Muscle weakness and hypoketotic coma on fasting

There is obviously continuing loss of protein, which is not being replaced. There are obviously obligatory losses of nitrogen from the body, meaning that there is a continuing need for a dietary intake of protein.

In the next weeks he was fed 30 g of protein /day for 1 week, then 40 g /day for the second week

30 g of protein /day is not enough to meet his requirements, and he is still in negative nitrogen balance, still losing protein from the body. However, at 40 g /day he is able to maintain nitrogen balance, suggesting that this is adequate to meet his requirements and permit replacement of obligatory losses.

He was then fed 100 g of protein /day for 2 weeks. Initially he was in positive nitrogen balance, then after the first week he returned to nitrogen equilibrium.

During the first week at 100 g of protein /day he is in positive nitrogen balance, with output less than intake, as he is replacing the body protein that was lost during the weeks with an inadequate intake of protein.

Note that once he has replaced the lost protein he returns to nitrogen equilibrium, with intake = output, and no change in total body protein.

At any level of intake above requirements he is in nitrogen equilibrium - as intake increases, so output of nitrogenous compounds also increases to match the intake.

The table below shows the results of experiments performed by Schoenheimer and colleagues in the 1940s, when he fed rats diet containing an adequate amount of protein for them to maintain nitrogen equilibrium, but labelled with 15N amino acids. Since they were in nitrogen equilibrium, they expected to recover more or less all of the labelled nitrogen in urine and faeces.

From data reported by Schoenheimer, R 1946. The Dynamic State of the Body Constituents, Cambridge Mass, Harvard University Press.

The unexpected finding from these experiments was that less than half of the labelled nitrogen was recovered in urine and faeces, and about half had been incorporated into newly synthesised body proteins. While we now know that there is continual synthesis of proteins in the body, at the time Schoenheimer performed the experiments this was not known. He coined the term dynamic equilibrium to describe the phenomenon of breakdown of existing proteins and replacement, with no change in the total body protein content.

This means that we can modify the diagram of nitrogen balance to include not only protein synthesis, but also protein catabolism.

The driving force for this dynamic equilibrium is the catabolism of tissue proteins, and in fact our current understanding was predicted by the French physiologist Magendie in 1829, when he wrote

"All parts of the body of man experience an intimate movement [we would now call this metabolism] that serves both to expel those molecules that can or ought no longer to compose the body and replace them with new ones"

The table below shows the results of feeding rats with [15N]labeled amino acids and measuring the label in different proteins over a period of time. The time taken for the label to half to half its maximum is called the half-life of that protein.

The enzymes with a short half-life are all key regulatory enzymes in metabolic pathways whose synthesis and catabolism are regulated in response to hormones. With a short half-life it is possible to change the amount of the enzyme in cells in a short time. Note that this is changing the amount of enzyme in a cell, and requires a few hours for the hormone effect to be seen, as opposed to changes in the activity of existing enzyme protein in response to hormone action, which is seen within minutes or seconds of the hormone being secreted.

The enzymes with a half-life of several days can be regarded as "house-keeping" enzymes, whose synthesis is not significantly altered in response to hormone action.

Collagen is a structural protein, and except in times of rapid growth, when the skeleton is being remodelled, turns over only very slowly.

The table below summarises a series of experiments in which young men were fed mixtures of amino acids in amounts that were adequate to permit them to maintain nitrogen balance, but with one amino acid at a time removed from the mixture.

If any one of nine amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan or valine) is omitted from the diet then it is not possible to maintain nitrogen balance. This suggests that these nine amino acids are dietary essentials that cannot be synthesised in the body, and must be provided in the diet.

The remaining 11 amino acids can be omitted from the diet without affecting nitrogen balance, suggesting that they can be synthesised in the body, and are not dietary essentials. They are generally known as non-essential or indispensable amino acids.

Two of the non-essential amino acids are only synthesised in the body from essential amino acids: cysteine from methionine and tyrosine from phenylalanine. This means that if the essential precursor is not provided in adequate amounts then the non-essential amino acids becomes a dietary requirement. Equally, if the non-essential amino acid is provided in the diet then the requirement for the essential precursor is reduced. This is important with the two sulphur amino acids methionine and cysteine, since most diets are limited by their content of [methionine + cysteine].

