Three experiments were carried out to obtain more knowledge about the use of feeding protein diet separately and protein-free diet to estimate the biological value of proteins.
Proteins were given in three experiments for one hour daily and free access to protein-free diet for the following 23-hour. In Expt. 1, casein and wheat gluten were eaten daily in an amount of 192 mg nitrogen (N) per male rat for 12 weeks after a training period. In Expt. 2, these two proteins in addition to potato protein and soya protein were given daily in an amount of 192 mg N per male rat for 16 weeks after a training period. In Expt. 3, casein and wheat gluten were given to appetite for three replicates of weanling male or female rats for 20 weeks. Liveweight, feed intake and carcass analysis of water, fat and N were recorded in the three experiments. Levels of free amino acids in blood (FAA) before and after the protein meal were estimated at different intervals in Expt. 2, and fasting levels (before giving the protein) of the same rats at different ages were determined in Expt. 3. Furthermore, FAA of some newborn rats and their mothers (Trial 3) on the day of birth were estimated.
As judged from the effect of the protein types on different parameters, casein and the third potato protein ranked alike in their nutritive value. They were superior in their nutritive value to wheat gluten and soya protein (heated). The wheat gluten was inferior in nutritive value to the soya protein in a relatively short experimental period, but similar in a relatively long experimental period.
On an equal amount of N, rats on inferior proteins increased in weight more slowly than rats on superior proteins. Prolongation of the experiment with restricted protein resulted in a similar total gain in weight, when the difference in the protein quality was not too large (wheat gluten and soya protein). Furthermore, rats on proteins with large difference in quality also gained similar total liveweight, when the proteins (casein and wheat gluten) were offered to appetite for a relatively long experiment (Expt. 3).
The ratio of energy to protein was higher on giving superior protein to appetite or in restricted amount than on inferior protein. When the protein was to appetite, rats seemed to adapt themselves to the deficiency of amino acids (AA) in wheat gluten as judged from the similar liveweight of the casein and wheatgluten group of the same sex in Expt. 3 by increasing the protein intake.
On restricted protein, the percentage and quantities of the component of the rats' bodies determined did not differ significantly between casein and potato groups and between wheat-gluten and soya group, whereas there were significant differences between the casein or potato group and wheat-gluten or soya group. These significant differences indicate that superior protein can increase rapidly not only quantities of body components but also some of their proportions. The ratio of N to water and N % are slightly, though significantly, affected by the quality of protein in a restricted amount over a relatively long period (Expt. 1 and 2). However this ratio and percentage varied generally in relatively narrow limits in the three experiments. Although there were no significant differences between the weights of the casein and wheat-gluten group of the same sex at the end of Expt. 3, significant differences between their body constituents were found in some cases but not between the amount or percentage N. In general, age influence was confirmed to be a decrease in water percentage and ail increase in nitrogen percentage, in fat percentage and in the quantities of the body components.
In most instances, higher levels of FAA were recorded after eating proteins than during fasting on the protein-free diet. Generally, the percentage increases in tyrosine or the branched amino acids (isoleucine, leucine and valine) were highest of FAA, whereas those of glycine and glutamic acid were lowest.
Detection of limiting amino acid(s) in the protein - casein, potato protein, wheat gluten and soya protein - from the levels of FAA before and after eating proteins was not completely successful. Free arginine and histidine levels decreased to a great extent in some instances after eating casein, potato protein, wheat gluten and soya protein in spite of being probably, not the most limiting AA in these four proteins. This probably indicates metabolism interrelations. Care must be taken, therefore, in the interpretation of levels of FAA as an index for limiting AA, i.e., they may not reflect the exact limiting order of the dietary AA. Nevertheless, the data show that wheat gluten is deficient in lysine and soya protein is more deficient than potato protein in methionine.
Markedly, fasting levels of free threonine and glutamic acid in blood of potato group and taurine in blood of wheat-gluten group were the highest values of the four groups on feeding 192 N in Expt. 2. Moreover, levels of free threonine, lysine and glutamic acid in blood of wheat-gluten group before and after feeding on the proteins were the lowest values. When fed on casein and wheat gluten to appetite (Expt. 3), free lysine and threonine were mostly lower in blood of males and females of wheat-gluten group than of casein group. Although there were sometimes rough relationships between the levels of FAA and AA contents in dietary proteins, rats modify the ratio between essential AA of dietary proteins to maintain almost constant ratio between essential FAA. Generally, variations in the levels of essential FAA due to feeding, ages, individuals or amount of protein were reduced on expressing them in percentages of the total essential amino acids.
At 12 weeks of age in Expt. 3, free lysine levels in blood of wheat-gluten group were markedly higher in females than in males. At 24 weeks of age, levels of free threonine in blood of wheat-gluten group were also markedly higher in females than in males. This suggests that females adapt themselves better for the deficiency of lysine and threonine in wheat-gluten diet than males.
With age, most FAA, though not the same, increased from birth to weaning at about 4 weeks old, from beginning to Week 8 of Expt. 3 (4-12 weeks old) as shown in males and females of casein and wheat-gluten group at 12 weeks of age, then decreased from Week 8 to 20 of Expt. 3 (12-24 weeks old) except for females of casein group at 24 weeks of age. Furthermore, most FAA of male rats also decreased in many cases in Expt. 2 with age from 10-21 weeks, in spite of the difference in the amount and type of the protein in Expt. 2 and Expt. 3. It is noteworthy that a markedly reversed trend (decrease) was found in level of free histidine from birth to weaning. Furthermore, levels of free lysine in blood of rats on deficient diet in lysine increased with age, whereas most free essential FAA decreased. Generally, intake of wheat gluten compared to casein tended to reduce the average levels of most individual FAA, when restricted or fed to appetite. Most fasting levels of FAA of wheat-gluten group were lower on restricted amount of gluten (Expt. 2) than on free access to the protein (Expt. 3).
Most essential FAA of newborn rats were higher than their mothers. Furthermore, rats post partum had a lower level of FAA than virgin rats.
Data of the experiments show that alteration in levels of FAA is affected by the interaction of protein type, age and sex in addition to other factors such as pregnancy and metabolic interrelationships.