Nitrogen balance and protein requirements

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Figure 9.1 shows an overview of protein metabolism; in addition to the dietary intake of about 80 g of protein, almost the same amount of endogenous protein is secreted into the intestinal lumen. There is a small faecal loss equivalent to about 10 g of protein per day; the remainder is hydrolysed to free amino acids and small peptides, and absorbed (section 4.4.3). The faecal loss of nitrogen is partly composed of undigested dietary protein, but the main contributors are intestinal bacteria and shed mucosal cells, which are only partially broken down, and the protective mucus secreted by intestinal mucosal goblet cells (see Figure 4.2). Mucus is especially resistant to enzymic hydrolysis, and contributes a considerable proportion of inevitable losses of nitrogen, even on a protein-free diet.

There is only a small pool of free amino acids in the body, in equilibrium with proteins that are being catabolized and synthesized. A small proportion of the amino acid pool is used for synthesis of a wide variety of specialized metabolites (including hormones and neurotransmitters, purines and pyrimidines). An amount of amino acids equivalent to that absorbed is oxidized, with the carbon skeletons being used for gluconeogenesis (sections 5.7 and 9.3.2) or as metabolic fuels, and the nitrogen being excreted mainly as urea (section

The state of protein nutrition, and the overall state of body protein metabolism, can be determined by measuring the dietary intake of nitrogenous compounds and

Metabolism Nitrogen Balance
Figure 9.1 An overview of protein metabolism.

the output of nitrogenous compounds from the body. Although nucleic acids also contain nitrogen (section 9.2.1), protein is the major dietary source of nitrogenous compounds, and measurement of total nitrogen intake gives a good estimate of protein intake. Nitrogen constitutes 16% of most proteins, and therefore the protein content of foods is calculated on the basis of mg N X 6.25, although for some foods with an unusual amino acid composition other factors are used.

The output of N from the body is largely in the urine and faeces, but significant amounts may also be lost in sweat and shed skin cells — and in longer-term studies the growth of hair and nails must be taken into account. Obviously, any loss of blood or tissue will also involve a loss of protein. Although the intake of nitrogenous compounds is mainly protein, the output is mainly urea (section, though small amounts of a number of other products of amino acid metabolism are also excreted, as shown in Table 9.1.

The difference between intake and output of nitrogenous compounds is known as nitrogen balance. Three states can be defined:

  • An adult in good health and with an adequate intake of protein excretes the same amount of nitrogen each day as is taken in from the diet. This is nitrogen balance or nitrogen equilibrium: intake = output and there is no change in the total body content of protein.
  • In a growing child, a pregnant woman or someone recovering from protein loss, the excretion of nitrogenous compounds is less than the dietary intake — there is a net retention of nitrogen in the body and an increase in the body content of protein. This is positive nitrogen balance: intake > output and there is a net gain in total body protein.
  • In response to trauma or infection (section or if the intake of protein is inadequate to meet requirements, there is net a loss of nitrogen from the body — the output is greater than the intake. This is negative nitrogen balance: intake < output and there is a loss of body protein.
Table 9.1 Average daily excretion of nitrogenous compounds in the urine


10-35 g

150-600 mol

Depends on the intake of protein


340-1200 mg

20-70 mmol

Depends on the state of acid-base balance

Amino acids,

1.3-3.2 g


peptides and



< 60 mg


Significant proteinuria indicates kidney damage

Uric acid

250-750 mg

1.5-4.5 mmol

Major product of purine metabolism


Male 1.8 g

Male 16 mmol

Depends on muscle mass

Female 1.2 g

Female 10 mmol


< 50 mg

< 400 mmol

Higher levels indicate muscle catabolism

246 Protein nutrition and metabolism 9.1.1 Dynamic equilibrium

The proteins of the body are continually being broken down and replaced. As shown in Table 9.2, some proteins (especially enzymes that have a role in controlling metabolic pathways) may turn over within a matter of minutes or hours; others last for longer before they are broken down, perhaps days or weeks. Some proteins only turn over very slowly — for example the connective tissue protein collagen is broken down and replaced so slowly that it is almost impossible to measure the rate — perhaps half of the body's collagen is replaced in a year.

This continual breakdown and replacement is dynamic equilibrium. Superficially, there is no change in body protein. In an adult there is no detectable change in the amount of protein in the body from one month to the next. Nevertheless, if an isotopically labelled amino acid is given, the process of turnover can be followed. As shown in Figure 9.2, the label rapidly becomes incorporated into newly synthesized proteins, and is gradually lost as the proteins are broken down. The rate at which the label is lost from any one protein depends on the rate at which that protein is broken down and replaced; the time for the labelling to fall to half its peak is the half-life of that protein.

