Download Nitrogen Balance and Protein Requirements: Definition and

Chapter 3.2
Nitrogen Balance and Protein Requirements: Definition
and Measurements
Paolo Tessari
Introduction
Nit rogen is a main body component and is
required for both tissue protein synthesis and the
production of several nitrogenous compounds
involved in a variety of functions (hormones,
immune mediators, neurotransmitters, antioxidant defences, etc.). Thus, the body nitrogen content should be both quantitatively and qualitatively normal, as well as normally maintained, to
ensure normal body functions.
Nitrogen homeostasis is a highly regulated
function. Nitrogen balance is commonly referred
to as the net difference between the intake (and/or
the effective absorption) of nitrogen contained in
the diet and its excretion. Since nitrogen is contained predominantly in proteins, this term pertains mainly to the balance of proteins and of
amino acids [1].
Nitrogen excretion and/or loss can occur
through different routes. The principal component
is in the urine as urea, ammonia and creatinine
(Table 1). Faecal and miscellaneous losses represent an additional route, which may be fairly constant and lower as an absolute amount [1].
Measurements of nitrogen balance usually
require an adaptive period of the subject of at
least 4–5 days [2], to ensure that equilibration has
been achieved and that acute changes do not
occur within the time span of measurement.
Apart from intake, the rate of nitrogen excretion is also affected by renal function, the hydration state and the anabolic/catabolic state of the
subject [3].
With prolonged fasting, total urinary nitrogen
and urea nitrogen excretion diminish, whereas
ammonia excretion increases relatively [4]. Such a
shift is related to the excretion of acid equivalents,
which are produced in excess by ketogenesis during fasting.
Nitrogen excretion cannot be reduced below a
certain amount despite reduction to zero intake.
This amount is called the ‘obligatory nitrogen
losses’ (ONL), which represent the nitrogen loss
that is measurable in subjects fed a protein-free
diet for a relatively short period of time (Table 2).
These losses have been estimated to be 36
mg/kg/day in the urine, 12 mg/kg/day in faeces and
8 mg/kg/day as miscellaneous nitrogen losses
(sweat, sebum, desquamations, nails, hairs and
saliva) [5]. Given the equivalence of 6.25 grams of
protein per gram of N, ONL thus correspond on
the whole to a protein amount of 0.35 g/kg/day [6].
Rand and Young recently pointed out a series
of limitations in the estimation of nitrogen bal-
Table 1. Urinary nitrogen excretion (‘azoturia’)
Table 2. Obligatory nitrogen losses
As urea N: urea excretion (in grams) × 0.46
since: Urea MW = 60; N2 MW = 28; then: urea N = Urea
× [28/60] i.e. × [0.46]
Urine: 36 mg/kg/day N
Faeces: 12 mg/kg/day N
Urea usually accounts for 70–90% of urinary nitrogen
excretion
As non-urea N: 2 g/day (ammonia, uric acid, creatinine,
nitrates, amino acids, etc.)
Miscellaneous N losses (sweat, sebum, desquamations,
nails, hairs and saliva): 8 mg/kg/day
Total (as protein equivalents): 0.35 g/kg/day
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Paolo Tessari
ance [7]. They state that: ‘Nitrogen balance estimates are highly dependent on the assumed
amount of N miscellaneous losses... further studies on these losses and on the factors that influence them are essential.’ They raised the following
points: (a) there is a slight difference between
large values for N intake and N losses; (b) it is well
recognised that the nitrogen balance technique
overestimates N intake and underestimates N losses. This is mainly due to the difficulty in the
assessment of the N gas losses after denitrification by the colonic microflora, of the N losses
through the skin (urea) and in the expired air
(ammonia) and of the nitrate content in food and
urine, which is not measured using the Kjeldahl
method.
The irreversible loss of amino acid nitrogen
corresponds to net protein (i.e. amino acid) catabolism. This occurs because nitrogen is firstly and
reversibly lost through deamination/transamination of the amino acids. If this step is followed by
another step irreversibly catabolising the amino
acid carbon skeleton (i.e. oxidation, hydroxylation, etc.), the nitrogen cannot be re-utilised for
amino acid re-synthesis (despite the reversibility
of transamination reactions), thus it enters the
urea cycle and is either excreted as such, or
included into ammonia. Therefore, the net nitrogen loss should theoretically correspond to the
irreversible catabolism of the amino acids. This
assumption has indeed been proven in 24-hour
studies using leucine tracer and nitrogen balance
measurements [8, 9]. Therefore, nitrogen loss is an
integrated measurement of oxidation/catabolism
of all amino acids and thus of net protein loss.
