Posted on

Dilutions For The Enzymatic Assays Biology Essay

With the coming of biotechnology, culturing of mammalian cells for bring forthing commercially good merchandises has become progressively popular. The cell civilization samples incorporating assorted metabolites – lactate & A ; ammonium and substrates – glucose & A ; glutamine are still being quantified utilizing commercially available enzymatic checks affecting clip and cost. This survey quantitatively compares the enzymatic checks to HPLC method for profiling of cell civilization constituents. The survey is based on batch culturing of Chinese Hamster Ovary ( CHO320 ) cells bring forthing human interferon-I? . Samples were collected daily from civilization flask incubated in 37°C agitating brooder ( no CO2 supplemented ) , 100rpm for 7 yearss. HPLC analysis and enzymatic checks were carried out on samples collected to profile the cell civilization metabolites and substrates. Consequences were analysed to quantitatively compare Agilent HPLC method to enzymatic checks. It was concluded that enzymatic checks and HPLC method are straight comparable for profiling glucose and lactate in the CHO320 cell civilization samples, therefore they can be faithfully profiled utilizing HPLC from farther on to salvage cost and clip. But, the ammonium concentration detected in cell civilization samples utilizing enzymatic checks and HPLC consequences are non coincident, therefore it can non be profiled faithfully utilizing HPLC. Glutamine profiling utilizing HPLC method could non be carried out due to HPLC dislocation, but a new Waters Column has been received to accurately profile ammonium and glutamine-glutamate concentrations in cell civilization samples.

AIM & A ; INTRODUCTION

HPLC application has been set up to observe the chief cell civilization metabolites – lactate and ammonium hydroxide, and substrates – glucose, glutamate and glutamine. Besides, enzymatic checks are used for mammalian cell civilization metabolite and substrate analysis. However, checks are expensive and clip consuming. Therefore, the purpose of this survey is to quantitatively compare HPLC method for glucose, lactate, ammonium hydroxide, glutamine and glutamate to commercially available enzymatic check kits. If the HPLC method sensing degree is comparable to the enzymatic checks, the hereafter metabolite/substrate analysis can be done with HPLC entirely. If the enzymatic check is found more accurate than HPLC, the following purpose is to scale down the check for 96-well format to cut down costs.

MATERIALS & A ; METHODS

Cell Culture. Cell civilization was carried out in a 125ml shingle flask ( Belco Glass ) with a on the job volume of 50ml, specially conditioned for batch operation. CHO320 cell line was cultured utilizing EXCELL CHO DHFR medium ( Sigma Aldrich ) supplemented with 4mM L-Glutamine ( Sigma ) and 1AµM Methotrexate ( Sigma ) with a get downing cell denseness of 0.3 * 106 cells/ml. The cell civilization work was carried out in purely unfertile clean room environment and the civilization flask was maintained in 37°C brooder for 7 yearss with uninterrupted agitation at the velocity of 100rpm ( no CO2 supplemented ) .

Sampling. Cell civilization samples of 2.5 milliliters were collected daily for 7 yearss from the cell civilization flask in 15ml extractor tubings and were spun down in extractor for 5 proceedingss at 200g velocity. The spun sample was filtered through 0.22 Aµm unfertile filter utilizing syringe and aliquoted into 1.5ml micro-centrifuge tubings harmonizing to table 1 below and stored in -80°C deep-freeze for analysis.

Table 1. Volume of cell civilization samples aliquoted in micro-centrifuge tubings for enzymatic checks and HPLC analysis

A

Enzymatic Kits

HPLC

Glutamine-Glutamate

600 AµL

500 AµL

Ammonia

100 AµL

500 AµL

Glucose

100 AµL

Lactate

100 AµL

Enzymatic Assaies

Glucose Assay 3. Lactate Assay

Ammonia Assay 4. Glutamine – Glutamate Assay

All checks were performed in conformity with the criterion operating processs of each kit. These checks work on the footing of an enzyme reaction where the substrate of each reaction is measured utilizing a spectrophotometer utilizing a specific wavelength. This optical density reading is so used to cipher the concentration of unknown substance in the sample e.g. Ammonia. Table 2 below inside informations all the dilutions done on the samples for the several checks.