Protein breakdown occurs at a more or less constant rate throughout the day, and an adult catabolizes and replaces some 3—6 g of protein per kilogram body weight per day. Turnover is also important in growing children, who synthesize considerably more protein per day than the net increase in body protein. Even children recovering from severe protein—energy malnutrition (see Chapter 8), and increasing their body protein rapidly, still synthesize 2—3 times more protein per day than the net increase.

Although an adult may be in overall nitrogen balance, this is the average of periods of negative balance in the fasting state and positive balance in the fed state. As discussed

Table 9.2 Half-lives of some proteins



Ornithine decarboxylase

Lipoprotein lipase

Tyrosine transaminase

Phosphoenolpyruvate carboxykinase

Tryptophan oxygenase

HMG CoA reductase


Alanine transaminase


Serum albumin


Lactate dehydrogenase Adult collagen Infant collagen

1.25 days 3.5 days 4-5 days 16 days 300 days

1 hours 1.5 hours

2 hours

2 hours

3 hours 12 hours

0.7-1 days

11 minutes

1-2 days and 150 days

Nitrogen Balance Metabolism

Figure 9.2 Determination of the half-life of body proteins using 15N-labelled amino acids.

half-life days

Figure 9.2 Determination of the half-life of body proteins using 15N-labelled amino acids.

in section, protein synthesis is energy expensive, and in the fasting state the rate of synthesis is lower than that of protein catabolism. There is a loss of tissue protein, which provides amino acids for gluconeogenesis (section 5.7). In the fed state, when there is an abundant supply of metabolic fuel, the rate of protein synthesis increases and exceeds that of breakdown, so that what is observed is an increase in tissue protein, replacing that which was lost in the fasting state.

As discussed in section 8.3, even in severe undernutrition, the rate of protein breakdown remains more or less constant, while the rate of replacement synthesis falls, as a result of the low availability of metabolic fuels. It is only in cachexia (section 8.4) that there is increased protein catabolism as well as reduced replacement synthesis. Mechanisms involved in tissue protein catabolism

The catabolism of tissue proteins is obviously a highly regulated process; as shown in

Table 9.1, different proteins are catabolized (and replaced) at very different rates.

Three different mechanisms are involved in the process:

  • Lysosomal cathepsins are proteases with a broad range of specificity, leading to complete hydrolysis of proteins to free amino acids. They hydrolyse proteins that have entered the cell by phagocytosis and are also involved in the hydrolysis of cell proteins after cell death, when they are released into the cytosol. In addition, a number of intracellular proteins contain the sequence Lys-Phe-Glu-Arg-Gly, which targets them for uptake into the lysosomes, where they undergo hydrolysis.
  • Calpain is a protease with a broad specificity for hydrophobic amino acids, resulting in partial proteolysis. It has a calcium-dependent regulatory subunit and is inhibited by a second protein, calstatin, so its activity in the cell is regulated. Both proteins turn over relatively rapidly, and there is an increase in the amount of mRNA for both proteins in the cell during fasting and starvation — this seems to be the result of increased gene expression in order to maintain a constant amount of both proteins despite the reduction in overall protein synthesis.
  • The ubiquitin—proteasome system catalyses ATP-dependent proteolysis. It is important in both protein turnover and antigen processing.
  • Ubiquitin is a small peptide (M 8,500) that forms a covalent bond from the carboxy terminus to the e-amino of lysine residues in target proteins — this is an ATP-dependent process, and multiple molecules of ubiquitin are attached to target proteins. It is not known what targets proteins for ubiquitination; at least four different ubiquitin-transferring enzymes are known.
  • The proteasome (also known as the multifunctional protease) is a multi-subunit complex that accounts for about 1% of the total soluble protein of cells. There are at least five types of subunit with specificity for esters of hydrophobic, basic and acidic amino acids.


It is the continual catabolism of tissue proteins that creates the requirement for dietary protein. Although some of the amino acids released by breakdown of tissue proteins can be re-used, most are metabolized, by pathways which are discussed in section 9.3, yielding intermediates that can be used as metabolic fuels and for gluconeogenesis (section 5.7) and urea (section, which is excreted. This means that there is a need for dietary protein to replace losses even in an adult who is not growing. In addition, relatively large amounts of protein are lost from the body in mucus, enzymes and other proteins, which are secreted into the gastrointestinal tract and are not completely digested and reabsorbed.