Only three of the non-essential amino acids can be regarded as completely dispensable, since they are synthesised from common metabolic intermediates:

• alanine is synthesised from pyruvate
• aspartic acid is synthesised from oxaloacetate
• glutamic acid is synthesised from alpha-ketoglutarate

The capacity to synthesise other non-essential amino acids may be inadequate to meet requirements at times of high demand. For example:

• glutamine in response to surgical trauma and sepsis
• arginine at times of high protein intake or rapid growth
• glycine with high intakes of some xenobiotics which are excreted as glycine conjugates, and in rapid growth because of the requirement for collagen synthesis
• proline in severe trauma because of the requirement for collagen synthesis

Protein losses in response to trauma

In response to physical trauma of various kinds, and fever, there are considerable losses of tissue protein. The catabolic loss may be 6 - 7% of total body protein over 10 days. The table below shows these losses over 10 days in various conditions.

[From data reported by Cuthbertson, DP. Physical injury and its effects on protein metabolism, pp 373-414 in Human Protein Metabolism Vol 2 (Munro HN & Allison JB, Eds), Academic Press, New York, 1964]

By measurement of nitrogen balance, knowing their food intake and measuring urine and faeces loss of nitrogenous compounds

Old physiology textbooks said that this was mobilisation of tissue protein reserves for repair. This is obviously incorrect, since this is net catabolism of protein and loss of nitrogenous metabolites in the urine.

Two mechanisms explain this catabolic loss of protein:

1) Infection and trauma lead to increased secretion of the stress hormone cortisol.

Cortisol acts in the liver to increase gluconeogenesis in two ways:
a) induction of key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase
b) induction of two enzymes that initiate the catabolism of essential amino acids: tryptophan dioxygenase and tyrosine transaminase. This results in depletion of the liver, then the whole body. pool of these two amino acids, leaving a mixture of amino acids that cannot be utilised for protein synthesis, but will be catabolised.

2) Infection and trauma lead to increased synthesis of a variety of acute phase response proteins, many of which have a higher content of essential amino acids than the average pattern of tissue protein synthesis. Again this leads to depletion of the liver, then the whole body, pool of essential amino acids, leaving a mixture that cannot be utilised for protein synthesis., but will be catabolised.

Changes in protein turnover throughout the day

The table below shows the results of studies of protein synthesis and catabolism in healthy subjects in nitrogen balance, in experiments involving constant infusion of [15N]labelled amino acids.

[From data reported by Clugston GA and Garlick PJ (1982). The response of protein and energy metabolism to food intake in lean and obese man. Hum Nutr Clin Nutr 36C(1): 57-70]

Although an adult is in overall nitrogen balance day by day, this is the average of periods of positive nitrogen balance after a meal and negative balance in the fasting state. In the table above there is positive balance of +0.7 mmol /hour in the fed state but negative balance of -1.45 mmol /hour in the fasting state

In the fed state there is an ample supply of amino acids from dietary protein, and an ample supply of metabolic energy.

Protein synthesis. is energy expensive. There is a cost of 4 x ATP or GTP per amino acid incorporated in ribosomal protein synthesis., equivalent to 2.80 kJ /g of protein synthesised. Allowing for the cost of active transport of amino acids into the cell increases this to 3.59 kJ /g of protein synthesised. Allowing for the ATP cost of mRNA synthesis. increases this to 4.19 kJ /g or protein synthesised.

In the fasting state there is a need to conserve energy, since body reserves of glycogen and fatty acids are being used as metabolic fuels, so the rate of protein synthesis falls.

Tissue protein catabolism continues at almost the same rate as in the fed state, but instead of being used for replacement protein synthesis, the amino acids are used for gluconeogenesis and energy-yielding metabolism.

The table below shows the Km values of key enzymes involved in the catabolism of amino acids.

• The concentration of free amino acids in the liver in the fasting state is 30 - 50 µmol /L
• The amino acyl tRNA synthetases have values of Km of 1 - 50 µmol /L

[From data reported by Young VR &Marchini JS (1990). Mechanisms and nutritional significance of metabolic responses to altered intakes of protein and amino acids, with reference to nutritional adaptation in humans. Am J Clin Nutr 51(2): 270-289]

There is no storage of free amino acids in the body. Once the amino acyl tRNA synthetases are saturated with their substrates, amino acids will be substrates for the catabolic enzymes. This means that after a moderately protein-rich meal, although there is an increase in protein synthesis, there is also an increase in amino acid catabolism.

