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12 Determination of Net Protein Utilization, Digestibility and Biological Value (Pellet and Young 1980)

12 Determination of Net Protein Utilization, Digestibility and Biological Value (Pellet and Young 1980)

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7.12



Determination of Net Protein Utilization, Digestibility. . .



111



and non-proteinaceous of the body – a measure of nitrogen intake absorption and

retention would be an ideal practical approach.

Based on nitrogen balance studies, net protein utilization (NPU), digestibility

(D) and biological value (BV) are calculated.

Principle

The nitrogen content of the test diet is determined. The feed consumption over a

5-days-period is measured, leading to a calculation of the total N-intake. The faecal

and urinary nitrogens on these 5 days are also determined. From these values

calculations can be made, to know the “N retained” and “N absorbed”. NPU, D

and BV are then calculated as follows:

N retained

N intake

N absorbed



N intake



NPU ¼



BV ¼



N retained

N absorbed



Materials

1.

2.

3.

4.

5.

6.

7.



Wistar male rats (weighing ~60 g)

Metabolic cages

Balance for weighing rats

Diet

Plastic container with tight lids

Sieves

N-free mixture

Sucrose – 9.0%

Cellulose powder – 5.2%

Soybean oil – 5.2%

N-free starch – 80.6%



Procedure

Preparation of diets: Five hundred gram diet (dry weight basis) is prepared for each

treatment. Initially, determine the protein and dry matter content in the test sample,

then calculate the amount of sample required to give 7.5 g N on dry weight basis.

To this add 20 g mineral and 5 g vitamin mixture to make up 500 g diet (dry wt. basis)

and rest of the amount from N-free diet after determining the moisture in N-free diet.

1. Weigh 40.0 g diet on dry weights basis (actual weight will vary depending upon

the moisture in the diet) into plastic boxes for each rat sufficient for preliminary

period for 4 days, and tightly close with the lid. Thus each animal receives

150 mg N and 10 g dry matter per day throughout the test period.

2. Put cages on the rack without funnel.



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Qualitative and Quantitative Estimations of Amino Acids and Proteins



3. Weigh the rats in the beginning of the experiment, divide the rate into groups of

five, such that average weight of the group differs by no more than 0.5 g and

record the weight (lesser the difference better the standardization).

4. Transfer diet equivalent to 10 g dry weight to the feed box of respective cage.

In all the experiments, group 1 is always allotted for casein diet.

5. Press the feed with suitable flat surface.

6. Place plastic bowls below each rat cage for the collection of urine and faeces.

7. Feed every rat once a day in the morning and check for the water in bottles.

8. On the last day of the preliminary period, when all the diet from diet box had

been transferred to the rat cage feed box, weigh 50 g equivalent of dry weight

diet and transfer to respective plastic boxes. Apply Vaseline grease to nylon net

as well as Perspex funnel.

9. At the end of the preliminary period of 4 days, again weigh the rats and clean

the rat cage feed box as well. Clean inside of the rat cage with lukewarm water.

10. Put both the greased Perspex funnel and nylon net in proper position.

11. Transfer 10.0 g dry weight equivalent diet from respective boxes to cage feed

box and follow as in preliminary period for the 5 days of N-balance period.

12. Put flask containing 35 mL 5% H2SO4 and funnel with glass wool below the

Perspex funnel to collect the urine and beaker containing 50 mL 5% H2SO4

below nylon net to collect faeces.

13. During the 5 days of the N-balance period daily weighing of the feed from diet

box and its transfer to cage feed boxes is followed. Any of the feaces

remaining, at the neck of the nylon netting is also transferred to beaker with

the help of forceps. If by chance faeces have fallen on the funnel, these are also

removed to respective beakers.

14. Every morning spray the net in situ with small quantity of 20% citric acid from

a plastic wash bottle to prevent N losses and then finally wash the glass wool

with small quantity of 5% H2SO4 for the same reason.

15. Thus following the procedure, during the N-balance period of 5 days, urine and

faeces are collected, respectively, in flasks and beakers.

16. At the end of N-balance period, remove all the water bottles and prevent the

access of rats to feed box 3 h before the termination of the experiment.

17. Weigh the rats and transfer to bigger cages.

18. Transfer any remaining feed in the feed boxes and spill tray to the respective

diet boxes.

19. Wash bottom lids and lower portion of the case, funnel and nylon net with

approximately 75 mL of lukewarm water, using a soft brush through a large

glass funnel down the urine flask with funnel and glass wool. Further, wash funnel

with glass wool 3 times with water to ensure that all N has been washed out.