Current estimates of protein requirements are based on studies of the amount required to maintain nitrogen balance. If the intake is not adequate to replace the protein that has been broken down, then there is negative nitrogen balance — a greater output of nitrogen from the body than the dietary intake. Once the intake is adequate to meet requirements, nitrogen balance is restored. The proteins that have been broken down can be replaced, and any surplus intake of protein can be used as a metabolic fuel.

Such studies show that for adults the average daily requirement is 0.6 g of protein per kilogram body weight. Allowing for individual variation, the reference intake (section 11.1.1) is 0.75 g/kg body weight, or 50 g/day for a 65 kg adult. Average intakes of protein by adults in developed countries are considerably greater than requirements, of the order of 80—100 g/day. The reference intake of protein is sometimes called the safe level of intake, meaning that it is safe and (more than) adequate to meet requirements, not implying that there is any hazard from higher levels of intake.

Protein requirements can also be expressed as a proportion of energy intake. The energy yield of protein is 17 kJ/g, and the reference intake of protein represents some 7—8% of energy intake. In Western countries protein provides 14—15% of energy intake.

It is unlikely that adults in any country will suffer from protein deficiency if they are eating enough food to meet their energy requirements. As shown in Figure 9.3, the major dietary staples that are generally considered as sources of carbohydrate also provide significant amounts of protein. Even among people in Western countries who eat meat, fish and eggs (which are generally regarded as rich protein sources) about 25% of protein intake comes from cereals and cereal products, with an additional 10% from fruit and vegetables.

Only cassava, yam and possibly rice provide insufficient protein (as a percentage of energy) to meet adult requirements. The shortfall in protein provided by a diet based on yam or rice would be made up by small amounts of other foods that are sources of protein — this may be either small amounts of meat and fish or legumes and nuts, which are rich vegetable sources of protein. With diets based largely on cassava there is a more serious problem in meeting protein requirements.

cassava yam rice barley potato rye oatmeal maize pasta wheat

12 15

protein, % energy

Figure 9.3 Protein as percentage of energy in dietary staples. Protein requirements of an adult are met when the diet provides 7—8% of energy from protein. Of the major dietary staples, only cassava, yam and (marginally) rice fail to pro-vide this much protein. Protein requirements of children

Because children are growing, and increasing the total amount of protein in the body, they have a proportionally greater requirement than adults. A child should be in positive nitrogen balance while he or she is growing. Even so, the need for protein for growth is relatively small compared with the requirement to replace proteins which are turning over. Table 9.3 shows protein requirements at different ages. Children in Western countries consume more protein than is needed to meet their requirements, but in developing countries protein intake may well be inadequate to meet the requirement for growth.

A protein-deficient child will grow more slowly than one receiving an adequate intake of protein — this is stunting of growth. As discussed in section 8.2, the protein— energy deficiency diseases, marasmus and kwashiorkor, result from a general lack of food (and hence metabolic fuels), not a specific deficiency of protein. Protein losses in trauma and infection - requirements for convalescence

One of the metabolic reactions to a major trauma, such as a burn, a broken limb or surgery, is an increase in the net catabolism of tissue proteins. As shown in Table 9.4, apart from the loss of blood associated with injury, as much as 750 g of protein (about 6—7% of the total body content) may be lost over 10 days. Even prolonged bed rest results in a considerable loss of protein, because there is atrophy of muscles that are not used. Muscle protein is catabolized as normal, but without the stimulus of exercise it is not completely replaced.

This protein loss is mediated by the hormone cortisol, which is secreted in response to stress, and the cytokines that are secreted in response to trauma; four mechanisms are involved:

  • Tryptophan dioxygenase and tyrosine transaminase. This results in depletion of the tissue pools of these two amino acids, leaving an unbalanced mixture of amino acids that cannot be used for protein synthesis (section 9.2.3).
  • In response to cytokine action there is an increase in metabolic rate, leading to an increased rate of oxidation of amino acids as metabolic fuel, so reducing the amount available for protein synthesis.
  • Cytokines cause an increase in the rate of protein catabolism, as occurs in cachexia (section 8.4).
  • A variety of plasma proteins synthesized in increased amount in response to cytokine action (the so-called acute-phase proteins) are richer in two amino acids, cysteine and threonine, than most tissue proteins. This leads to depletion of tissue pools of these two amino acids, again leaving an unbalanced mixture of amino acids that cannot be used for protein synthesis.