Protein Requirements
Dietary requirements for protein, amino acid and
nitrogen depend on the metabolic demand that
must be satisfied. They are conditioned by both
the amount of proteins needed and their quality.
Protein quality in turn depends on the amount of
essential amino acids (EAA), but also of the nonessential (NEAA) ones [10, 11]. The link between
protein quality and EAA is obvious: since the EAA
cannot, by definition, be synthesised by the body,
they must be introduced with the diet in a proportion that will fit with the organism’s metabolic
needs. On the other hand, in the absence of
dietary NEAA, despite the theoretical capability of
the body to synthesise them, nitrogen will be
needed for their de novo synthesis. This nitrogen
in turn must be derived either from EAA catabolism (thus increasing their requirement above theoretical values) or from the diet. In this respect,
although NEAA can theoretically be replaced, they
are required in nutrition as well.
An evaluation of dietary protein quality must
therefore consider not only the quality of the protein itself, but also the various processes involved
in amino acid and nitrogen homeostasis, which
may vary as regards the individual amino acids
and the individual metabolic conditions of a subject.
Nitrogen balance can be used to derive estimates of human nitrogen (i.e. protein) requirements [1, 12]. The usual approach is based upon
the regression of nitrogen balance (i.e. the equilibrium between intake and loss) on intake. The subject is adapted for a few days to a diet of a given
protein (and energy) content, and nitrogen balance is measured at the end of adaptation. Diets
with varying amounts of proteins (and energy)
are tested. Requirement is then defined as the
intake level that would produce a zero (or a slightly positive) nitrogen balance.
An intake of 0.6 g/kg/day of well-balanced proteins is considered sufficient to achieve a zero (i.e.
at equilibrium) nitrogen balance [6] (Table 3). A
safety amount is considered to be 0.75 g/kg/day.
These values represent the minimum recommended protein intake, derived also from studies investigating the metabolic response to a range of protein intakes between 0.75 and 2 g/kg/day.
Amino acid requirement may increase in many
physiological conditions (Table 3). In children
[13], the requirement for growth must be integrated in addition to the requirement for maintenance. In the first 6 months of life, a suggested
intake is of ≈1.7 g/kg/day, with a further allowance
of +25% (+2 SD), leading therefore to a total of ≈2
g/kg/day. Beyond the sixth month of life, suggest-
3.2 Nitrogen Balance and Protein Requirements: Definition and Measurementss
Table 3. Daily protein requirements by age
Adult, weight stable, moderate activity: 0.75 g/kg
Children:
first 6 months: 2 g/kg
beyond sixth month: 1.6 g/kg
Between 7 and 14 years: 1 g/kg
Beyond 14 years: 0.75 g/kg
ed intake is 1.6 g/kg/day, resulting from a +50%
increase, beyond a suggested intake of 0.8
g/kg/day of the adult, due to individual variability
in growth, plus a +30% increase due to variability
in utilisation efficiency, +25% (= 2 SD). Between 7
and 14 years, the recommended intake is 1
g/kg/day, and beyond 14 years it is the same as for
an adult.
In pregnancy [14], the total nitrogen deposition over the entire period up to delivery is estimated to be ≈925 g. Average rates of nitrogen
retention are 0.11 g/kg/day in the first trimester,
0.52 g/kg/day in the second, and 0.92 g/kg/day in
the third. In practice, due to a 70% efficiency in
nit rogen ut ilisat ion, and the st ill par t ially
unknown effective nitrogen retention in the first
trimester, it is suggested to increase the dietary
protein intake by 10–12 g/day in each trimester.
During lactation, an extra protein intake of
15–20 g/day in the first 6 months, and of 12 g/day
in the subsequent months, is advisable [15].
In the elderly, the maintenance of nitrogen
equilibrium by a diet containing 0.8 g/kg/day and
a normal energy intake may be difficult, because
of a lower efficiency in nitrogen utilisation for
anabolic purposes [16].
A surplus of dietary proteins is also recommended for individuals who exercise regularly
[17]. Amino acids are oxidised as substrates during prolonged submaximal exercise. In addition,
both endurance and resistance training exercise
increase skeletal muscle protein synthesis and
breakdown in the post-exercise recovery period.
