Dogmas and controversies in the handling of nitrogenous wastes: The effect of feeding and fasting on the excretion of ammonia, urea and other nitrogenous waste products in rainbow trout
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4K1
* Author for correspondence (e-mail: kajimur{at}mcmaster.ca)
Accepted 23 January 2004
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Summary |
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Key words: amino acid, protein, nitrogen excretion, fish, nitrogen quotient
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Introduction |
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The implication of these findings is that a considerable amount of excreted nitrogen is likely to be found in waste products other than ammonia and urea. While the importance of alternative nitrogen products such as creatine, creatinine, trimethylamine (TMA), trimethylamine oxide (TMAO), uric acid, amino acids and protein has been investigated sporadically, there has been no thorough, systematic examination of these compounds in a single set of experiments. The primary goal of the current research was to determine the nature and relative importance of these minor nitrogen end products in both fed and fasting rainbow trout.
The breakdown of protein, as with all fuels, is associated with an increase
in metabolic rate as oxygen is consumed. Therefore, the ingestion of food is
followed by an increase in metabolic rate in most animals
(Kleiber, 1961;
Garrow, 1974
). According to
standard metabolic theory (Kleiber,
1992
), the excretion of waste nitrogen
(
N) can be related to the
consumption of oxygen
(
O2) to provide
a quantitative measure of protein utilisation, the nitrogen quotient
(NQ=
N/
O2).
From knowledge of the typical composition and metabolism of fish protein, an
NQ of 0.27 represents the condition in which aerobic respiration is fueled
entirely by protein (van den Thillart and
Kesbeke, 1978
), so under experimental conditions, the percent of
metabolism fueled by protein oxidation can be calculated by the ratio of the
measured NQ to 0.27. Traditionally, NQ has been calculated from the sum of
ammonia-N + urea-N excretion. On this basis, protein has been estimated to
contribute 1436% in fasted salmonids
(Brett and Zala, 1975
;
Wiggs et al., 1989
; Lauff and
Wood,
1996a
,b
;
Alsop and Wood, 1997
;
Kieffer et al., 1998
), with
similar values usually determined in other species (1634%;
Kutty and Peer Mohamed, 1975
;
Jobling, 1980
;
Alsop et al., 1999
). Values may
increase greatly, sometimes close to 100%, in actively feeding fish (reviewed
by Wood, 2001
).
This calculation assumes that all the nitrogen excretion products result from the oxidation of protein. This is a valid conclusion when examining the production of ammonia and urea, both of which are oxidized waste products. However, as stated above, it is likely that a significant proportion of nitrogen excretion is achieved by alternative N-forms, some of which may not be oxidized. Considerable excretion of amino acids and protein, for example, could greatly influence the nitrogen output of the fish, without having contributed to oxygen consumption. Alternatively, there may be significant excretion of other oxidized N-products that are missed by the measurement of only ammonia-N + urea-N. The second focus of the present research was to critically re-examine measures of protein utilisation, using the knowledge gained regarding the nature and concentration of nitrogen excretory products determined under the primary research aim.
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Materials and methods |
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Experimental protocols
Series 1 quantification of the components of nitrogen excretion
After the initial holding period, juvenile rainbow trout (1949 g)
were anesthetised by immersion in neutralised tricaine methanesulphonate
(MS-222; 100 mg l1), weighed and then transferred
individually to aerated containers (22 cmx22 cm) filled with 2 liters of
dechlorinated Hamilton tap water (Day 0) thermostatted to the acclimation
temperature. This holding water was changed every other day. Individual fish
were randomly allocated into one of four groups: sutured fasting
(N=16), sutured feeding (N=16), unsutured fasting
(N=12) and unsutured feeding (N=12). Feeding groups were fed
2% of their body mass every other day, while fasting fish were not fed at all.
On Day 9, fish designated to the sutured groups were anesthetised by
neutralised MS-222 and the anus was sutured closed with a needle and silk
thread (Ethicon, Somerville, NJ, USA). On day 10, fish in both sutured and
unsutured feeding groups were fed 2% of their body mass as usual. The sutured
fish ate normally, and virtually all food was consumed by both groups. After 4
h, both groups of sutured fish were placed individually in clean containers
with 1.5 liters of dechlorinated Hamilton tap water for 24 h, and samples for
nitrogen analysis were collected. Unsutured groups were transferred to
identical clean conditions at the same time, and again water samples were
taken for nitrogen analysis. For both unsutured groups, water samples were
filtered through cheese cloth to remove any feces. Water samples were taken at
the beginning and end of the 24-h period and frozen at 20°C for
later analysis of nitrogen waste products.
