarrouk’s medium for culture condition manipulation.


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NaNO3

30 mM as in regular Zarrouk’s medium was considered as control. Five different

concentrations (15, 25, 35, 45 and 55 mM) of nitrate added to medium.

K2HPO4

3 mM as in regular Zarrouk’s medium was considered as control. Five different

concentrations (1.5, 2.5, 3.5, 4.5 & 5.5mM) of phosphate added to medium.

NaHCO3

0.2 M as in regular Zarrouk’s medium was considered as control. Five different

concentrations (0.06, 0.12, 0.18, 0.24 & 0.3M) of carbonate added to medium.

For sulphate effect

K2SO4 (6 mM), MgSO4 (0.8 mM) and FeSO4 (0.036 mM) as in regular Zarrouk’s

medium were considered as controls.

K2SO4+MgSO4+FeSO4 were added in a dose dependent manner [K2SO4 (4.5, 5.5, 6.5,

7.5 and 8.5 mM); MgSO4 (0.65, 0.75, 0.85, 0.95 and 1.05 mM); FeSO4 (0.015, 0.025,

0.035, 0.045 and 0.055 mM)].

Calcium

0.4 mM as in regular Zarrouk’s medium was considered as control. Five different

concentrations (0.2, 0.6, 0.8, 1.0, 1.2 mM) of calcium added to medium.


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Selenium

The effect of selenium stress was examined by adding calculated amount of Sodium

selenite in the culture medium to have varying concentrations (0.15, 0.30, 0.45, 0.6, 0.75

mM) of the selenium.

Glucose

The effect of exogenous glucose stress was examined by adding calculated amount in

the culture medium to have varying concentrations (5, 7.5, 10, 12.5, 15 mM) of the

glucose.

pH stress

The effect of pH on the growth S.platensis CFTRI, Mysore was examined by growing

the test strain at different pH scale like - 8, 10 and 12. pH of the control was 9. The

different pH values were adjusted by adding N/10 HCl or N/10 NaOH into the culture

medium.

3. material and methods - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/25200/8/08_chapter...

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