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Methodology for Amino Acids and Proteins

 
     
 
Protein efficiency and net protein ratios
 
The nutritive value of a protein is best determined by in vivo experiments with rat which give more practical values then the mere chemical composition. However, the results of animal experiments have certain limitations due to various reasons. Yet, the animal experiments have been widely used to evaluate protein quality, along with the animal techniques, since there are no other better alternatives. Rat is used as the experimental animal because the results obtained with rat system have been found to agree many a time with the results obtained with human volunteers.

Measuring the growth rate of young animals fed with a test food over a period of time offers the simplest method to evaluate the nutritive value of proteins. The determination of Protein Efficiency and Net Protein Ratio (PER and NPR) are routinely carried out in many laboratories and these ratios are derived from the weight gain of the test animals.

 
When the growth method is used, PER and NPR are considered as the basic animal experiments. But precise information can be drawn only from nitrogen balance studies such as the estimation of Relative Net Protein Ratio.

As a word of caution, in animal experiments strict standardization of the experimental procedures is required to detect small differences in protein quality. The room temp between 22 and 25°C and a relative humidity between 50% and 65% are recommended for rats. The Wistar strains are frequently used. Healthy male rats should be selected. Feed consumption measurements should be accurate.

 
 


Principle
After four weeks of feeding with the test protein the ratio of weight gain to protein consumed is calculated as PER. The ratio of the weight gain in test animal plus the weight loss in control animal to the protein consumption by test animal gives the NPR.
 
 
Materials
Weanling male rats of Wistar strain, 20-23 days of age. Ten animals for each diet. Body weight 64-68g.
Basal diet on an air-dried basis:
Corn starch                                           80%
Corn oil or cotton seed oil                  10%
Non-nutritive cellulose                         5%
Salts*                                                       4%
Vitamin mixture*     &nb

sp;                             1%

Incorporate the protein food under test into the diet at the expense of corn starch to give 10 per cent (9.7-10.3) protein.
Individual cages provided with feeders.
 
 
Procedure
1.
Randomize 10 rats for each diet and feed them for four weeks with diet and water ad libitum.
2.
Record the food consumption and body weight at weekly and ten-day intervals.
3.
For PER Determination: In addition to the test group, maintain a reference group of rats on a diet consisting of the basal ration with casein  at the level of 10 per cent protein.
4.
For NPR Determination: in addition to the test group, maintain a control group of rats, matched with the test animals with respect to weights, on a diet consisting of the unmodified basal ration.
 
 
Calculation
1. PER: At four weeks, calculate the PER for each food and reference casein as
PER =
Wt. gained of test animal
Protein consumed
2. For corrected PER, proceed as follows:
Corrected PER = PER x
2.5
Determined PER for reference casein
3. NPR Determination: At ten days calculate the NPR for each food as follows
NPR =
Wt. gained of test animal + Weight loss of control animal
Protein consumed by test animal

Notes

1.The weights of animal is test and control groups should be matched to within 1g for the NPR determination.
2. The animal weight and protein consumption should be in grams.
3. Accurate determination of food spillage and separation of food from faecal and urinary contamination are essential.
4. Salt mixture
Salt
Weight in grams
Calcium carbonate (CaCO3)
78.6
Calcium citrate (Ca3C12H10O14.4H2O)
308.3
Calcium hydrogen phosphate (CaHPO4.2H2O)
112.8
Dipotassium hydrogen phosphate (K2HPO4)
218.8
Potassium chloride (KCl)
124.7
Sodium chloride (NaCl)
77.1
Magnesium sulphate (MgSO4)
38.3
Magnesium carbonate (MgCO3)
35.2
Ammonium ferric citrate (Brown, 20.5-22.5% Fe)
15.3
Manganese sulphate (MnSO4.H2O)
0.201
Copper sulphate (CuSO4.5H2O)
0.078
Potassium iodide (KI)
0.041
Aluminum ammonium sulphate (AlNH4(SO4)2.12H2O)
0.507
 
5. Composition of vitamins mixture: Prepare a vitamin mixture containing the following amounts in 1g.
Vitamin A, 1,000IU; Vitamin D, 100IU; Vitamin E, 10IU; Vitamin K (menadione), 0.5mg; thiamine, 0.5mg; riboflavin 1mg; pyridoxine, 0.4mg; pantothenic acid, 4mg; niacin, 4mg; choline, 200mg; inositol, 25mg; para-aminobenzoic acid, 10mg; Vitamin B12, 2mg; biotin 0.02mg; and folic acid, 0.2mg. ADD sufficient cellulose to make 1g.
 
 


References
1. Pellet, P L and Young, V R (1980) In: Nutritional Evaluation of Protein Foods. UN Univ Fd Nutr Bull Supp 4.


 
     
 
 
     




     
 
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