Does the nitrogen balance cover the various components
of human protein needs?
By Daniel Tomé, AgroParisTech, Paris
(Translated and adapted from a Cholé-Doc nr. 121 September-October 2010, kindly provided by the French Dairy
Board (CNIEL)
Introduction ..................................................................................... 1
Nitrogen balance and safe levels of protein intake ................................. 1
Protein requirements and protein metabolism....................................... 2
Muscle… .......................................................................................... 3
Bone… ............................................................................................. 3
Sarcopenia and osteoporosis .............................................................. 4
Protein quality .................................................................................. 4
Conclusion ....................................................................................... 5
Introduction
Proteins are an indispensable component of food, ensuring intake of nitrogen and
the amino acids used for synthesis and maintenance of a wide number of
proteins in the body. They are also used as precursors of other non-protein
nitrogen molecules, including, hormones, neuro-peptides, the nucleic acids,
glutathione, or creatine. After deamination, the carbon skeleton of an amino acid
also follows the energy metabolism pathway, in particular through the
gluconeogenesis metabolic pathway where certain amino acids act as
predominant precursors. It can be seen that protein intake affects a great
number of body functions. However, it remains difficult to define which functions
are good markers for measuring protein needs.
Nitrogen balance and safe levels of protein intake
Despite certain limitations, the nitrogen balance – the difference between the dietary intake
of nitrogen (mainly protein) and its excretion via urine, hair, skin, or perspiration - remains the
main reference method for determining protein needs. Nitrogen is considered as a good
marker as 95 percent of bodily and dietary nitrogen can be linked to proteins. Healthy adults
excrete the same amount as is ingested when their protein need is fulfilled and so the
nitrogen balance is in equilibrium (zero). The balance becomes negative when protein intake
is inadequate (i.e. energy output exceeds energy intake). In practice, the amount of protein
required by a healthy adult with an energy balance has been defined as the minimum protein
intake required enabling achievement of a nitrogen balance (FAO/WHO/UNU, 2007). Based
on this, the average nitrogen requirement for an adult male is estimated at 105 milligrams
(mg) N/kg/d (i.e. 0.66 g/kg/d of protein using the conversion coefficient for nitrogen in protein
at N x 6.25) and the 97.5th percentile at 133 mg N/kg/d (i.e. 0.83 g/kg/d of protein).
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However, due to the lack of statistical force of available data on the nitrogen balance in
humans, it is not possible to establish differences in needs according to age, sex or diet
types (Rand et al, 2003).
Under these conditions, an intake of 0.83 g/kg/d of good quality protein is considered
sufficient to cover the needs of a healthy adult. This value is being progressively used as the
recommended intake value by various national and international bodies. According to the
current hypotheses on amino acids and protein metabolism, a tendency exists to consider
the nitrogen balance as providing a “safe” minimum intake value, i.e. ensuring nitrogen
homeostasis in the body. However, it has yet to be demonstrated that the nitrogen balance is
sufficiently comprehensive and omnipresent and that all of the physiological and metabolic
functions depending directly on protein intake are optimised concomitantly when the nitrogen
balance is zero. In order to address this issue, discoveries of additional sensitive markers for
protein intake are required. Different markers associated with protein intake can be
considered as complementary criteria for protein requirements and some of these markers
could indicate a higher requirement than that derived from the nitrogen balance. This
question can be illustrated through analyses of the relationships between protein intake and
protein renewal, muscle metabolism and bone metabolism.
Protein requirements and protein metabolism
Protein metabolism represents a specific and major pathway of amino acid metabolism. In a
man weighing 70 kilograms (kg), proteins account for 10-12 kg, of which approximately 40
percent are located in the skeletal muscle, 15 percent in the skin, 15 percent in the blood and
10 percent in the visceral zone. When the number of proteins found in the body is high
(25,000), four proteins (collagen, myosin, actin and haemoglobin) represent half of these and
collagen alone represents 25 percent. Proteins are in constant renewal through protein
synthesis and proteolysis. In an adult male weighing 70 kg, this happens at a rate of 250-300
g/d of proteins which exchange with free amino acids in the human body (about 100g). It
should be noted that daily body protein renewal represents 2-3 times the usual food protein
intake (80-100 g/d).
