Nitrogen Balance and Protein Requirements: Definition and Measurements
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Chapter 3.2
Nitrogen Balance and Protein Requirements: Definition
and Measurements
Paolo Tessari
least 4–5 days [2], to ensure that equilibration has
Introduction been achieved and that acute changes do not
Nit rogen is a main body component and is occur within the time span of measurement.
required for both tissue protein synthesis and the Apart from intake, the rate of nitrogen excre-
production of several nitrogenous compounds tion is also affected by renal function, the hydra-
involved in a variety of functions (hormones, tion state and the anabolic/catabolic state of the
immune mediators, neurotransmitters, antioxi- subject [3].
dant defences, etc.). Thus, the body nitrogen con- With prolonged fasting, total urinary nitrogen
tent should be both quantitatively and qualitative- and urea nitrogen excretion diminish, whereas
ly normal, as well as normally maintained, to ammonia excretion increases relatively [4]. Such a
ensure normal body functions. shift is related to the excretion of acid equivalents,
Nitrogen homeostasis is a highly regulated which are produced in excess by ketogenesis dur-
function. Nitrogen balance is commonly referred ing fasting.
to as the net difference between the intake (and/or Nitrogen excretion cannot be reduced below a
the effective absorption) of nitrogen contained in certain amount despite reduction to zero intake.
the diet and its excretion. Since nitrogen is con- This amount is called the ‘obligatory nitrogen
tained predominantly in proteins, this term per- losses’ (ONL), which represent the nitrogen loss
tains mainly to the balance of proteins and of that is measurable in subjects fed a protein-free
amino acids [1]. diet for a relatively short period of time (Table 2).
Nitrogen excretion and/or loss can occur These losses have been estimated to be 36
through different routes. The principal component mg/kg/day in the urine, 12 mg/kg/day in faeces and
is in the urine as urea, ammonia and creatinine 8 mg/kg/day as miscellaneous nitrogen losses
(Table 1). Faecal and miscellaneous losses repre- (sweat, sebum, desquamations, nails, hairs and
sent an additional route, which may be fairly con- saliva) [5]. Given the equivalence of 6.25 grams of
stant and lower as an absolute amount [1]. protein per gram of N, ONL thus correspond on
Measurements of nitrogen balance usually the whole to a protein amount of 0.35 g/kg/day [6].
require an adaptive period of the subject of at 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 Urine: 36 mg/kg/day N
× [28/60] i.e. × [0.46]
Faeces: 12 mg/kg/day N
Urea usually accounts for 70–90% of urinary nitrogen
excretion Miscellaneous N losses (sweat, sebum, desquamations,
nails, hairs and saliva): 8 mg/kg/day
As non-urea N: 2 g/day (ammonia, uric acid, creatinine,
nitrates, amino acids, etc.) Total (as protein equivalents): 0.35 g/kg/day74 Paolo Tessari
ance [7]. They state that: ‘Nitrogen balance esti- cannot, by definition, be synthesised by the body,
mates are highly dependent on the assumed they must be introduced with the diet in a propor-
amount of N miscellaneous losses... further stud- tion that will fit with the organism’s metabolic
ies on these losses and on the factors that influ- needs. On the other hand, in the absence of
ence them are essential.’ They raised the following dietary NEAA, despite the theoretical capability of
points: (a) there is a slight difference between the body to synthesise them, nitrogen will be
large values for N intake and N losses; (b) it is well needed for their de novo synthesis. This nitrogen
recognised that the nitrogen balance technique in turn must be derived either from EAA catabo-
overestimates N intake and underestimates N loss- lism (thus increasing their requirement above the-
es. This is mainly due to the difficulty in the oretical values) or from the diet. In this respect,
assessment of the N gas losses after denitrifica- although NEAA can theoretically be replaced, they
tion by the colonic microflora, of the N losses are required in nutrition as well.
through the skin (urea) and in the expired air An evaluation of dietary protein quality must
(ammonia) and of the nitrate content in food and therefore consider not only the quality of the pro-
urine, which is not measured using the Kjeldahl tein itself, but also the various processes involved
method. in amino acid and nitrogen homeostasis, which
The irreversible loss of amino acid nitrogen may vary as regards the individual amino acids
corresponds to net protein (i.e. amino acid) catab- and the individual metabolic conditions of a sub-
olism. This occurs because nitrogen is firstly and ject.
