á141ñPROTEIN—BIOLOGICAL ADEQUACY TEST
This test is intended for the evaluation of the biological adequacy,as an index to the completeness of the mixture of amino acids contained,of Protein Hydrolysate Injection.

Depletion Diet—
Parts by Weight
Dextrin 83.9
Corn Oil 9.0
Salt Mixture 4.0
Agar 2.0
Cod Liver Oil 1.0
Choline Chloride 0.15
Inositol 0.10
Calcium Pantothenate 0.002
Niacinamide 0.0015
Riboflavin 0.0003
Pyridoxine 0.00025
Thiamine 0.0002
p-Aminobenzoic Acid 0.0002
Folic Acid 0.0002
Menadione 0.0002
Biotin 0.00002
Salt Mixture— Prepare the salt mixture specified in the Depletion Dietas follows:
Sodium Chloride 139.3g
Potassium Biphosphate 389.0g
Magnesium Sulfate,Anhydrous 57.3g
Calcium Carbonate 381.4g
Ferrous Sulfate 27.0g
Manganese Sulfate 4.01g
Potassium Iodide 0.79g
Zinc Sulfate 0.548g
Cupric Sulfate 0.477g
Cobaltous Chloride 0.023g
Place a portion of the weighed quantity of sodium chloride in a suitable mortar and add,with grinding,the potassium iodide.Set aside the mixture,and mix in a similar manner all the other salts with the remainder of the sodium chloride,adding finally the previously mixed sodium chloride and potassium iodide.Reduce the entire mixture to a fine powder (see Powder Fineness á811ñ).

Control Nitrogen Supplement Mixture— Place 50g of calcium caseinate and 46g of anhydrous dextrose in a beaker,add sufficient water to make a paste,and finally add 1000mLof water.Heat the solution between 70and 82for 5minutes with stirring,and cool.Determine nitrogen on an aliquot using Nitrogen Determination—Method Ior Method IIá461ñ.Store in a refrigerator.Mix before removing portions for analysis or use.

Depletion and Control Periods— Select a group of not less than six male rats 2to 4months of age and each weighing between 190g and 225g.Place the rats in individual cages with free access to water and the Depletion Dietfor 12days.Weigh the depleted rats,and discard any rat that weighs more than 90%of its starting weight.
For the next 3days substitute as drinking water the Control Nitrogen Supplement Mixturein a quantity equivalent to 0.12g of nitrogen per rat per day,diluted with water to 20mL,and offered at the same time each morning either in a dish suitable for preventing spillage or in a reservoir fitted with a drinking tube.Remove all drinking water from the cages of the depleted rats during each feeding,and return it after the supplement has been consumed or is removed.On the third day,weigh each rat.Discard any rats that have not consumed all of the Control Nitrogen Supplement Mixture.
For the next 3days,replace the Control Nitrogen Supplement Mixturewith water ad libitum,and continue the rats on the Depletion Diet.Weigh the rats,and discard any that have not lost weight since the previous weighing.

Procedure— Assemble not less than six rats that have completed the depletion and control periods.For 5days maintain the assembled rats on the Depletion Dietwith a daily supplement of 20mL,accurately measured,of a solution containing the Protein Hydrolysate Injection in an amount equivalent to 0.12g of nitrogen offered each morning in the same way as the Control Nitrogen Supplement Mixturewas offered previously.Withhold water for at least 2hours prior to offering the supplement and for 4hours afterward.Then if the supplement has been consumed,offer water ad libitum.
On the afternoon of the fifth day,weigh each rat,and compare the respective final and starting weights.Not fewer than 80%of the group of rats used gain weight or maintain their weight during the test.