The lost protein has to be replaced during recovery, and patients who are

Table 9.3 Reference nutrient intakes for protein


Recommended protein intake (g/day)

4-6 months


7-9 months


10-12 months


1-1.5 years


1.5-2 years


2-3 years


3-4 years


4-5 years


5-6 years


6-7 years


7-8 years


8-9 years


9-10 years



10 years


11 years


12 years


13 years


14 years


15 years


16 years


17 years





10 years


11 years


12 years


13 years


14 years


15 years


16 years


17 years




Source: FAO/WHO/UNU (1985) Energy and protein requirements. Report of a Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report Series 724, Geneva.

Source: FAO/WHO/UNU (1985) Energy and protein requirements. Report of a Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report Series 724, Geneva.

convalescing will be in positive nitrogen balance. However, this does not mean that a convalescent patient requires a diet that is richer in protein than usual. As discussed in section 9.1.2, average protein intakes are twice requirements; a normal diet will provide adequate protein to permit replacement of the losses due to illness and hospitalization.

Table 9.4 Protein losses (g) over 10 days after trauma or infection

Tissue loss

Blood loss



Fracture of femur




Muscle wound





35% burns










Typhoid fever





From data reported by Cuthbertson DP (1964), in Human Protein Metabolism, Vol. II, Munro HN and Allison JB (eds), New York, Academic Press, pp. 373-414.

From data reported by Cuthbertson DP (1964), in Human Protein Metabolism, Vol. II, Munro HN and Allison JB (eds), New York, Academic Press, pp. 373-414.

9.1.3 Essential amino acids

Early studies of nitrogen balance showed that not all proteins are nutritionally equivalent. More of some is needed to maintain nitrogen balance than others. This is because different proteins contain different amounts of the various amino acids (section 4.4.1). The body's requirement is not simply for protein, but for the amino acids which make up proteins, in the correct proportions to replace the body proteins.

As shown in Table 9.5, the amino acids can be divided into two main groups, with each group further subdivided:

  • The nine essential or indispensable amino acids, which cannot be synthesized in the body. If one of these is lacking or provided in inadequate amount, then regardless of the total intake of protein it will not be possible to maintain nitrogen balance, as there will not be an adequate amount of the amino acid for protein synthesis.
  • Two amino acids, cysteine and tyrosine, can be synthesized in the body, but only from essential amino acid precursors — cysteine from methionine and tyrosine from phenylalanine. The dietary intakes of cysteine and tyrosine thus affect the requirements for methionine and phenylalanine — if more of either is provided in the diet, then less will have to be synthesized from the essential precursor.
  • For premature infants, and possibly also for full-term infants, a tenth amino acid is essential — arginine. Although adults can synthesize adequate amounts of arginine to meet their requirements, the capacity for arginine synthesis is low in infants and may not be adequate to meet the requirements for growth.
  • The non-essential or dispensable amino acids, which can be synthesized from metabolic intermediates, as long as there is enough total protein in the diet. If one of these amino acids is omitted from the diet, nitrogen balance can still be maintained.
  • Only three amino acids, alanine, aspartate and glutamate, can be considered to be truly dispensable; they are synthesized from common metabolic intermediates (pyruvate, oxaloacetate and a-ketoglutarate respectively; section

Table 9.5 Essential and non-essential amino acids


Essential precursor












Cysteine Tyrosine










  • The remaining amino acids are generally considered as non-essential, but under some circumstances the requirement may outstrip the capacity for synthesis:
  • A high intake of compounds that are excreted as glycine conjugates will increase the requirement for glycine considerably.
  • In response to severe trauma there is an increased requirement for proline for collagen synthesis for healing,
  • In surgical trauma and sepsis the requirement for glutamine increases significantly — a number of studies have shown considerably improved healing after major surgery if additional glutamine is provided.

The requirements for essential amino acids for growth (expressed as proportion of total protein intake) are higher than the requirement to maintain N balance in adults, and younger children, with a faster growth rate, have a higher requirement for essential amino acids as a proportion of total protein than do older children with a lower rate of growth. Table 9.6 shows various estimates of essential amino acid requirements and the 'reference pattern' of the amount of each amino acid that should ideally be present per gram of dietary protein.