reversibly lost through deamination/transamina- Nitrogen balance can be used to derive esti-
tion of the amino acids. If this step is followed by mates of human nitrogen (i.e. protein) require-
another step irreversibly catabolising the amino ments [1, 12]. The usual approach is based upon
acid carbon skeleton (i.e. oxidation, hydroxyla- the regression of nitrogen balance (i.e. the equilib-
tion, etc.), the nitrogen cannot be re-utilised for rium between intake and loss) on intake. The sub-
amino acid re-synthesis (despite the reversibility ject is adapted for a few days to a diet of a given
of transamination reactions), thus it enters the protein (and energy) content, and nitrogen bal-
urea cycle and is either excreted as such, or ance is measured at the end of adaptation. Diets
included into ammonia. Therefore, the net nitro- with varying amounts of proteins (and energy)
gen loss should theoretically correspond to the are tested. Requirement is then defined as the
irreversible catabolism of the amino acids. This intake level that would produce a zero (or a slight-
assumption has indeed been proven in 24-hour ly positive) nitrogen balance.
studies using leucine tracer and nitrogen balance An intake of 0.6 g/kg/day of well-balanced pro-
measurements [8, 9]. Therefore, nitrogen loss is an teins is considered sufficient to achieve a zero (i.e.
integrated measurement of oxidation/catabolism at equilibrium) nitrogen balance [6] (Table 3). A
of all amino acids and thus of net protein loss. safety amount is considered to be 0.75 g/kg/day.
These values represent the minimum recommend-
ed protein intake, derived also from studies inves-
Protein Requirements tigating the metabolic response to a range of pro-
tein intakes between 0.75 and 2 g/kg/day.
Dietary requirements for protein, amino acid and Amino acid requirement may increase in many
nitrogen depend on the metabolic demand that physiological conditions (Table 3). In children
must be satisfied. They are conditioned by both [13], the requirement for growth must be integrat-
the amount of proteins needed and their quality. ed in addition to the requirement for mainte-
Protein quality in turn depends on the amount of nance. In the first 6 months of life, a suggested
essential amino acids (EAA), but also of the non- intake is of ≈1.7 g/kg/day, with a further allowance
essential (NEAA) ones [10, 11]. The link between of +25% (+2 SD), leading therefore to a total of ≈2
protein quality and EAA is obvious: since the EAA g/kg/day. Beyond the sixth month of life, suggest-3.2 Nitrogen Balance and Protein Requirements: Definition and Measurementss 75
Table 3. Daily protein requirements by age recommended intakes of proteins for strength and
endurance exercising athletes are 1.6–1.7 g/kg/day
Adult, weight stable, moderate activity: 0.75 g/kg
and 1.2–1.4 g/kg/day, respectively. It is presently
Children: first 6 months: 2 g/kg estimated that most athletes consume adequate (if
beyond sixth month: 1.6 g/kg not excessive!) amounts of proteins. Recent
research has also pointed out that the timing and
Between 7 and 14 years: 1 g/kg
nutritional amount of a meal ingested after exer-
Beyond 14 years: 0.75 g/kg cise have synergistic effects on net protein accu-
mulation 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
ed intake is 1.6 g/kg/day, resulting from a +50%
bout or the training session.
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 Protein Requirement and Energy Intake
and 14 years, the recommended intake is 1
It has been proposed that protein requirement is,
g/kg/day, and beyond 14 years it is the same as for
within a certain limit, inversely dependent on
an adult.
energy intake, i.e. the more energy is ingested, the
In pregnancy [14], the total nitrogen deposi-
less protein is needed (Table 4). This is because
tion over the entire period up to delivery is esti- proteins can be used also as energ y sources
mated to be ≈925 g. Average rates of nitrogen (beyond their structural, regulatory and function-
retention are 0.11 g/kg/day in the first trimester, al role). Therefore, if their use to produce energy
0.52 g/kg/day in the second, and 0.92 g/kg/day in varies, their requirement also varies. Furthermore,
the third. In practice, due to a 70% efficiency in alternative energy substrates, such as the carbohy-
nit rogen ut ilisat ion, and the st ill par t ially drates, can stimulate insulin secretion, which in
unknown effective nitrogen retention in the first turn spares endogenous proteins [18].
trimester, it is suggested to increase the dietary A relationship between protein requirement
protein intake by 10–12 g/day in each trimester. and energy intake is reported in Table 4. The
During lactation, an extra protein intake of reported amount should be increased by 2 SD for
15–20 g/day in the first 6 months, and of 12 g/day safe allowances.