Series 2 respirometry
After the initial holding period, juvenile rainbow trout (1843 g)
were divided into three tanks (211 liters each, N=104 per tank) and
fed a ration of 1%, 3% or 5% body mass day1 for 7 days. A
further tank contained fish that were initially fed on the 3% ration but
thereafter starved for 13 days. On day 8 for fed fish and day 14 for the
fasting group, a subset of fish were placed in Blazka-style swimming
respirometers (3 liter) under flow-through conditions. Thus, the fed fish had
been starved since the previous evening, 12 h). The water velocity was
set to 10 cm s1, a speed at which the fish would maintain
orientation but not swim actively. Fish were allowed to settle for one hour,
then water flow was shut off while the current was maintained, and initial
water samples were taken. The oxygen partial pressure
(PO2) of the water in the respirometer was
measured sequentially until saturation had dropped to
70%, using a
temperature-controlled Cameron E101 oxygen electrode connected to a Cameron
OM-200 O2 meter. The appropriate solubility coefficients from
Boutilier et al. (1984
) were
used to convert PO2 to O2
concentration. Thereafter, aeration was resumed but the system remained closed
for 3 h, when final samples were taken. Fish were then removed and weighed.
The water samples taken at the beginning and end of the 3-h period were frozen
at 20°C for later analysis of nitrogen waste products (total
nitrogen, ammonia and urea).
Analysis of different nitrogen products
Total nitrogen (total-N) was analysed on an Antek 7000V nitrogen analyser
using ammonium sulfate as a standard. The efficiency with which the Antek
7000V nitrogen analyser detected N in various compounds was assessed using 50
µmol l1 ammonia-N [25 µmol l1
(NH4)2SO4] as a reference standard, with the
unknowns made up in the same concentration range. Similarly, the degrees of
detection of the same compounds by the ninhydrin reagent used for total amino
acid analysis and the salicylate/hypochlorite assay used for ammonia analysis
were also evaluated.
Protein concentration in water samples was measured by the dye-binding
method of Bradford (1976) using
Sigma reagent and bovine serum albumin (Sigma) as standards. Special attention
was devoted to protein because it was incompletely detected by the total
nitrogen analyser (see Results; Table
1) and also because it may precipitate out of solution. To solve
the analytical problem, all protein was removed from water samples prior to
total-N analysis. This was achieved by acidification (12 µl of 5 mol
l1 HCl, to 3 ml water sample) followed by ultrafiltration at
5000 g at 4°C, using MicrosepTM Centrifugal devices
with a 1000 Da molecular mass cutoff, and finally neutralisation of the
filtrate with 5 mol l1 KOH prior to total-N analysis. Tests
demonstrated that protein removal was >95%. The N concentration determined
on the total nitrogen analyser was then added to protein-N measured by the
Bradford assay to yield total-N.
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To deal with the precipitation problem, several preliminary tests were performed. We found that protein was often stratified in the water within the experimental containers. Therefore, in the standard measurement protocol, all the water was collected and thoroughly stirred in a large beaker prior to sub-sampling and analysis. We were also concerned that some excreted proteins may adhere to the wall of the experimental container and therefore not be detected in the water samples. Containers were therefore rinsed with 1 mol l1 KOH (20 ml) and then wiped with KOH-soaked filter paper to remove container-bound protein. This fraction was analysed separately for protein.
The ammonia content of the water was measured by the
salicylate/hypochlorite method (Verdouw et
al., 1978) using (NH4)2SO4
standards, and the amino acid content of the water was measured by ninhydrin
assay (Moore, 1968
), using
Sigma reagent and glycine as a standard. However, it was found that these
methods partially detect both ammonia and amino acids (see Results;
Table 1). To determine actual
ammonia and amino acid levels, the following treatment was performed. KOH (5
mol l1, 25 µl) was added to a 5-ml water sample. This
basic water sample (pH=12.3) was then aerated for 4 h to remove volatilised
ammonia, to give an ammonia-free water sample. This sample was then
neutralised by HCl and analysed for amino acid-N by the ninhydrin method.
Ammonia-N content was calculated as the difference between untreated and
ammonia-free samples by the salicylate-hypochlorite method.
Urea-N levels were measured with the diacetyl-monoxime method
(Rahmatullah and Boyde, 1980).
The sum of nitrite and nitrate levels was measured with the hydrazine sulfate
method (Rand, 1975
).
Creatinine, creatine, TMA, TMAO and uric acid levels in water samples were
very low. To increase the resolution of these nitrogenous products, water
samples (generally 50 ml) were concentrated using a rotary evaporator and then
reconstituted in a small volume of distilled water (1.2 ml). As samples are
concentrated by evaporation, the remaining solution becomes basic and may
promote precipitation of nitrogenous waste products. Prior to evaporation, 1
mol l1 HCl (100:l) was added to maintain an acidic solution
and promote N solubilisation. Creatinine levels were measured using a Sigma
Diagnostics kit (No. 555). Creatine was converted to creatinine by a
modification of the method of Smith
(1929). Creatine content was
calculated as the difference between samples of newly converted creatinine and
creatinine levels in unmodified samples. TMA and TMAO levels were measured by
the ferrous sulfate and EDTA method
(Wekell and Barnett, 1991
).
Uric acid was measured using the appropriate Sigma Diagnostics kit (No.