According to available data, the effect of an increase in protein intake on protein renewal is
complex and does not allow a conclusion to be reached regarding its use as a protein
requirement marker. An increase in protein intake above the recommended safe level alters
protein renewal and increases oxidation of amino acids. Compared to the protein renewal
observed at the safe level of protein intake, an increase in protein intake is associated with
stimulation of proteolysis during fasting, and with a strong inhibition when nourished,
whereas protein synthesis for the whole body is only slightly altered (Fouillet et al, 2008;
Harber et al, 2005; Morens et al, 2003; Forslund et al, 1998; Pacy et al, 1994; Price et al,
1994). However it should be noted that renewal is variable depending on the proteins under
consideration, and that intestinal proteins are renewed more rapidly than those found in
peripheral tissue in general (Masanes et al, 1999). Under these conditions, protein renewal
measurement for the entire body represents an average of different tissues in the body with
individual variations or even contrary measurements depending on the level of protein intake.
Therefore more specific body tissues should be evaluated to determine the impact of protein
intake.
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Muscle…
Muscle is the major protein containing tissue found in the body and could therefore be used
as a sensitive marker for protein intake. Muscle protein anabolism is stimulated by eating and
contractile activity. Protein ingestion is required for the synthesis of muscle protein, as well
as function and muscle mass maintenance. Protein synthesis and muscle protein mass are
therefore sensitive to protein deficiency. In contrast, protein consumption higher than the
recommended safe intake level does not seem to impact on muscle mass in a healthy
subject in the absence of exercise (Bolster et al, 2005; Chevalier et al, 2009; Taillandier et al,
2005; Almurshed et al, 2000). Branched-chain lateral amino acids (valine, isoleucine and
especially leucine) seem to play a vital role during in vitro protein synthesis. However, in vivo
results obtained from animals and human tests are contradictory and do not demonstrate
that an increase in leucine intake (i.e. above the basic requirement as defined according to
current criteria), further aids protein synthesis and muscle mass development. To date
according to current knowledge, no clear evidence exists to demonstrate that the ingestion of
regular leucine supplements further stimulates protein synthesis effectively when the intake
of protein and indispensable dietary amino acids is adequate (Sherwin, 1978; Tessari et al,
1985; Schwenk and Haymond, 1987; Nair et al, 1992a; Koopman et al, 2005; Balage and
Dardevet, 2010). However, the intake of amino acids and leucine remain a sensitive criterion
for muscle. The use of rapid absorption proteins such as whey proteins illustrated interesting
results allowing increase of post-prandial amino acid and leucine muscle contents.
Bone…
Proteins and calcium are the main components of bone structure. It has been demonstrated
that protein intake lower than the recommended safe level increases bone fragility and the
risk of fracture (Dawson-Hughes, 2003). A number of epidemiological studies also
demonstrate a positive correlation between bone mineral density and protein intake for
values higher than the safe level of protein intake also (Promislow et al, 2002, Devine et al,
2005). This result leads certain authors to recommend higher intake of protein than the
recommended safe level in order to optimise the relationship between ingestion and mineral
bone density. The relation between protein intake above the recommended level and the
risk of fracture remains less evident and under dispute (Frassetto et al, 2000). An increase
in protein intake is often associated with increased in urinary calcium excretion. This was
initially interpreted as bone absorption activation in order to provide calcium to neutralise acid
production from sulphur-containing amino acids catabolism. However, this supposition has
yet to be confirmed. It is believed that the increase in calciuria is due to increased calcium
absorption resulting in its increased excretion through urine (Kerstetter et al, 2003a,
Kerstetter et al, 2003b). The body’s acid-base balance is a complex phenomenon and is
highly localized and regulated. Urine acidity produced by the catabolism of sulphurcontaining amino acids is not directly correlated to plasma acidity. Moreover, mobilisation of
bone calcium is not a major process for acid regulation in the body which takes place mainly
through respiratory and renal pathways (Fenton et al, 2009a; Bonjour, Chevalley, 2009).
Under these conditions, the risk of bone resorption does not seem to be a major limiting
factor for protein intake (Pye et al, 2009; Fenton et al, 2009b).