There is irreversible loss of amino acids as the concentrations rise above that at which the amino acyl tRNA synthetases are saturated.

In the fed state, amino acids in excess of immediate requirements for protein synthesis are catabolised.

In the fasting state, amino acids arising form tissue protein catabolism are metabolised as metabolic fuels and used for gluconeogenesis.

The table below shows protein turnover, measured in experiments using constant infusion of [15N]labelled amino acids, and energy expenditure, calculated from measurement of oxygen consumption, in the fasting state and after high-carbohydrate and high-protein meals.

[From data reported by Robinson SM et al. (1990). Protein turnover and thermogenesis in response to high-protein and high-carbohydrate feeding in men. Am J Clin Nutr 52(1): 72-80]

After a high-protein meal there is an increase in protein turnover. This is not an increase in net protein synthesis, since the rate of catabolism also increases. Remember that at any intake of protein above requirements you maintain nitrogen balance. A high protein diet does not lead to an increase in body protein content.

Both protein synthesis and catabolism are ATP-expensive processes. This accounts for much of the increase in energy expenditure.

(Most tissue proteins are catabolised in the proteasome, after they have been targeted by addition of multiple copies of the small peptide ubiquitin. The process of ubiquitination of proteins requires ATP, as does unfolding of native proteins in the proteasome).

A high-protein diet is also relatively low in carbohydrate, and amino acids will be used for gluconeogenesis. THis again is an ATP-expensive process (See the exercise Breathless after sprinting for the energy cost of gluconeogenesis).

Key points from this exercise:

• Under normal conditions in an adult, urinary excretion and faecal losses of nitrogenous compounds are equivalent to the dietary intake of nitrogenous compounds (mainly protein). This is the condition of nitrogen balance or equilibrium.
• The total flux of protein through the gut is considerably greater than the dietary intake. The average dietary intake is ~ 90 g per day, but some 200 g of endogenous protein enters the intestinal lumen from shed intestinal mucosal cells and secreted proteins.
• In growth or recovery from protein losses, the intake of nitrogenous compounds is greater than urinary and faecal losses. This is positive nitrogen balance - an increase in the total body content of protein.
• If the dietary intake of protein is inadequate to meet requirements, and in pathological conditions that involve tissue protein loss, the urinary and faecal losses of nitrogenous compounds is greater than the intake. This is negative nitrogen balance.
• Estimates of protein requirements are based on studies feeding varying levels of protein to determine the intake at which nitrogen balance can just be achieved.
• There is continual turnover of proteins in the body, with catabolism and replacement synthesis, even when there is no change in the total body protein content - this is dynamic equilibrium. Different proteins turn over at very different rates; key regulatory enzymes have a half life of only a few hours.
• The requirement is not just for protein, but for nine essential amino acids that cannot be synthesised in the body and must be provided in the diet.
• In response to trauma and infection there is a considerable loss of protein from the body. This is partly the result of induction of key enzymes of gluconeogenesis and amino acid catabolism in response to cortisol, and partly due to the synthesis of acute phase proteins that contain disproportionately large amounts of essential amino acids. The resultant pool of amino acids depleted in one or more of the essential amino acids cannot be used for protein synthesis but will be catabolised.
• Even in nitrogen balanced, there is cycling between positive balance after a meal and negative balance in the fasting state. In the fasting state the rate of protein synthesis falls, partly to conserve metabolic energy and partly because amino acids from protein catabolism are being used as substrates for gluconeogenesis. In the fed state the rate of protein synthesis increases, and this protein is replaced.
• In the fed stated, amino acids in excess of immediate requirements for protein synthesis are catabolised; there is no storage of free amino acids in the body.
• There is an increase on protein turnover, but not net protein synthesis, in response to a high protein meal. This leads to an increase in energy expenditure.
• High-protein diets are effective for weight loss partly because of the increased energy expenditure associated with increased protein turnover, and partly because of the energy cost of gluconeogenesis form amino acids.