Table 2. Dilutions for the Enzymatic Assays

A

Ammonia

Glucose

Lactate

Gln-Glu

D0

1:5A

1:100

1:100

A 1:2

D1

A 1:5

A 1:100

A 1:100

1:2

D2

A 1:10

A 1:100

A 1:100

A 1:2

D3

A 1:10

A 1:100

A 1:100A

A 1:2

D4

A 1:10

A 1:80

1:200

A 1:2

D5

A 1:10

A 1:70

A 1:300

A –

D6

A 1:15

A 1:70

A 1:400

A –

D7

A 1:15

A 1:70

A 1:400

A –

HPLC Analysis

Metabolite sensing utilizing HPLC method was carried out utilizing Agilent HPLC 1100 series and Supelcogel C-610H ( saccharide ) column equipped with a guard column. A diluted H2SO4 Mobile stage solution ( 1.35ml H2SO4 in 5L ultra-pure H2O ) was used for isocratic elution / additive gradient. Agilent Chemstation – online package was used to put up the method for HPLC analysis of samples and the Agilent Chemstation – offline package was used to analyze the informations and obtain the consequences of sample and standard analysis.

Ammonium, Glucose and Lactate criterions of different concentrations ( 2g/L, 1g/L, 0.5 g/L and 0.25 g/L ) were prepared utilizing extremist pure H2O harmonizing to the criterion operating process, filtered through 0.22Aµm filter utilizing syringe and transferred into HPLC phials. The cell civilization samples were filtered in similar manner and transferred to phials. An internal criterion of 30 g/L isopropanol solution was prepared and used to take or minimise the background mistake from the consequences.

The HPLC a ) lines blushing, B ) equilibration of column, degree Celsius ) stabilisation of sensor, vitamin D ) sample and standard burden and vitamin E ) sequence table creative activity stairss were carried out in conformity with the criterion runing process set-up for HPLC metabolite sensing at the Laboratory of Integrated Bioprocessing ( Refer Pages 011-014, Lab Manual 1197 ) . Once the HPLC tally of criterions and samples was finished, the information was collected utilizing Agilent Chemstation – offline package and was analysed on the footing of constituent ‘s peak keeping clip, peak country and the extremum tallness ( Refer Pages 015-021, Lab Manual 1197 ) .

RESULTS & A ; DISCUSSION

Enzymatic Assay Results

1. Glucose Assay 3. Lactate Assay

2. Ammonium Assay 4. Glutamine-Glutamate Assay

Figure 1-3 below shows the Standard Curves prepared for the several enzymatic checks with first-class arrested development values of around 0.99. The corresponding criterion curve equations were used for the computations for metabolites and substrates in the cell civilization samples.

Figure 1. Standard Curve for Glucose utilizing Sigma Aldrich Kit

Figure 2. Standard Curve for Glutamine utilizing Sigma Aldrich Kit

Figure 3. Standard Curve for Lactate utilizing Sigma Aldrich Kit

Figure 4 & A ; 5 below diagrammatically shows the concentration of different metabolites and substrates in the CHO320 cell civilization samples changing over a 7-day civilization period.

Figure 4. Relationship between Glucose & A ; Lactate concentrations within a 7-day CHO320 batch cell civilization

Figure 4 shows the relationship between the glucose and lactate concentrations within the civilization ; on the other manus, contrast between L-glutamine and ammonium concentrations is shown in Figure 5. As expected, there was a lessening in glucose concentration from 35mM to 13mM ( 5.25 g/L to 2.48 g/L ) in the CHO320 cell civilization with clip due to ingestion of glucose as C and energy beginning via the glycolysis tract in the batch civilization and its transition to breastfeed and therefore increase in lactate concentration from 0mM to 9.31mM was observed.

Theoretically, harmonizing to the procedure of anaerobiotic glycolysis ( absence of air ) 1 mole of glucose gets converted into pyruvate and so pyruvate to 1 mole of lactate utilizing lactate dehydrogenase. This phenomenon is non observed in the above CHO320 cell civilization survey, as 1 mole of glucose is converted to ~0.6 moles of lactate ( Refer Table 3 below ) harmonizing to the consequences utilizing enzymatic checks. The ground for this uncomplete anaerobiotic glycolysis could be the air which was present in the cell civilization flask at the clip of get downing the civilization ; besides, air could be exchanged within the flask during mundane sampling.