Early studies of essential amino acid requirements were based on the amounts required to maintain nitrogen balance in young adults. Interestingly, for reasons that are not clear, these relatively short-term studies did not show any requirement for histidine. More recent studies have measured the rate of whole-body protein turnover using isotopically labelled amino acids. These have shown that the maximum rate of protein turnover is achieved with intakes of the essential amino acids some threefold higher than are required to maintain nitrogen balance. What is not clear is whether the maximum rate of protein turnover is essential, or even desirable. Protein quality and complementation

A protein that contains at least as much of each of the essential amino acids as is required will be completely useable for tissue protein synthesis, whereas one that is

Table 9.6 Estimates of essential amino acids requirements and reference pattern for adults


Protein Amino acid turnover oxidation

Require- Require- require- requirement Pattern ment Pattern ment ment (mg/kg (mg/g (mg/kg (mg/g (mg/kg (mg/kg bw) protein) bw) protein) bw) bw)

Histidine Isoleucine Leucine Lysine

Methionine +

cysteine Phenylalanine + tyrosine

0 10 14 12


25 22

10 14 12 13

16 13 19 16 17

33 65 70 27

66 50 22






















Sources: WHO/FAO (1973) Energy and protein requirements: Report of a joint FAO/WHO ad hoc expert committee. WHO Technical Reports Series 522, WHO, Geneva. FAO/WHO/UNU (1985) Energy and protein requirements. Report of a Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report Series 724, Geneva. Young VR, Bier DM and Pellett PL (1989) American Journal of Clinical Nutrition 50: 80—92. Young VR (1994) Journal ofNutrition 124: 1517—1523S.

Sources: WHO/FAO (1973) Energy and protein requirements: Report of a joint FAO/WHO ad hoc expert committee. WHO Technical Reports Series 522, WHO, Geneva. FAO/WHO/UNU (1985) Energy and protein requirements. Report of a Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report Series 724, Geneva. Young VR, Bier DM and Pellett PL (1989) American Journal of Clinical Nutrition 50: 80—92. Young VR (1994) Journal ofNutrition 124: 1517—1523S.

relatively deficient in one or more of the essential amino acids will not. More of such a protein will be required to maintain nitrogen balance or growth.

The limiting amino acid of a protein is that essential amino acid which is present in lowest amount relative to the requirement. In cereal proteins the limiting amino acid is lysine, while in animal and most other vegetable proteins it is methionine. (Correctly, the sum of methionine plus cysteine, as cysteine is synthesized from methionine and the presence of cysteine lowers the requirement for methionine.)

The nutritional value or quality of individual proteins depends on whether or not they contain the essential amino acids in the amounts that are required. A number of different ways of determining protein quality have been developed:

  • Biological value (BV) is the proportion of absorbed protein retained in the body. A protein that is completely useable (e.g. egg and human milk) has a BV of 0.9— 1; meat and fish have a BV of 0.75—0.8, wheat protein 0.5 and gelatine (which completely lacks tryptophan) a BV of 0.
  • Net protein utilization (NPU) is the proportion of dietary protein that is retained in the body (i.e. it takes account of the digestibility of the protein). By convention it is measured at 10% dietary protein, at which level the protein synthetic mechanism of the animal can utilize all of the protein so long as the balance of essential amino acids is correct.
  • Protein efficiency ratio (PER) is the gain in weight of growing animals per gram of protein eaten.
  • Relative protein value (RPV) is the ability of a test protein, fed at various levels of intake, to support nitrogen balance, compared with a standard protein.
  • Chemical score is based on chemical analysis of the protein; it is the amount of the limiting amino acid compared with the amount of the same amino acid in egg protein (which is completely useable for tissue protein synthesis). Protein score (or amino acid score) uses a reference pattern of amino acid requirements as the standard.

Although protein quality is important when considering individual dietary proteins, it is not particularly relevant when considering total diets, because different proteins are limited by different amino acids, and have a relative excess of other essential amino acids. This means that the result of mixing different proteins in a diet is to give an unexpected increase in the nutritional value of the mixture. Wheat protein is limited by lysine and has a protein score of 0.6; pea protein is limited by methionine and cysteine and has a protein score of 0.4. A mixture of equal amounts of these two individually poor-quality proteins has a protein score of 0.82 — as high as that of meat.

The result of this complementation between proteins that might individually be of low quality means that most diets have very nearly the same protein quality, regardless of the quality of individual protein sources. The average Western diet has a protein score of 0.73, while the poorest diets in developing countries, with a restricted range of foods, and very little milk, meat or fish, have a protein score of 0.6.

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  • Tuomo
    Is normal diet adequate to permit replacement of lost protein in response to trauma?
    2 years ago
  • DINA
    What is the total amount of endogenous protein that is been lost by the body in grammes?
    2 years ago
  • ines
    Why do fevers require negative nitrogen balance?
    1 year ago
  • Phillipp
    What is the unusual state of nitrogen balance of healthy infants children and pregnant women?
    1 year ago

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