In studies using nitrogen balance, it has been confirmed that protein requirements for individuals
engaged in regular exercise are increased. Current
recommended intakes of proteins for strength and
endurance exercising athletes are 1.6–1.7 g/kg/day
and 1.2–1.4 g/kg/day, respectively. It is presently
estimated that most athletes consume adequate (if
not excessive!) amounts of proteins. Recent
research has also pointed out that the timing and
nutritional amount of a meal ingested after exercise have synergistic effects on net protein accumulation in body tissues after exercise. It has been
suggested that athletes who engage in strenuous
activity should consume a meal rich in amino
acids and carbohydrates soon after the exercise
bout or the training session.
Protein Requirement and Energy Intake
It has been proposed that protein requirement is,
within a certain limit, inversely dependent on
energy intake, i.e. the more energy is ingested, the
less protein is needed (Table 4). This is because
proteins can be used also as energ y sources
(beyond their structural, regulatory and functional role). Therefore, if their use to produce energy
varies, their requirement also varies. Furthermore,
alternative energy substrates, such as the carbohydrates, can stimulate insulin secretion, which in
turn spares endogenous proteins [18].
A relationship between protein requirement
and energy intake is reported in Table 4. The
reported amount should be increased by 2 SD for
safe allowances.
Table 4. Relationship between dietary protein requirement
(in grams of protein per kg of body weight), titrated to
the achievement of zero nitrogen balance, and energy
intake (in kJ per kg of body weight) in a weight-stable
healthy adult man [19, 20]
Protein
requirement
(g/kg)
Safe allowance
(+2 SD)
(g/kg)
Energy
intake
(kJ/kg)
0.78
1.02
9.57
0.56
0.74
10.77
0.51
0.62
11.48
0.42
0.50
13.64
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Paolo Tessari
The Fate of Dietary Protein Nitrogen During
the Postprandial Phase
The diurnal cycle of feeding and fasting is accompanied by concurrent changes in protein turnover.
Protein feeding is necessary to replenish the body
protein stores that would be wasted during fasting
[21–24]. Because of this, nitrogen retention calculated on a daily basis is lower than that derived
just from the postprandial phase [21], and, conversely, dietary protein utilisation calculated as
the daily gain is lower than the postprandial gain.
Dietary proteins, once ingested, are digested in
the gut and thereafter absorbed as either free
amino acids or dipeptides [25]. The absorbed
amino acids are subjected to a variable first-pass
extraction by splanchnic organs (mainly the liver)
[26–28] and then they travel as such through the
extracellular spaces before being used by the cells,
either for catabolism or for protein synthesis. A
minor fract ion of amino acids are excreted
unmodified into the urine [29].
The acute nitrogen deposition during the postprandial phase is likely to be the most critical in
terms of the net deposition of proteins in the tissues, more than the rate of protein synthesis
occur r ing in the postabsor pt ive per iods.
Therefore, the assessment of the postprandial utilisation of dietary proteins is a key step to understand net body protein deposition. It also represents an important conditioning factor of the rate
of whole-body protein turnover [30].
The key steps of the fate of dietary nitrogen
are: (1) the amount of nitrogen that is actually
absorbed; (2) the amount that is deaminated and
then recovered mainly in the form of urea; and (3)
the amount that is retained in the body.
As regards point (1), nitrogen digestibility
within the ileum and the short-term retention of
dietary protein nitrogen can be measured by the
use of 15N-labelled proteins. By this technique,
therefore, it is possible to assess the metabolic
utilisation of dietary nitrogen in humans, i.e. the
amount that is effectively absorbed [31–35].
As concerns point (2), assuming that wholebody protein turnover is ≈300 g, and that daily
protein intake is ≈100–110 g/day, it has been calculated that ≈80 g of the total proteins turned over
(i.e. ≈27% of total) are lost through the oxidative/urea-producing pathways, and ≈14 g within
the ileum [21, 22]. The amounts of dietary nitrogen entering the anabolic (i.e. protein synthesis)
and oxidative pathways are 70–80 and 13–20
g/day, respectively, i.e. contributing by 30–40% to
total anabolism and by 15–25% to total oxidation
(Fig. 1).
This indicates that dietary nitrogen (and proteins) is preferentially directed toward anabolic
pathways. Such a preferential orientation of
dietary nitrogen toward body protein synthesis is
strictly linked to the adequacy (i.e. quality) of the
dietary protein amino acid composition with
respect to that of body protein.