Figure 5. Relationship between Glutamine & A ; Ammonium concentrations within a 7-day CHO320 batch cell civilization

As expected, there was an addition in ammonium concentration during the 7-day CHO320 cell civilization period. This can be justified due to the non-enzymatic dislocation of glutamine into pyroglutamate and ammonium during cell civilization. Theoretically, it is assumed that in anaerobiotic conditions, 1 mole of L-Glutamine is non-enzymatically broken down to 2 moles of ammonium and 0 moles of pyroglutamate. Harmonizing to our consequences obtained from CHO320 cell civilization samples utilizing enzymatic checks, it is observed, that 1 mole of Glutamine was converted to ~1.5 moles of ammonium ( Refer Table 3 ) . This could be due to formation of little measures of pyroglutamate due to the influence of air which could hold been introduced into the cell civilization flask during mundane sampling as described above.

Table 3 below numerically shows the changing concentrations of cell civilization metabolites – lactate and ammonium hydroxide, and substrates – glucose, glutamate and glutamine quantitatively measured through the enzymatic checks over 7-day cell civilization period.

Table 3. Numeric informations set demoing changing concentrations of cell civilization metabolites and substrates quantitatively measured utilizing enzymatic checks

Samples ( Days )

Glucose Concentration ( millimeter )

Lactate Concentration ( millimeter )

Glutamine Concentration ( AµM/ml )

Ammonium Concentration ( AµM/ml )

0

29.1966849

-0.276995305

3.902079882

1.022748092

1

34.91444426

2.962441315

1.840603718

1.67519084

2

32.62734052

5.098591549

1.811154059

2.036679389

3

26.87948769

10.49765258

1.538744708

2.98889313

4

16.88123311

11.88732394

0.434382477

3.932290076

6

15.69795786

6.685446009

-0.044174489

5.730916031

7

13.7810038

9.314553991

0.220872446

6.427442748

HPLC Results

Duplicate sets of criterions of glucose, lactate and ammonium were prepared at four different concentrations of 2 g/L, 1 g/L, 0.5 g/L and 0.25 g/L utilizing glucose, calcium lactate and ammonium chloride chemical pulverizations ( Sigma Aldrich ) with extremist pure H2O. An internal criterion of 30g/L isopropanol solution was prepared and used to take / minimise the background mistake from the consequences.

The consequences analysis of standard sets 1 & A ; 2 from Agilent Chemstation – offline package are detailed below in Table 4A, 4B giving the information in the signifier of Rapport Area and Height which are further used for fixing the standard curves for glucose, lactate and ammonium severally.

Table 4A. HPLC analysis of glucose, lactate and ammonium criterions set 1 of changing concentrations utilizing two parametric quantities: Peak Area and Peak Height

STANDARD SET 1

Glucose Concentration ( g/L )

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

0.25

13.128

118624.2

2579.7

0.04036971

0.033950613

Isopropyl alcohol

28.667

2938445.7

75983.9

1

1

0.5

13.129

117353.4

4255.9

0.040727627

0.056264311

Isopropyl alcohol

28.669

2881420

75641.2

1

1

1

13.127

206717.1

7745.4

0.072060502

0.102352601

Isopropyl alcohol

28.662

2868660.3

75673.7

1

1

2

13.129

396490.9

15572.6

0.137577453

0.205554456

Isopropyl alcohol

28.651

2881946.8

75759

1

1

Lactate Concentration ( g/L )

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

0.25

18.069

87239.8

3045.9

0.029689097

0.040086124

Isopropyl alcohol

28.667

2938445.7

75983.9

1

1

0.5

18.072

164532.1

6119.4

0.057101047

0.080900356

Isopropyl alcohol

28.669

2881420

75641.2

1

1

1

18.071

297415.5

11421.2

0.10367749

0.15092694

Isopropyl alcohol

28.662

2868660.3

75673.7

1

1

2

18.073

591810.3

22986.6

0.205350876

0.303417416

Isopropyl alcohol

28.651

2881946.8

75759

1

1

Ammonium Concentration ( g/L )

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

0.25

7.995

6209.9

550.7

0.002113328

0.007247588

Isopropyl alcohol

28.667

2938445.7

75983.9

1

1

0.5

8.538

115894.2

8327.8

0.04022121

0.110096085

Isopropyl alcohol

28.669

2881420

75641.2

1

1

1

8.552

370537.4

26158.1

0.129167403

0.345669632

Isopropyl alcohol

28.662

2868660.3

75673.7

1

1

2

8.601

918870.6

64842.4

0.318836767

0.855903589

Isopropyl alcohol

28.651

2881946.8

75759

1

1

Table 4B. HPLC analysis of glucose, lactate and ammonium criterions set 2 of changing concentrations utilizing two parametric quantities: Peak Area and Peak Height

STANDARD SET 2

Glucose Concentration ( g/L )