20. Transfer urine plus washings quantitatively to a graduated 500 mL flask and

make up the volume. Mix well.

21. To beaker containing faeces, add 4 times 25 mL conc. H2SO4 at hourly

intervals. After each addition stir and mix thoroughly with spatula and allow

it to cool.



7.13



Protein Estimation by Lowry’s Method



113



22. This process if followed 4 times, the resultant faeces solution would become

homogenous. Transfer this mixture to 500 mL volumetric flask and make up the

volume.

23. Weigh the remaining feed in diet boxes and record in the notebook.

24. Determine N in urine and faeces by taking out 25 mL sample of urine and

50 mL sample of faces by micro-Kjeldahl method. Samples are analysed in

duplicate.

25. Calculate the total amount of N excreted in urine and in faeces by each rat

during balance period.

Determination of metabolic and endogenous nitrogen: Feed a separate group

with 4% freeze-dried, ether-extracted egg protein as the protein source. Since egg

protein at this level is completely utilized by rats, nitrogen in the urine and faeces

must be of endogenous origin.

Collect the determined nitrogen in the urine and faeces as described, and

incorporate in the calculations.

Calculations

NPU ¼





N retained I F Fk ị U Vk ị



N intake

I

N absorbed I F Fk ị



N intake

I



and

BV ẳ



N retained

I Fk ị U Uk ị



N absorbed

I À ðF À Fk Þ



where I is the intake nitrogen (nitrogen in the diet), F ¼ faecal nitrogen,

Fk ¼ endogenous faecal nitrogen, U ¼ urinary nitrogen and Uk ¼ endogenous

urinary nitrogen.



7.13



Protein Estimation by Lowry’s Method (Lowry et al. 1951)



Protein can be estimated by method described by Lowry and colleagues. The

method is sensitive enough to give a moderately constant value and hence largely

followed. Protein content of enzyme extracts is usually determined by this method.

Principle

The blue colour developed by the reduction of the phosphomolybdicphosphotungstic components in the Folin-Ciocalteau reagent by the amino acids

tyrosine and tryptophan present in the protein plus the colour developed by the



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Qualitative and Quantitative Estimations of Amino Acids and Proteins



biuret reaction of the protein with the alkaline cupric tartrate are measured in the

Lowry’s method.

Reagents

1. Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide.

2. Reagent B: 0.5% copper sulphate (CuSO4Á5H2O) in 1% potassium sodium

tartrate.

3. Reagent C: alkaline copper solution: Mix 50 mL of A and 1 mL of B prior to use.

4. Reagent D: Folin-Ciocalteau reagent – reflux gently for 10 h a mixture

consisting of 100 g sodium tungstate (Na2WoO4Á2H2O), 25 g sodium molybdate

(Na2MoO4Á2H2O), 700 mL water, 50 mL of 85% phosphoric acid, and 100 mL

of concentrated hydrochloric acid in a 1.5-L flask. Add 150 g lithium sulphate,

50 mL water and a few drops of bromine water. Boil the mixture for 15 min

without condenser to remove excess bromine. Cool, dilute to 1 L and filter. The

reagent should have no greenish tint. (Determine the acid concentration of the

reagent by titration with 1 N NaOH to a phenolphthalein end-point). FolinCiocalteau reagent can be purchased commercially. Store refrigerated in amber

bottles. A good quality reagent is straw yellow in colour.

5. Protein solution (stock standard): weigh accurately 50 mg of bovine serum

albumin and dissolve in distilled water and make up to 50 mL in a standard flask.

6. Working standard: dilute 10 mL of the stock solution to 50 mL with distilled

water in a standard flask. One millilitre of the solution contains 200 mg protein.

Procedure

Extraction of protein from sample: Extraction is usually carried out with buffers

used for the enzyme assay. Weigh 500 mg of the sample and grind well with a pestle

and mortar in 5–10 mL of the buffer. Centrifuge and use the supernatant for protein

estimation.

Estimation of protein:

1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 mL of the working standard into a series of test

tubes.

2. Pipette out 0.1 and 0.2 mL of the sample extract in two other test tubes.

3. Make up the volume to 1 mL in all the test tubes. A tube with 1 mL of water

serves as the blank.

4. Add 5 mL of reagent C to each tube including the blank. Mix well and allow to

stand for 10 min.