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 Table 4. Relationship between dietary protein requirement
of a lower efficiency in nitrogen utilisation for (in grams of protein per kg of body weight), titrated to
the achievement of zero nitrogen balance, and energy
anabolic purposes [16]. intake (in kJ per kg of body weight) in a weight-stable
A surplus of dietary proteins is also recom- healthy adult man [19, 20]
mended for individuals who exercise regularly
[17]. Amino acids are oxidised as substrates dur- Protein Safe allowance Energy
requirement (+2 SD) intake
ing prolonged submaximal exercise. In addition,
(g/kg) (g/kg) (kJ/kg)
both endurance and resistance training exercise
increase skeletal muscle protein synthesis and 0.78 1.02 9.57
breakdown in the post-exercise recovery period. 0.56 0.74 10.77
In studies using nitrogen balance, it has been con- 0.51 0.62 11.48
firmed that protein requirements for individuals 0.42 0.50 13.64
engaged in regular exercise are increased. Current76 Paolo Tessari
The Fate of Dietary Protein Nitrogen During (i.e. ≈27% of total) are lost through the oxida-
the Postprandial Phase tive/urea-producing pathways, and ≈14 g within
the ileum [21, 22]. The amounts of dietary nitro-
The diurnal cycle of feeding and fasting is accom- gen entering the anabolic (i.e. protein synthesis)
panied by concurrent changes in protein turnover. and oxidative pathways are 70–80 and 13–20
Protein feeding is necessary to replenish the body g/day, respectively, i.e. contributing by 30–40% to
protein stores that would be wasted during fasting total anabolism and by 15–25% to total oxidation
[21–24]. Because of this, nitrogen retention calcu- (Fig. 1).
lated on a daily basis is lower than that derived This indicates that dietary nitrogen (and pro-
just from the postprandial phase [21], and, con- teins) is preferentially directed toward anabolic
versely, dietary protein utilisation calculated as pathways. Such a preferential orientation of
the daily gain is lower than the postprandial gain. dietary nitrogen toward body protein synthesis is
Dietary proteins, once ingested, are digested in strictly linked to the adequacy (i.e. quality) of the
the gut and thereafter absorbed as either free dietary protein amino acid composition with
amino acids or dipeptides [25]. The absorbed respect to that of body protein.
amino acids are subjected to a variable first-pass The maintenance of nitrogen homeostasis
extraction by splanchnic organs (mainly the liver) involves a complex series of changes in whole-
[26–28] and then they travel as such through the body protein turnover, amino acid oxidation, urea
extracellular spaces before being used by the cells, production and nitrogen excretion, during the
either for catabolism or for protein synthesis. A fasting, fed, postprandial and postabsorptive peri-
minor fract ion of amino acids are excreted ods of the day. Whole-body processes also repre-
unmodified into the urine [29]. sent the additive result of the metabolism of indi-
The acute nitrogen deposition during the post- vidual organs and tissues, which may be different-
prandial phase is likely to be the most critical in ly affected during physiological and pathological
terms of the net deposition of proteins in the tis- conditions. Therefore, whole-body measurements
sues, more than the rate of protein synthesis are crude, although comprehensive, estimates of
occur r ing in the postabsor pt ive per iods. body protein metabolism, but rarely can they pro-
Therefore, the assessment of the postprandial util- vide information on regional protein turnover.
isation of dietary proteins is a key step to under- The usual daily protein consumption is nor-
stand net body protein deposition. It also repre- mally greater than the theoretical requirement
sents an important conditioning factor of the rate based on nitrogen balance estimates [36]. Since
of whole-body protein turnover [30]. body proteins cannot be stored in the body, mech-
The key steps of the fate of dietary nitrogen anisms exist to dispose of the protein ingested in
are: (1) the amount of nitrogen that is actually excess. Thus, the effects of increased protein loads
absorbed; (2) the amount that is deaminated and on whole-body nitrogen balance and protein
then recovered mainly in the form of urea; and (3)
the amount that is retained in the body.