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

0.25

13.122

65938.5

2026.4

0.022943297

0.026865569

Isopropyl alcohol

28.617

2873976.7

75427.4

1

1

0.5

13.121

114127.3

3761.9

0.039117978

0.049629157

Isopropyl alcohol

28.594

2917515.3

75800.2

1

1

1

13.119

195857.8

7448.9

0.067689556

0.098519086

Isopropyl alcohol

28.559

2893471.5

75608.7

1

1

2

13.122

433494.5

15590.7

0.146646206

0.205007265

Isopropyl alcohol

28.533

2956056.7

76049.5

1

1

Lactate Concentration ( g/L )

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

0.25

18.067

80619.4

2834

0.028051515

0.037572553

Isopropyl alcohol

28.617

2873976.7

75427.4

1

1

0.5

18.068

132572.8

5212.4

0.04544031

0.06876499

Isopropyl alcohol

28.594

2917515.3

75800.2

1

1

1

18.071

278317.2

10913.8

0.096187987

0.144345823

Isopropyl alcohol

28.559

2893471.5

75608.7

1

1

2

18.076

604462.2

22894.2

0.204482614

0.301043399

Isopropyl alcohol

28.533

2956056.7

76049.5

1

1

Ammonium Concentration ( g/L )

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

0.25

8.002

2144.8

160.7

0.000746283

0.002130526

Isopropyl alcohol

28.617

2873976.7

75427.4

1

1

0.5

8.536

88862.7

6693.1

0.030458349

0.088299239

Isopropyl alcohol

28.594

2917515.3

75800.2

1

1

1

8.553

362655.2

25864.3

0.125335674

0.342081004

Isopropyl alcohol

28.559

2893471.5

75608.7

1

1

2

8.605

927195.1

65838.3

0.313659444

0.865729558

Isopropyl alcohol

28.533

2956056.7

76049.5

1

1

Using the consequences of standard sets 1 & A ; 2 from Table 4A & A ; 4B, standard curves were prepared for glucose, lactate and ammonium. The standard curves were compared to each other utilizing rapport country and resonance tallness informations and are illustrated in Figures 6, 7 and 8 below demoing graphs from two duplicate sets of criterions prepared.

Figure 6. Standard Curves for glucose prepared utilizing informations obtained from HPLC analysis of duplicate sets of criterions prepared

Figure 7. Standard Curves for lactate prepared utilizing informations obtained from HPLC analysis of duplicate sets of criterions prepared

Figure 8. Standard Curves for ammonium prepared utilizing informations obtained from HPLC analysis of duplicate sets of criterions prepared

The figures 6-8 presented above shows that about similar equations and arrested development values are obtained for both country and tallness resonances utilizing two duplicate sets of criterions prepared for glucose, lactate and ammonium, therefore demoing the efficaciousness and specificity of experimentation carried out for profiling cell civilization constituents.

The Table 5 below shows the numerical informations obtained from HPLC Chemstation – offline package for profiling of CHO320 cell civilization samples for lactate, ammonium and glucose profiling over 7-day period utilizing two parametric quantities – Peak Area and Peak Height. Furthermore, utilizing the standard curve equations obtained in figures 6-8 above, the existent concentrations of the cell civilization metabolites – lactate and ammonium and substrates – glucose were obtained and reported in g/L units in two duplicate sets of resonance country and resonance tallness.

Table 5. HPLC analysis of CHO320 cell civilization samples for metabolite: lactate and ammonium & A ; substrate: glucose profiling utilizing 2 parametric quantities: Peak Area and Height