686).
Calculations
The concentration of the various nitrogen end products was converted to
molar concentrations of N in the traditional manner. For example, urea
excretion was multiplied by 2 to yield urea-N, creatinine and creatine were
multiplied by 3 to yield creatinine-N and creatine-N, respectively. Based on
recovery tests, a small correction factor was applied to the ninhydrin
measurement of total amino acid-N, as explained in the Results. Protein
standard in Sigma bovine serum albumin contains 0.1550.165 g N
g1. For the purposes of this study, protein-N content was
regarded as 0.16 g N g1 (i.e. 731 moles N per mole
BSA). Total-N was the value determined by the nitrogen analyser on
deproteinised samples with the addition of separately measured protein-N, for
the reason described above. Unknown nitrogen was total-N minus the sum of all
forms of measured N.
For the respirometry experiments of series 2,
O2 was
calculated in the traditional manner from the change in molar concentration of
oxygen in the water, factored by individual fish mass, time and respirometer
volume. Nitrogen quotient (NQ) was calculated as the ratio of the nitrogen
excretion rate (
N) to
O2. This was
calculated using either total-N excretion rate, or ammonia-N + urea-N
excretion rate, or the excretion rate of the (estimated) sum of all oxidized
nitrogen compounds (all N-compounds, including unknown-N but specifically
excluding protein-N and amino acid-N) as the nitrogen products of interest.
The percentage contribution of protein as an aerobic fuel source was then
calculated using the following formula
(van den Thillart and Kesbeke,
1978
):
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Statistics
All values are expressed as means ±
S.E.M. In series 1, an independent
t-test was used to determine significance in nitrogen excretion
between fasting and feeding and between sutured and unsutured groups. A
one-way analysis of variance (ANOVA) followed by the LSD test was used to test
significant differences in oxygen consumption and nitrogen excretion between
groups of fish in series 2. For all analyses, P<0.05 was
considered significant.
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Results |
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The salicylate/hypochlorite assay
(Verdouw et al., 1978) used
for ammonia-N analysis proved to be relatively specific, the only
cross-reaction of any note being with glycine-N (20% recovery;
Table 1). Nevertheless, to
ensure the greatest accuracy, ammonia-N was measured as the difference between
untreated and ammonia-purged samples, using the Verdouw et al.
(1978
) assay. The ninhydrin
assay used for amino acid analysis, on the other hand, proved to be relatively
nonspecific, detecting ammonia-N with 82% efficiency
(Table 1). Therefore, this
assay was run only on water samples that had been purged of ammonia. In
addition, the ninhydrin reaction yielded widely varying recoveries for the
N-content in different amino acids (Table
1). In general, the assay appeared to detect only the
-amino group, in accord with theory, and therefore underestimated the
N-content for amino acids that contain 24 N per molecule. Furthermore,
the efficiency of detection of N in ß-amino acids was only
50%. To
correct for this, the assumption was made (see Discussion) that the profile
(percentage composition) of excreted amino acids would reflect the profile of
amino acids in the blood plasma of resting trout, as reported by Wood et al.
(1999
). By applying the ratio
of the detection efficiency for each class of amino acid by the total nitrogen
analyser (essentially 100%) to the corresponding detection efficiency by the
ninhydrin assay (variable %) and weighting the outcome according to the
relative contribution of each amino acid to the profile, a correction factor
(x1.226) was derived and applied to the ninhydrin measurement of total
amino acid-N. The adjustment is relatively small because of the quantitative
predominance of amino acids bearing single
-amino groups in trout
plasma.
The excretion rates of certain compounds by the fish were below the limits
of detection, despite the fact that pre-concentration was employed. These
included TMA, TMAO, uric acid, and nitrate + nitrite. In the nitrate + nitrite
case, while the background concentration (3.6 µmol
l1 N) was certainly detectable in our water, the increment
above this relatively high background was undetectable.
Table 4 gives the practical
detection limits and demonstrates that the total contribution of each of these
compounds to total-N excretion could have been no greater than a few nmol N
g1 h1, trivial relative to measured
total-N excretion rates, which routinely exceeded 500 nmol N
g1 h1.
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Series 1 nitrogen excretion rates, feeding and fasting
Based on these tests, unknown-N excretion in series 1 was calculated as:
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Trout in this experiment had been either fasted for 10 days or fed a 2% ration every other day for the same period.
The excretion rates for total-N, ammonia-N, urea-N, protein-N, amino
acid-N, creatine-N, creatinine-N and unknown-N for both fed and fasted trout,
with or without anal suturing to prevent fecal N-excretion, are shown in
Table 3, while
Fig. 1 portrays the relative
contributions of each component on a percentage basis. As in most teleosts,
the majority of nitrogen waste was excreted as ammonia-N in all the groups
(5368%), and urea-N was the next most important product (610%),
except in the fed, sutured group where it was surpassed by both protein-N
(11%) and amino acid-N (10%). In general, the contributions of protein-N and
amino acid-N excretion were approximately equal (311% range), while
creatinine-N and creatine-N made up <1% each. Thus, unknown-N amounted to
1220% of the total, while the two products measured traditionally
(ammonia-N and urea-N) accounted for 5877% of total-N excretion in
the various treatments (Fig.