75-80 g
As regards point (1), nitrogen digestibility OXIDATION
TOTAL
within the ileum and the short-term retention of PROTEIN g
- 20
dietary protein nitrogen can be measured by the TURNOVER 13 5%
-2
300 g 17
use of 15N-labelled proteins. By this technique,
206-211 g
therefore, it is possible to assess the metabolic PROTEIN
SYNTHESIS
DIETARY 0 g
utilisation of dietary nitrogen in humans, i.e. the PROTEIN 70-8
INTAKE %
amount that is effectively absorbed [31–35]. 3 4 -3 8
100-110 g
As concerns point (2), assuming that whole-
14 g
body protein turnover is ≈300 g, and that daily ILEAL LOSSES
protein intake is ≈100–110 g/day, it has been cal-
culated that ≈80 g of the total proteins turned over Fig. 1. Proportions of nitrogen turnover and utilisation3.2 Nitrogen Balance and Protein Requirements: Definition and Measurementss 77
turnover must be determined. These investiga- of essential amino acids generates the ineffective
tions should involve the study of nitrogen pools utilisation of dietary nitrogen. Furthermore,
likely to be modified by the level of nitrogen besides such an insufficient utilisation, it is
intake, the effects linked to the type of protein important to assess the amount of dietary and
ingested, as well as the effects of the nitrogen intestinal nitrogen that is absorbed as free amino
loads on the different nitrogen pathways [37]. acids or dipeptides, or excreted in the faeces, urine
An increase in protein intake is followed by or other routes. Finally, the assessment of the ana-
adaptive processes: (1) an increase in amino acid bolic utilisation for protein synthesis is a key step
oxidation and in the associated nitrogen excre- to measure amino acid retention in the body.
tion, mainly as urea, which is especially pro- As stated above, classic nitrogen balance stud-
nounced in the fed state; (2) a trend toward a dis- ies reflect the integrated net result of the diurnal
proportionate increase in nitrogen balance when cycling between the fasted and fed states (i.e.
nitrogen intake is increased [38], possibly linked phases of nitrogen accretion postprandially and
to an enhanced inhibition of protein breakdown of nitrogen losses postabsorptively).
by feeding and to an increase in protein synthesis Other factors may affect nitrogen retention.
[39]. This likely occurs because whole-body as Differences in the gastric emptying rate of dietary
well as tissue protein synthesis are sensitive to proteins may result in highly variable rates of
amino acid availability, whereas degradation may amino acid absorption in the small intestine [45].
be sensitive to an interactive effect by both the Also, differences in the rate of protein digestion
amino acid level and insulin [40]. Thus, high pro- and/or absorption result in relevant differences in
tein intakes are associated with a continuous, pos- amino acid oxidation and postprandial nitrogen
itive N balance approaching 1–3 g N/day [38, 39, accretion [46]. In this regard, the concept of net
41, 42]. However, it is not clear whether this postprandial protein utilisation (NPPU) has been
apparent retention is a real one or linked to intrin- proposed, which is calculated using true ileal
sic errors in calculating N balance. digestibility and true 15N-labelled protein deami-
Interestingly, the amplitude of diurnal body nation parameters, adding the dietary nitrogen
protein cycling increases with an increase in collected in the urine [22, 47] and that retained in
dietary protein intake, with no clear change in the the body in the form of urea.
mean daily protein turnover rate [43]. 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%,
Nitrogen Metabolism and Dietary Protein
respectively [47]. These data strongly suggest the
Characteristics existence of differences between the nutritional
Nitrogen balance data measured after adaptation value of proteins and their utilisation for anabolic
to different protein levels over periods of several purposes. These differences are valuable and
days is the usual approach to measure nitrogen should be taken into account when calculating
retention [2, 44]. Diets containing poor quality amino acid scores. Finally, differences in interor-
proteins are associated with an increase in nitro- gan amino acid metabolism may be due to the
gen losses, due to the inefficient utilisation of protein source-dependent difference, as shown in
indispensable amino acids in turn linked to unbal- pigs after the administration of either soy or
anced amino acid composition. The (relative) lack casein [48].78 Paolo Tessari
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