CHO320 Cell Culture Sample – Day 0

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Height

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Height

Glucose

13.131

1027040.3

40593.1

0.27250314

0.418002924

4.35389983

4.173810208

3.809366385

4.094847746

Lactate

18.104

40149.9

763.9

0.010652916

0.007866175

0.052634427

0.028517213

0.12832113

0.081409975

Ammonium

8.605

771866.9

56074.3

0.204798345

0.577418857

1.388148823

1.444665008

1.416794867

1.441712575

Isopropyl alcohol

28.376

3768911.8

97112

1

1

A

A

CHO320 Cell Culture Sample – Day 1

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Height

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Height

Glucose

13.131

984375.8

38855.8

0.34626274

0.515256476

5.614747689

5.162159308

4.851168642

5.045517848

Lactate

18.064

75939.5

2215.5

0.026712379

0.029379159

0.213550891

0.172320581

0.284998818

0.223035937

Ammonium

8.577

743204.6

52268

0.261428675

0.693112109

1.698622121

1.681498688

1.727779654

1.672406997

Isopropyl alcohol

28.742

2842858

75410.6

1

1

A

A

CHO320 Cell Culture Sample – Day 2

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Height

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Height

Glucose

13.136

939211.2

36252.9

0.326974329

0.4795687

5.285031272

4.799478664

4.578733466

4.696663739

Lactate

18.072

160369.3

4980.6

0.055830514

0.065885484

0.505315774

0.416346819

0.569078188

0.463367243

Ammonium

8.588

798434.6

56527.2

0.277964762

0.747765719

1.789280495

1.793379159

1.818587382

1.781387277

Isopropyl alcohol

28.722

2872431

75594.8

1

1

A

A

CHO320 Cell Culture Sample – Day 3

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Height

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Height

Glucose

13.133

840990.1

31890.8

0.289938868

0.420226777

4.651946468

4.196410336

4.055633734

4.116586286

Lactate

18.07

225429.1

7388.3

0.077718701

0.097356024

0.724636283

0.626711392

0.782621473

0.670546572

Ammonium

8.586

814020.3

57271.9

0.280640788

0.754674889

1.80395169

1.807522803

1.833282747

1.795164285

Isopropyl alcohol

28.717

2900577.3

75889.5

1

1

A

A

CHO320 Cell Culture Sample – Day 4

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Height

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Height

Glucose

13.135

767333.8

29029.8

0.266958763

0.383321757

4.259124146

3.821359324

3.731055968

3.755833406

Lactate

18.073

223872.1

7858.8

0.077886076

0.103770919

0.726313388

0.669591705

0.784254401

0.712777611

Ammonium

8.59

806629.8

57116.6

0.280630012

0.754191744

1.803892608

1.806533765

1.833223567

1.794200885

Isopropyl alcohol

28.72

2874353.3

75732.2

1

1

A

A

CHO320 Cell Culture Sample – Day 6

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Height

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Height

Glucose

13.131

598122.6

22773.5

0.205588136

0.299924405

3.210053601

2.973825253

2.864239204

2.940610018

Lactate

18.072

204781.5

6925.9

0.070387989

0.091213315

0.651182251

0.585650503

0.711102329

0.630107408

Ammonium

8.589

817861.1

57358.4

0.28111718

0.755403604

1.806563488

1.809014543

1.835898848

1.796617357

Isopropyl alcohol

28.666

2909324.5

75930.8

1

1

A

A

CHO320 Cell Culture Sample – Day 7

Time ( Mins )

Area

Height

Rapport Area ( X / Isopropanol )

Rapport Height ( X / Isopropanol )

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 1: Compound Concentrations ( g/L ) – utilizing Rapport Height

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Area

STD 2: Compound Concentrations ( g/L ) – utilizing Rapport Height

Glucose

13.125

495248.9

18879.3

0.169125818

0.247803419

2.586766117

2.444140442

2.349234716

2.43111847

Lactate

18.068

169262.9

6083.3

0.057802706

0.079847375

0.525077211

0.509674965

0.58831908

0.555282256

Ammonium

8.585

801200.6

56946.7

0.273607285

0.747463465

1.765390815

1.792760419

1.794658345

1.780784576

Isopropyl alcohol

28.663

2928286.8

76186.6

1

1

The informations obtained from the HPLC Chemstation – offline package for profiling of CHO320 cell civilization samples for lactate, ammonium and glucose utilizing two parametric quantities – Peak Area and Peak Height have been diagrammatically represented in Figures 9-11 below. The figures clearly indicate that the consequences obtained via both methods, that is, utilizing peak country and top out tallness give about precisely same concentration of the constituent. Therefore, it can be concluded that both methods, that is, informations analysis utilizing peak country and extremum heigh, t are dependable as both ways provides coincident consequences.

Figure 9. HPLC analysis of CHO320 cell civilization samples for glucose profiling utilizing two parametric quantities in duplicate sets: Peak Area and Peak Height

Figure 10. HPLC analysis of CHO320 cell civilization samples for lactate profiling utilizing two parametric quantities in duplicate sets: Peak Area and Peak Height

Figure 11. HPLC analysis of CHO320 cell civilization samples for ammonium profiling utilizing two parametric quantities in duplicate sets: Peak Area and Peak Height

Comparison – HPLC vs. Enzymatic Assays

The quantitative comparing of HPLC for profiling glucose, lactate and ammonium to commercially available enzymatic kits in CHO320 cell civilization samples is presented below in Table 6. The tabular array shows the constituents ( glucose, lactate and ammonium ) in similar units for both methods ( HPLC and enzymatic checks ) for the easiness of comparing.