1). The relative contributions of unknown-N, amino-acid-N,
protein-N and creatinine-N all tended to increase with feeding, with
corresponding decreases in the relative contributions of ammonia-N and urea-N
(Fig. 1).
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In fasted fish, anal suturing had little effect on the pattern or rate of nitrogen excretion, although protein-N excretion and creatine-N excretion were both significantly higher in the sutured group (Table 3). In fed fish, the rates of total-N, ammonia-N, urea-N and creatinine-N excretion were all significantly higher in unsutured versus sutured animals, while the reverse was true for protein-N excretion. There was no difference in either amino acid-N or unknown-N excretion between sutured and unsutured fish in either fasting or fed groups.
Feeding clearly increased total-N excretion rate, even when the anus was sutured. For example, in the sutured group, total-N excretion was 1.4 times higher in the fed group than in the fasted group. Amino acid-N, protein-N, creatinine-N and unknown-N excretion rates in sutured fed fish were 23 times higher than those in the fasted groups. There were significant differences in total-N, amino acid-N, protein-N, creatinine-N and unknown-N excretion between the fasted and fed sutured groups (Table 3).
In unsutured fish, the rate of total-N excretion in the fed group was two times that of fasted animals. The largest contributor was ammonia-N excretion, which almost doubled. Amino acid-N, protein-N and creatinine-N excretion rates in the fed group were 35 times higher than those in the fasted group. There were significant differences in total-N, ammonia-N, urea-N, amino acid-N, protein-N, creatinine-N and unknown-N excretion between the fasted and fed unsutured groups (Table 3).
Series 2 ration and respirometry
In this series, ammonia-N, urea-N and total-N excretion rates were
measured, in addition to oxygen consumption rates, in fish that had been
either fasted for 13 days or fed 1%, 3% or 5% daily rations throughout the
preceding week. Thus, unknown-N was total-N [ammonia-N + urea-N].
Fed groups had higher
O2 values than
the fasted group, a difference that was significant for the 1% and 5% ration
groups (Fig. 2). However,
O2 did not
increase with increasing ration, as there were no significant differences
detected among the fed groups.
N, when expressed as the
sum of ammonia-N + urea-N, followed a very similar pattern to
O2, being higher
in all fed groups than in the fasted fish, although there were no significant
differences among the 1%, 3% or 5% ration treatments. However, when expressed
as total-N excretion,
N
exhibited a somewhat different pattern, increasing steadily with ration,
although only the value at 5% ration was significantly different from the
fasted group. Clearly, the proportion attributable to unknown-N excretion
products increased with food ration. In fasted fish, the percentage of unknown
nitrogen (i.e. not ammonia-N or urea-N) was 9%, and this increased to 17%, 25%
and 63%, respectively, in trout fed 1%, 3% and 5% daily rations. The impact on
calculated NQ is illustrated in Fig.
3 (see Discussion).
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Discussion |
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One significant discovery was the considerable excretion of nitrogen in the
form of amino acids (Table 3).
We employed the ninhydrin method of Moore
(1968) to detect amino acids
en masse, and the detection efficiencies that we recorded at quite
low levels (50 µmol l1) were very comparable with those
reported by other authors (Moore and Stein,
1948
,
1954
;
Fisher et al., 2001
). The
assay detects only
-amino nitrogen groups with full efficiency;
consequently, the ninhydrin assay will underestimate the nitrogen content of
those amino acids with single (i.e. tryptophan, asparagine, glutamine, lysine)
and multiple side-chain nitrogen groups [i.e. histidine (two), arginine
(three), as well as ß-amino acids]. By contrast, the total nitrogen
analyser detects the N-content of such amino acids with close to 100%
efficiency. Thus, the occurrence of these amino acids in the excreted amino
acid pool would create an artificial discrepancy between these two methods and
thus promote a higher value for the unknown portion of nitrogenous waste. To
correct for this, a correction factor (x1.226) was applied, based on the
assumption that the profile (percentage composition) of excreted amino acids
would reflect the profile of amino acids in the blood plasma of resting trout,
as reported by Wood et al.
(1999
). Nevertheless, if this
assumption is in gross error, a significant discrepancy may still exist,
especially in the case of fed fish, where amino acid-N excretion reached
7.29.6% of total-N, nearly twice that of fasting animals
(Fig. 1). However, when we
compared this correction factor (x1.226) calculated from the plasma
amino acid profile of fasted trout (Wood
et al., 1999
) with calculations based on the data of Espe et al.
(1993
) for Atlantic salmon
(Salmo salar) at fast (x1.346) or at various times (6 h, 12 h,
24 h) after feeding (x1.314, 1.413, 1.395), it seems probable that the
error would be quite small.
