The virtual laboratory: Nitrogen balance and protein requirements
copyright © 1982 - 2006 David A Bender
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As you run the program, you are asked after each simulated experiment whether you wish to save the results to print out. If you save results, they are saved in a file called simout.txt in your temporary file area. When you close the program you are given the option of printing out the results you have chosen to save for printing.
Nitrogen balance and protein requirements
Estimating protein requirements from studies of N balance
Essential and non-essential amino acids
Amino acid requirements
Nitrogen balance and protein requirements
An adult's average intake of protein is about 80 g per day; an additional 70 g enters the gut in digestive enzymes, shed intestinal cells and mucus secreted to protect the intestinal wall against digestive enzymes. Most of this protein is hydrolysed to amino acids and dipeptides in the intestinal lumen, and dipeptides are hydrolysed in the brush border of the mucosal cells. There is a faecal loss of protein and other nitrogenous compounds equivalent to about 10 g of protein per day. Some of this is indigestible dietary protein, some is mucin, the main protein of mucus, which is resistant to enzymic hydrolysis; much is bacterial protein.
The absorbed amino acids are used for protein synthesis, to replace proteins that are constantly turning over; in a growing child, or an adult recovering from protein loss, there is also nett protein synthesis.
Some of the amino acids released by catabolism of tissue proteins are re-utilised, but in general they are deaminated, and their carbon skeletons are used for gluconeogenesis or as metabolic fuels. Similarly, newly absorbed amino acids in excess of the need for protein synthesis are deaminated, and their carbon skeletons used for gluconeogenesis or fatty acid synthesis. The urea formed from the amino groups of these amino acids is the main nitrogenous compound excreted in the urine.
A relatively small proportion of the body's free amino acid pool is used for synthesis of a wide variety of other compounds, which are later metabolised to yield other nitrogenous compounds that are excreted mainly in the urine.
It is relatively easy to measure total nitrogenous compounds in foods, urine and faeces, by means of the Kjeldahl reaction, which is a catalytic reduction in concentrated sulphuric acid of all nitrogenous compounds to ammonium sulphate. Alkalisation of the resultant reaction mixture permits distillation of the ammonia, which can then be determined by titration or other methods.
Therefore it is relatively simple to determine the total dietary intake of nitrogenous compounds (mainly protein) and the total output of nitrogenous compounds (in urine, faeces, and perhaps also sweat and shed skin cells).
As shown in the diagram above, for an adult, in whom the total amount of protein in the body is not changing, output of nitrogen in urine and faeces is approximately equal to dietary intake. This is N balance or N equilibrium - the difference between intake and output of nitrogenous compounds.
N balance
intake = output : N equilibrium
There is no change in total body protein. This is the normal state in an adult.
intake > output: positive N balance
There is an increase in total body protein. This is the normal state in growth (including pregnancy), and in an adult recovering from a loss of body protein in response to trauma or undernutrition.
intake < output: negative N balance
There is a nett loss of body protein. This is never normal, but reflects either a response to trauma or infection, or an intake that is inadequate to meet the need to replace tissue proteins that are turning over.
Estimating protein requirements from studies of N balance
Our estimates of protein requirements have been determined by studying groups of people fed different amounts of protein, in order to determine the minimum intake that will permit them to maintain N equilibrium. At intakes below their requirement, they will show negative N balance.
Apart from periods of recovery from protein loss, it is possible to maintain N equilibrium at any level of protein intake above the minimum requirement. Intake in excess of requirements does not lead to positive N balance, but simply leads to excretion of more end-products of amino acid catabolism, so that balance is maintained.
This program permits you to vary a subject's protein intake week by week, and see the daily N balance; for a subject overall in N equilibrium there is normally slight fluctuation between marginally negative and marginally positive balance from day to day.
The graph below shows the results of such a study in an adult. Initially the subject was in N equilibrium (averaged out over a week), at an intake of 70 g of protein per day.
During a week with a protein-free diet there was significant negative N balance; the lost protein was replaced during 2 weeks at an intake of 70 g /day, shown as a period of positive N balance; once the lost protein had been replaced, the subject returned to N equilibrium.
Reducing the intake to 50 g /day still permitted maintenance of N equilibrium (again averaged out over a week, not considering only the results from a single day). However, 40 g /day was not adequate to maintain N balance.
A growing child should be in positive N balance, since growth involves increasing the total amount of protein the the body. The graph below shows the results of varying a child's protein intake; an adequate intake will result in positive N balance, to the extent of the grey shaded area. An intake that only supports N equilibrium in not adequate for a growing child.
Essential and non-essential amino acids
Non-essential amino acids
Some of the amino acids can readily be synthesised in the body, from more or less common metabolic intermediates. These are known as non-essential, or dispensable, amino acids, since there is no need for them to be provided in the diet. As long as there is enough total protein in the diet, these amino acids can be synthesised.
If people are fed a mixture of amino acids lacking one (or more) of the non-essential amino acids, they can still maintain N equilibrium.
Essential amino acids
Other amino acids that are required for protein synthesis cannot be synthesised in the body, but must be provided in the diet.
If people are fed a mixture of amino acids lacking one of the essential amino acids, they cannot maintain N equilibrium, but show negative N balance.
This program permits you to simulate experiments in which your subject is fed a mixture of amino acids - this may be either a complete mixture, or lacking one amino acid. Again you feed each mixture for a week and see the daily N balance.
Not all non-essential amino acids are completely dispensable
Although it is possible for a healthy subject to maintain N equilibrium when any of the non-essential amino acids is excluded from the diet, there are conditions under which the capacity to synthesise some of the non-essential amino acids is less than the need for them. Such conditions include rapid growth in infants, and recovery from traumatic protein loss in adults.
Amino acid requirements
The need for protein is really the need for individual amino acids, and the nutritional value of any given protein depends on its content of the various essential amino acids, relative to the requirement for those amino acids.
This program permits you to investigate amino acid requirements by simulating experiments in which your subject is fed a mixture of amino acids containing plentiful amounts of all but the one under investigation. By varying the amount of this amino acid for a week at a time you can determine the minimum amount that is required to maintain N equilibrium.