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Sarcopenia and osteoporosis
The loss of both muscle mass (sarcopenia) and minerals in the bones (osteoporosis) are
degenerative processes frequently associated with ageing. It is suspected that these
phenomena are partly due to a decrease in the effectiveness of the metabolic response by
muscle and bone tissue to protein ingestion and that, under these conditions, the protein
requirement for protein synthesis could be higher for the elderly in comparison to young
adults. Data referring to protein intake levels required by the elderly are limited, with some
data indicating a negative nitrogen balance in elderly people with a protein intake of 0.8
g/kg/d. Reduced muscle surface is noted at this level of intake, without significant alteration
of the metabolism of leucine or total body composition. Several other studies also conclude
that there is a higher protein need in older subjects when compared to young adults. This is
linked to a reduction in the effectiveness of protein use by elderly (Wolfe et al, 2008; GaffneyStomberg et al, 2009; Thalacker-Mercer et al, 2010). However these results are not found
systematically. According to certain recent studies, improved body composition in older
subjects requires the linkage of adequate protein intake with physical exercise (Campbell et
al, 2008, Campbell and Leidy, 2007, Iglay et al, 2009).
Protein quality
These few illustrations highlight the main issue of markers and criteria related to better
definition of the relationship between the quantity and quality of protein intake. The
traditional approach evaluates protein quality on the basis of capacity at a safe level of
dietary ingestion to cover requirements of indispensable amino acids, a limiting factor for
protein synthesis. It follows that protein quality is derived from the indispensable amino acids
content. This criterion could potentially distinguish between different protein sources due to
the substantial differences in indispensable amino acid composition of different protein
sources. As a general rule, proteins from animal origins are richer in indispensable amino
acids than proteins derived from plants.
It has been demonstrated that tissue retention of amino acids (or net protein use) is higher
when the indispensable amino acids content of food proteins is high for equivalent protein
intake, including protein intake above the recommended safe level and an indispensable
amino acids intake above the basic requirement (Morens et al, 2003; Juillet et al, 2008;
Fouillet et al, 2009). Such results indicate that the concept of protein quality is still
meaningful for intake that is higher than the determined safe intake level. It also questions
the traditionally-accepted idea that surplus indispensable amino acids above the basic
dietary need no longer contribute to quality of the intake. Based on data regarding average
nitrogen balance, an average coefficient for net protein use at 50 percent in adults can be
derived (Rand et al, 2003). Considering the post-prandial phase - the sensitive phase of
food protein use - in greater detail, a net post-prandial protein use coefficient (NPPU) can be
measured. This approach highlights the variation between different sources of proteins with
values of 74 percent, 70 percent and 66 percent respectively for milk, soya and wheat
proteins (Bos et al, 2005; Fouillet et al, 2009).
The upper limit of protein intake tolerable is also an important issue. Intake analyses
demonstrate that for numerous populations of industrialized countries, intakes are
higher than the safe level of 0.83 g/kg/day, with an average intake of protein by
adults of 0.9 to 1.2 g/kg/day. Under these conditions, it is important to define an
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adequate margin between the safe level of protein intake and the upper limit of
intake. However, few pertinent markers have been identified. When considering the
ammonia produced by amino acid break down as a limiting factor, the capacity of the
urea cycle to eliminate the ammonia can be taken as a marker. Following this
approach and taking safety factors into account, a protein intake between 0.83
g/kg/day and 2.3 g/kg/day represents an adequate margin for adult intake. This is
nevertheless a theoretical recommendation that relies on functional markers that
have yet to be defined.
Conclusion
Today, available clinical observations do not enable several national and international bodies
and agencies to consider additional markers for use to correct the safe level of protein intake
derived from the nitrogen balance, despite an almost unanimous agreement regarding the
limits of this approach. Several questions remain unanswered:
1)
The concept of food protein quality at the recommended safe level of protein intake;
2)
The influence of the quantity and quality of food protein on metabolism and
physiology on several tissues and organs;
3)
The relation between protein metabolism and energy metabolism and the influence of
the quality of food protein on this relationship;
4)
The influence of physiological conditions and ageing on different processes related to
protein intake;
5)
The definition of criteria and markers to define the tolerable upper level of protein.
Daniel Tomé, AgroParisTech, Paris
Translation by CNIEL (Susan Owens) and
IDF (Sandra Tuijtelaars and Catherine Shiels)
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