Espe et al. (1993) reported
that plasma amino acid levels were significantly higher at 6-h and 12-h
post-prandium in Atlantic salmon, while in the channel catfish (Ictalurus
punctatus) it has further been proposed that this surge may facilitate
the loss of amino acids by leakage across the branchial membrane
(Brown and Cameron, 1991
).
This suggests that a significant amount of amino acid-N excretion noted here
probably passes though the gills, which would constitute a significant
energetic loss. Presumably, this is an unavoidable feature of gill design and
does not have direct adaptive value. We are aware of no data on renal amino
acid-N losses, but total urinary N-losses are small (see below). The lack of
difference between amino acid-N excretion rates in sutured and unsutured fish,
regardless of feeding or fasting (Table
3), indicates that the amino acid-N excretion was not sourced from
the gastrointestinal tract.
Protein-N excretion was also surprisingly high
(Table 3) and, like amino
acid-N excretion, was also greater in fed fish than in fasted fish, reaching
11.2% in the fed sutured treatment (Fig.
1). This is probably related to the increased nitrogen intake
associated with eating. Like amino acid-N, it does not appear to be excreted
via the gastrointestinal tract because protein-N excretion rates were
actually higher in sutured than in unsutured fish, regardless of feeding or
fasting (Table 3). Again, the
`purpose' of excreting a valuable energy resource may seem counterintuitive.
However, in the kelp bass (Paralabrax sp.), Bever et al.
(1981) found that a significant
portion of the [14C] radioactivity from labeled amino acids
injected into the bloodstream quickly appeared in body mucus. It is possible
that most protein-N excretion in rainbow trout is that detected from the
shedding of mucus and may be performing a role secondary to the excretion of
nitrogen (e.g. protection; Shephard,
1994
). Such a hypothesis is supported by the finding that the rate
of protein-N excretion in sutured fish was higher than that in unsutured fish,
suggesting that stress caused by handling, suturing and/or anesthesia
stimulated mucus production by the skin. The ability of the Bradford assay to
detect different protein is known to vary with protein composition
(Sapan et al., 1999
) but, to
our knowledge, the capacity of the Bradford assay to detect mucus
glycoproteins has not been specifically investigated. However, given that the
mechanism of dye interaction with protein appears to depend mainly on
interaction with protonated amino groups
(Sapan et al., 1999
), there is
no mechanistic reason to believe that our results either under- or
overestimate the contribution of protein to overall nitrogen excretion,
although this possibility cannot be dismissed.
The use of anal sutures permitted an investigation of the importance of the
gut as a route of excretion. In fasting fish, there were only minor
differences in nitrogenous waste products between sutured and unsutured groups
(Table 3). It is unlikely that
the gastrointestinal tract is an important route for nitrogen excretion in
rainbow trout under fasting conditions, where the gills (and/or the skin)
predominate. Although nitrogen in the urine was not measured in the present
study, it is unlikely to contribute significantly to nitrogen excretion based
on salmonid literature reviewed by Wood
(1995). For example, Fromm
(1963
) reported that total
nitrogen excretion rate in the urine was only 3.3% of the branchial excretion
rate in starved rainbow trout.
Nitrogen excretion increased with feeding, even when the anus was sutured.
In fed fish, the difference between nitrogen excretion in sutured and
unsutured fish indicates nitrogen excretion via the gut and/or
nitrogen that was not absorbed from the ingested food and therefore passed out
in the feces. The contamination by any N that was present as particulate
matter in the feces was minimised by sieving the water with cheesecloth, but
clearly this would not eliminate any soluble forms. Wood
(1995) reviewed the
aquacultural literature on fecal-N excretion in salmonids and concluded that
the true or `metabolic component' was probably less than 50 µmol N
kg1 h1, with the remainder, which is
generally a larger fraction in fed animals, originating from non-absorbed
N-compounds. However, this conclusion was based on experiments using largely
indirect measurement techniques. In the present study, we cannot eliminate the
possibility that significant amounts of ammonia-N and urea-N (totaling far
more than 50 µmol N kg1 h1; cf.
Table 3) may have been excreted
across the gut wall in fed fish (i.e. true `metabolic-N excretion'), perhaps
as a result of local metabolism in the tissues of the gastrointestinal tract.
Indeed, it seems unlikely that this gastrointestinal N-output originated
directly from non-absorbed N-compounds, because it was not present in the form
of protein-N or amino acid-N. More likely, if of `non-absorbed' origin, it was
produced by bacterial conversion to ammonia-N, urea-N and creatinine-N.
Regardless, the finding that 47% of total ammonia-N output, 32% of total urea-N output and 52% of total creatinine-N output were sourced from the gut implies an important role for this pathway in the total nitrogen budget of fed fish. This subject is worthy of future study. Notably, there was no difference in these parameters (except creatinine-N) between sutured fed fish and sutured fasted fish. This suggests that the rates of ammonia-N and urea-N excretion from the gill (and skin) were not changed with feeding, in contrast to the surge in these products from the gastrointestinal tract.