The maintenance of nitrogen homeostasis
involves a complex series of changes in wholebody protein turnover, amino acid oxidation, urea
production and nitrogen excretion, during the
fasting, fed, postprandial and postabsorptive periods of the day. Whole-body processes also represent the additive result of the metabolism of individual organs and tissues, which may be differently affected during physiological and pathological
conditions. Therefore, whole-body measurements
are crude, although comprehensive, estimates of
body protein metabolism, but rarely can they provide information on regional protein turnover.
The usual daily protein consumption is normally greater than the theoretical requirement
based on nitrogen balance estimates [36]. Since
body proteins cannot be stored in the body, mechanisms exist to dispose of the protein ingested in
excess. Thus, the effects of increased protein loads
on whole-body nitrogen balance and protein
75-80 g
OXIDATION
TOTAL
PROTEIN
TURNOVER
300 g
13
-
20
17
g
-2
5%
206-211 g
DIETARY
PROTEIN
INTAKE
100-110 g
70-8
0 g
3 4 -3 8
14 g
PROTEIN
SYNTHESIS
%
ILEAL LOSSES
Fig. 1. Proportions of nitrogen turnover and utilisation
3.2 Nitrogen Balance and Protein Requirements: Definition and Measurementss
turnover must be determined. These investigations should involve the study of nitrogen pools
likely to be modified by the level of nitrogen
intake, the effects linked to the type of protein
ingested, as well as the effects of the nitrogen
loads on the different nitrogen pathways [37].
An increase in protein intake is followed by
adaptive processes: (1) an increase in amino acid
oxidation and in the associated nitrogen excretion, mainly as urea, which is especially pronounced in the fed state; (2) a trend toward a disproportionate increase in nitrogen balance when
nitrogen intake is increased [38], possibly linked
to an enhanced inhibition of protein breakdown
by feeding and to an increase in protein synthesis
[39]. This likely occurs because whole-body as
well as tissue protein synthesis are sensitive to
amino acid availability, whereas degradation may
be sensitive to an interactive effect by both the
amino acid level and insulin [40]. Thus, high protein intakes are associated with a continuous, positive N balance approaching 1–3 g N/day [38, 39,
41, 42]. However, it is not clear whether this
apparent retention is a real one or linked to intrinsic errors in calculating N balance.
Interestingly, the amplitude of diurnal body
protein cycling increases with an increase in
dietary protein intake, with no clear change in the
mean daily protein turnover rate [43].
Nitrogen Metabolism and Dietary Protein
Characteristics
Nitrogen balance data measured after adaptation
to different protein levels over periods of several
days is the usual approach to measure nitrogen
retention [2, 44]. Diets containing poor quality
proteins are associated with an increase in nitrogen losses, due to the inefficient utilisation of
indispensable amino acids in turn linked to unbalanced amino acid composition. The (relative) lack
of essential amino acids generates the ineffective
utilisation of dietary nitrogen. Furthermore,
besides such an insufficient utilisation, it is
important to assess the amount of dietary and
intestinal nitrogen that is absorbed as free amino
acids or dipeptides, or excreted in the faeces, urine
or other routes. Finally, the assessment of the anabolic utilisation for protein synthesis is a key step
to measure amino acid retention in the body.
As stated above, classic nitrogen balance studies reflect the integrated net result of the diurnal
cycling between the fasted and fed states (i.e.
phases of nitrogen accretion postprandially and
of nitrogen losses postabsorptively).
Other factors may affect nitrogen retention.
Differences in the gastric emptying rate of dietary
proteins may result in highly variable rates of
amino acid absorption in the small intestine [45].
Also, differences in the rate of protein digestion
and/or absorption result in relevant differences in
amino acid oxidation and postprandial nitrogen
accretion [46]. In this regard, the concept of net
postprandial protein utilisation (NPPU) has been
proposed, which is calculated using true ileal
digestibility and true 15N-labelled protein deamination parameters, adding the dietary nitrogen
collected in the urine [22, 47] and that retained in
the body in the form of urea.
Using this approach, the NPPU values for milk
protein and soy protein, measured over 8 h after
the ingestion of a standard meal by healthy human
subjects, were reported between 80 and 72%,
respectively [47]. These data strongly suggest the
existence of differences between the nutritional
value of proteins and their utilisation for anabolic
purposes. These differences are valuable and
should be taken into account when calculating
amino acid scores. Finally, differences in interorgan amino acid metabolism may be due to the
protein source-dependent difference, as shown in
pigs after the administration of either soy or
casein [48].
77
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Paolo Tessari
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