5. Then add 0.5 mL of reagent D, mix well and incubate at room temp in the dark

for 30 min. Blue colour is developed.

6. Take the readings at 660 nm.

7. Draw a standard graph and calculate the amount of protein in the sample.



7.14



Polycrylamide-Sodium Dodecyl Sulphate Slab Gel Electrophoresis. . .



115



Calculation

Express the amount of protein mg/g or 100 g sample.

• If protein estimation is desired in a sample with high phenolic or pigment

content, extract should be prepared with a reducing agent preferably cysteine

and NaCl. Precipitate the protein with TCA, separate the protein and dissolve in

2N NaOH and proceed.

• If the protein concentration of the sample is high (above 50 mg/mL) measure the

colour intensity at 550 nm.

• For complete enzyme extraction, sometimes the chemicals like ethylenediamine

tetraacetic acid (EDTA), magnesium salts and mercaptoethanol are included.

This method of protein (mercaptoethanol) compounds as they interfere with this

procedure. When these chemicals are present in the extract, precipitate the

protein by adding 10% TCA, centrifuge and dissolve the precipitate in 2 N

NaOH and proceed for protein estimation.

• Rapid mixing as the Folin reagent is added is important for reproducibility.



7.14



Polycrylamide-Sodium Dodecyl Sulphate Slab

Gel Electrophoresis (SDS-PAGE) of Proteins

(Laemmli 1970)



Electrophoresis of proteins in polyacrylamide gels is carried out in buffer gels

(non-denaturing) as well as in sodium dodecyl sulphate (SDS) containing

(denaturing) gels. Polyacrylamide gel is more convenient than in any other medium

such as paper and starch gel. Electrophoretic procedures are rapid and relatively

sensitive requiring only micro-weights of proteins. Separation in buffer gels relies

on both the charge and size of the protein, whereas it depends only upon the size in

the SDS-gels. Analysis and comparison of proteins in a large number of samples is

easily made on polyacrylamide gel slabs.

Polyacrylamide gels are formed by polymerising acrylamide with cross-linking

agent (bisacrylamide) in the presence of a catalyst (persulphate ion) and chain

initiator (TEMED; N,N,N,N-tetramethylethylene diamine). Solutions are normally

degassed by evacuation prior to polymerization since oxygen inhibits polymerization. The porosity of the gel is determined by the oxygen inhibited polymerization.

The porosity of the gel is determined by the relative proportion of acrylamide

monomer to bisacrylamide. Gels are usually referred to in terms of the total

percentage of acrylamide and bis present, and most protein separations are

performed using gels in the range 7–15%. A low percentage gel (with large pore

size) is used to separate high molecular weight proteins and vice versa. At high

concentrations of persulphate and TEMED the rate of polymerization is also high.

Among a number of methods commonly used, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) that facilitates characterization of

polypeptides and determination of their molecular weight is described.



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Qualitative and Quantitative Estimations of Amino Acids and Proteins



Principle

SDS is an anionic detergent which binds strongly to, and denatures, proteins.

The number of SDS molecules bound to a polypeptide chain is approximately

half the number of amino acid residues in that chain. The protein–SDS complex

carries net negative charges, hence moves towards the anode and the separation is

based on the size of the protein (charge to mass ratio).

Reagents

1. Stock acrylamide solution

Acrylamide 30% – 30 g

Bisacrylamide 0.8% – 0.8 g

DD Water to – 100 mL

2. Separating gel buffer

1.875 M Tris–HCl – 22.7 g (pH 8.8)

Water to – 100 mL

Stacking gel buffer

0.6 M Tris–HCl – 7.26 g (pH 6.8)

Water to – 100 mL

3. Polymerising agents

(a) Ammonium – 0.5 g/10 mL, prepare fresh before use persulphate 5%

(b) TEMED – fresh from the refrigerator

4. Electrode buffer (may be used 2–3 times)

0.05 M Tris – 12 g

0.192 M Glycine – 28.8 g (pH 8.2–8.4)

0.1% SDS – 2 g

Water to – 2 L

5. Sample buffer (5Â concentration)

Tris–HCl Buffer, pH 6.8 – 5 mL

SDS – 0.5 g

Sucrose – 5 g

Mercaptoethanol – 0.25 mL

Bromophenol Blue – 1 mL

(0.5% W/V solution in water)

Water to – 10 mL

Dilute to 1Â concentration and use. Store frozen in small aliquots.

6. Sodium dodecyl sulphate 10% solution – store at room temperature and use



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