In series 1, despite investigating the presence of a wide range of potential nitrogenous excretory products, a significant proportion (1220%) of total nitrogen remained unaccounted for. Table 5 summarises previous studies investigating the nature of nitrogen waste products in fish and highlights the ubiquity of the unknown nitrogen fraction.
In many studies, a possible explanation for the discrepancy between total
nitrogen and the sum of individually measured nitrogen components may lie with
methodology used, unless the efficiency of N-detection by the various assay
methods has been checked and taken into account. In the present study, the
only important error occurred with the ninhydrin method
(Table 1), which tends to
underestimate the N-content of certain amino acids, and this was taken into
account by the use of a correction factor. However, Doi et al.
(1981) and Fisher et al.
(2001
) have reported that the
ninhydrin assay detects not only amino acids but also small peptides (di- and
tripeptides) to a limited extent. Thus, some of the detected amino acid-N may
actually have represented peptide-N. On the other hand, if there is
substantial excretion of small peptides, then this could account for part of
the discrepancy, as di- or tripeptides will have additional undetectable N
groups. For example, tests in the present study demonstrated that the N
content in two ubiquitous dipeptides (the `buffer peptides', carnosine and
anserine) were detected with only 821% efficiency
(Table 1). If these compounds
were excreted to a significant extent, they would elevate the discrepancy
between `amino acids/peptides' as analysed by the total nitrogen analyser and
as measured via the ninhydrin method.
The proposition that some of this unknown nitrogen component may exist in
the form of small peptides is not without merit. For example, some fish
species contain large amounts of carnosine, while others, such as salmonids,
are known to contain large amounts of anserine (histidine-related dipeptide;
ß-alanyl--methyl-L-histidine) in their tissues (Abe,
1983
,
1991
;
Van Waarde, 1988
). For
example, Espe et al. (1993
)
reported that anserine levels in the epaxial muscle of the Atlantic salmon
were 6300-fold higher than any amino acid. In fact, anserine
concentrations in white muscle of rainbow trout have been measured at levels
approaching 20 µmol g1 and are even higher in other
salmonids (Van Waarde, 1988
).
Anserine levels in the kidney (a possible excretion route) were at least 2
µmol g1 wet mass (Abe,
1991
). Interestingly, Abe
(1991
) found that the anserine
levels in the white muscle of rainbow trout were considerably lower than
reported by Van Waarde (1988
),
potentially underestimating the potential importance of renal anserine. A
further important peptide may be
-methyl-L-histidine, which is
also found in high levels in trout tissues
(Abe, 1991
). This entity is of
particular interest given that the demethylating enzyme responsible for
converting
-methyl-L-histidine to L-histidine is
considered not to exist in trout, suggesting that
-methyl-L-histidine may be excreted without reutilisation
(Abe, 1991
). The importance of
these nitrogen-containing compounds lies not only in their relative abundance
but also in the fact that they contain multiple nitrogen groups. Anserine
contains four, while
-methyl-L-histidine has three nitrogen
groups. It is reasonable to speculate that some of the unknown-N excreted by
rainbow trout may exist undetected in these forms. Other forms of nitrogen,
for example `rotting compounds' such as putrescine, may also contribute to the
unknown component. Examination of unknown nitrogen using HPLC is likely to
provide insight in future investigations.
In series 2 of the present study, both oxygen consumption
(Fig. 2A) and nitrogen
excretion (Fig. 2B) increased
with feeding. Furthermore, unknown-N production also increased with feeding,
with unknown-N in fish fed 5% body mass day1 accounting for
63% of total-N (Fig. 2B), a
much higher value than in the series 1 results for trout on much lower daily
ration (Fig. 1; Tables
3,
5) or those generally reported
in other studies (Table 5;
although see De Boeck et al.,
2001).
Fig. 3 presents NQ, and
associated protein utilisation values, calculated three different ways, and
illustrates the marked effect that using various assumptions can have on the
results. When NQ is calculated in the traditional manner based on the sum of
measured ammonia-N + urea-N excretion, the value increases from 0.14 to
0.160.21 in fed fish. Thus, the estimated use of protein in
aerobic metabolism increases from
50% to
6080%. The former is
somewhat higher than generally reported for fasted trout, but the latter
values are very representative of values for fed salmonids calculated in
earlier studies on the same basis (see Introduction). When total-N excretion
is used in the calculation, there is little change in fasted fish, but the
discrepancy progressively increases with ration such that the theoretical
maximum value (NQ=0.27, representing 100% protein utilisation) is reached at
3% ration, and this value is greatly exceeded at 5% ration. This strongly
suggests that the contribution of non-oxidized N-products increases greatly at
high ration. Thus, NQ based on ammonia-N + urea-N excretion underestimates
protein oxidation, while NQ based on total-N excretion overestimates it.