Table 6. Numeric comparing of HPLC and enzymatic checks for glucose, lactate and ammonium profiling of CHO320 cell civilization samples

HPLC

ENZYMATIC ASSAYS

Sample

Glucose Concentration- millimeter

Lactate Concentration – millimeter

Ammonium Concentration – AµM/ml

Sample

Glucose Concentration -mM

Lactate Concentration – millimeter

Ammonium Concentration – AµM/ml

0

24.18833333

0.561797753

0.081620675

0

29.1966849

-0.276995305

1.022748092

1

31.16666667

2.359550562

0.099236648

1

34.91444426

2.962441315

1.67519084

2

29.38888889

5.730337079

0.105108639

2

32.62734052

5.098591549

2.036679389

3

25.83333333

8.08988764

0.105695838

3

26.87948769

10.49765258

2.98889313

4

23.66666667

8.202247191

0.105695838

4

16.88123311

11.88732394

3.932290076

6

17.83333333

7.303370787

0.106283037

6

15.69795786

6.685446009

5.730916031

7

14.38888889

5.842696629

0.103934241

7

13.7810038

9.314553991

6.427442748

The tabular array above clearly indicates that the HPLC and enzymatic methods are straight comparable to each other for profiling glucose and to some extent lactate as good in the CHO320 cell civilization samples. Thus they can be faithfully profiled utilizing HPLC from farther on to salvage cost and clip. But, the ammonium concentration detected in the cell civilization samples utilizing the enzymatic checks does non match to the HPLC consequences. Therefore, it can non be profiled faithfully utilizing HPLC.

The informations presented above in the tabular array 6 is diagrammatically represented in the figures 12 and 13. The Figure 12 shows the relationship between the glucose and lactate concentrations within the civilization comparing both the HPLC and enzymatic methods. On the other manus, contrast between ammonium concentrations utilizing two methods is shown in Figure 13.

A lessening in glucose concentration from 35mM to 13mM was detected utilizing enzymatic check and a lessening from 32mM to 14mM was detected utilizing HPLC method in the CHO320 cell civilization with clip ( mention Table 6 & A ; Figure 12 ) . Besides, due to ingestion of glucose as C and energy beginning via the glycolysis tract in the batch civilization and its transition to breastfeed, an addition in lactate concentration from 0mM to 11.88mM was detected utilizing enzymatic check, whereas, an addition from 0mM to 8.2mM was detected utilizing HPLC analysis.

Figure 12. Graphical comparing of HPLC and enzymatic checks for glucose & A ; lactate profiling of CHO320 cell civilization samples

Non-enzymatic dislocation of L-glutamine in CHO320 cell civilization system is assumed to bring forth pyroglutamate and ammonium. Therefore, as expected, there was an addition in ammonium concentration detected in samples from 1.02AµM/ml to 6.02AµM/ml utilizing enzymatic checks, whereas, merely a little addition from 0.08AµM/ml to 0.103AµM/ml was detected utilizing HPLC analysis. This shows that the Supelcogel saccharide column used in this survey is rather sensitive to observe glucose & A ; lactate concentrations, but non specific for observing right ammonium concentrations in samples.

Figure 13. HPLC and assay comparing for ammonium profiling of CHO320 samples

Decision

Commercially available enzymatic check kits are really sensitive & A ; specific for mammalian cell civilization metabolites and substrates analysis. However, checks are expensive & A ; clip devouring. Thus, an alternate method of HPLC – Supelcogel carbohydrate column for profiling cell civilization constituents has been compared to enzymatic checks for bettering analysis efficiency. It has been concluded that enzymatic checks & A ; HPLC method are straight comparable for profiling glucose & A ; lactate in the CHO320 cell civilization samples therefore they can be faithfully profiled utilizing HPLC from farther on to salvage cost and clip. But, the ammonium concentration detected in cell civilization samples utilizing enzymatic checks and HPLC consequences are non coincident, therefore it can non be profiled faithfully utilizing HPLC. Glutamine profiling utilizing HPLC method could non be carried out due to HPLC dislocation, but a new Waters Column have been received to accurately profile ammonium and glutamine-glutamate concentrations in cell civilization samples. Besides, in the interim, the enzymatic checks for profiling ammonium and glutamine could be scaled down to 96-well format to cut down costs and sample measure demands.

Leave a Reply

Your email address will not be published.