The third, and presumably best, way would be to calculate NQ from the sum of all oxidized N-products. Unfortunately, the alternative N-products were not measured in the experiments of series 2. However, an estimate of the sum of all oxidized N-products can be obtained by applying the percentage contribution data from series 1 (Fig. 1) for the sum of ammonia-N, urea-N, creatinine-N, creatine-N and unknown-N (i.e. specifically excluding protein-N and amino acid-N) to the measured total-N data of series 2 (from Fig. 2B). This should be most accurate for fasted fish and those fed a 1% daily ration, because the former treatment was duplicated in series 1 while the latter was approximated by the 2% ration fed every other day in series 1. The results of these model calculations (Fig. 3) indicate only modest changes in the estimation of protein utilisation (relative to calculations based on ammonia-N + urea-N) for these two treatments but much larger differences at the higher rations. Most notably, the NQ still stays above the theoretical maximum value at the 5% ration level. Multiple explanations are possible, but the most likely is that the contribution of non-oxidized N-products is increasing greatly at high ration level, such that percentage estimates based on data taken at low ration level are not applicable. This is an important area for future investigation. Despite many years of intensive research on the nitrogen metabolism of teleost fish, it is clear that a more comprehensive understanding of protein and amino acid excretion, of the nature of minor nitrogen end products and of how their output varies with feeding is required.
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Abe, H. (1983). Distribution of free L-histidine and related dipeptides in the muscle of fresh-water fishes. Comp. Biochem. Physiol. B 76, 35-39.[CrossRef]
Abe, H. (1991). Interorgan transport and catabolism of carnosine and anserine in rainbow trout. Comp. Biochem. Physiol. B 100,717 -720.[CrossRef][Medline]
Alsop, D. H. and Wood, C. M. (1997). The interactive effects of feeding and exercise on oxygen consumption, swimming performance and protein usage in juvenile rainbow trout. J. Biol. Chem. 200,2337 -2346.
Alsop, D. H., Kieffer, J. D. and Wood, C. M. (1999). The effect of temperature and swimming speed on instantaneous fuel use and nitrogenous waste excretion of Nile tilapia. Physiol. Biochem. Zool. 72,474 -483.[CrossRef][Medline]
Beamish, F. W. H. and Thomas, E. (1984). Effects of dietary protein and lipid on nitrogen losses in rainbow trout (Salmo gairdneri). Aquaculture 41,359 -371.[CrossRef]
Bever, K., Chenoweth, M. and Dunn, A. (1981). Amino acids, gluconeogenesis, and glucose turnover in kelp bass (Paralabrax sp.). Am. J. Physiol. 240,R246 -R252.[Medline]
Boutilier, R. G., Hening, T. A. and Iwama, G. K. (1984). Appendix: physicochemical parameters for use in fish respiratory physiology. In Fish Physiology, vol.10A (ed. W. S. Hoar and D. J. Randall), pp.403 -430. Orlando: Academic Press.
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Brett, J. R. and Zala, C. A. (1975). Daily pattern of nitrogen excretion and oxygen consumption of sockeye salmon (Oncorhynchus nerka) under controlled conditions. J. Fish. Res. Bd. Canada 32,2479 -2486.
Brown, C. R. and Cameron, J. N. (1991). The induction of specific dynamic action in channel catfish by infusion of essential amino acids. Physiol. Zool. 64,276 -297.
Cockcroft, A. C. and Du Preez, H. H. (1989). Nitrogen and energy loss via nonfaecal and faecal excretion in the marine teleost Lithognathus lithognathus. Mar. Biol. 101,419 -425.
De Boeck, G., Alsop, D. and Wood, C. M. (2001). Cortisol effects on aerobic and anaerobic metabolism, nitrogen excretion, and whole-body composition in juvenile rainbow trout. Physiol. Biochem. Zool. 74,858 -868.[CrossRef][Medline]
Doi, E., Shibata, D. and Matoba, T. (1981). Modified colorimetric ninhydrin methods for peptidase assay. Anal. Biochem. 118,173 -184.[Medline]
Espe, M., Lied, E. and Torriessen (1993). Changes in plasma and muscle free amino acids in Atlantic salmon (Salmo salar) during absorption of diets containing different amounts of hydrolyzed cod muscle protein. Comp. Biochem. Physiol. A 105,555 -562.[CrossRef]
Fisher, G. H., Arias, I., Quesada, I., D'Aniello, S., Errico, F., Di Fiore, M. M. and D'Aniello, A. (2001). A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues. Amino Acids 20,163 -173.[CrossRef][Medline]
Fromm, P. O. (1963). Studies on renal and extra-renal excretion in a freshwater teleost, Salmo gairdneri.Comp. Biochem. Physiol. 10,121 -128.[CrossRef][Medline]
Garrow, J. S. (1974). Energy Balance and Obesity in Man. Amsterdam, London: Elsevier Science.
Fivelstad, S., Thomassen, J. M., Smith, M. J., Kjartansson, H. and Sando, A.-B. (1990). Metabolite production rates from Atlantic salmon (Salmo salar L.) and Arctic char (Salvelinus alpinus L.) reared in single pass land-based brackish water and sea-water systems. Aquacult. Eng. 9, 1-21.[CrossRef]
Jobling, M. (1980). Effects of starvation on proximate chemical composition and energy utilization of plaice, Pleuronectes platessa. J. Fish. Biol. 17,325 -334.
Kieffer, J. D., Alsop, D. and Wood, C. M.
(1998). A respirometric analysis of fuel use during aerobic
swimming at different temperatures in rainbow trout (Oncorhynchus
mykiss). J. Exp. Biol.
201,3123
-3133.
Kleiber, M. (1961). The Fire of Life: an Introduction to Animal Energetics. New York, London: John Wiley & Sons.
Kleiber, M. (1992). Respiratory exchange and metabolic rate. In Handbook of Physiology (ed. S. R. Geiser), pp. 927-938. Bethesda: American Physiological Society.
Korsgaard, B., Mommsen, T. P. and Wright, P. A. (1995). Nitrogen excretion in teleostean fish: adaptive relationships to environment, ontogenesis, and viviparity. In Nitrogen Metabolism and Excretion (ed. P. J. Walsh and P. A. Wright), pp. 259-288. Boca Raton: CRC Press.
Kutty, M. N. and Peer Mohamed, M. (1975). Metabolic adaptations of mullet Rhinomucil corsula (Hamilton) with special reference to energy utilization. Aquaculture 5, 253-270.[CrossRef]
Lauff, R. F. and Wood, C. M. (1996a). Respiratory gas exchange, nitrogenous waste excretion, and fuel usage during starvation in juvenile rainbow trout. J. Comp. Physiol. B 165,542 -551.[Medline]
Lauff, R. F. and Wood, C. M. (1996b). Respiratory gas exchange, nitrogenous waste excretion, and fuel usage during aerobic swimming in juvenile rainbow trout. J. Comp. Physiol. B 166,501 -509.
McCarthy, J. J. and Whitledge, T. E. (1972). Nitrogen excretion by anchovy (Engraulis mordax) and jack mackerel (Trachurus symmetricus). Fish. Bull. 70,395 -401.
Mommsen, T. P. and Walsh, P. J. (1991). Urea synthesis in fishes: evolutionary and biochemical perspectives. In Biochemistry and Molecular Biology of Fishes, vol.1 (ed. P. W. Hochachka and T. P. Mommsen), pp.137 -163. New York: Elsevier.
Mommsen, T. P. and Walsh, P. J. (1992). Biochemical and environmental perspectives on nitrogen metabolism in fishes. Experientia 48,583 -592.
Moore, S. (1968). Amino acid analysis: aqueous
dimethyl sulfoxide as solvent for the ninhydrin reaction. J. Biol.
Chem. 243,6281
-6283.
Moore, S. and Stein, W. H. (1948). Photometric
ninhydrin method for use in the chromatography of amino acids. J.
Biol. Chem. 176,367
-388.
Moore, S. and Stein, W. H. (1954). A modified
ninhydrin reagent for photometric determination of amino acids and related
compounds. J. Biol. Chem.
211,907
-913.
Olson, K. R. and Fromm, P. O. (1971). Excretion of urea by two teleosts exposed to different concentrations of ambient ammonia. Comp. Biochem. Physiol. A 40,999 -1007.[CrossRef][Medline]
Rahmatullah, M. and Boyde, T. R. C. (1980). Improvements in the determination of urea using diacetyl monoxime; methods with and without deproteinisation. Clin. Chim. Acta 107, 3-9.[CrossRef][Medline]
Rand, M. C. (ed.) (1975). Standard Methods for the Examination of Water and Wastewater. 14th edition. pp. 423. Washington, DC: APHA-AWWA-WPCF.
Sapan, C. V., Lundblad, R. L. and Price, N. C. (1999). Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29, 99-108.[Medline]
Shephard, K. L. (1994). Functions for fish mucus. Rev. Fish Biol. Fish. 4, 401-429.
Smith, H. W. (1929). The excretion of ammonia
and urea by the gills of fish. J. Biol. Chem.
81,727
-742.
van den Thillart, G. and Kesbeke, F. (1978). Anaerobic production of carbon dioxide and ammonia by goldfish Carassius auratus (L.). Comp. Biochem. Physiol. B 156,511 -520.
Van Waarde, A. (1988). Biochemistry of non-protein nitrogenous compounds in fish including the use of amino acids for anaerobic energy production. Comp. Biochem. Physiol. B 91,207 -228.[CrossRef]
Verdouw, H., Van Echted, C. J. A. and Dekkers, E. M. (1978). Ammonia determination based on indophenol formation with sodium salicylate. Water Res. 12,399 -402.[CrossRef]
Walsh, P. J. (1997). Nitrogen metabolism and excretion. In The Physiology of Fishes. 2nd edition (ed. D. H. Evans), pp. 199-214. Boca Raton: CRC Press.
Walsh, P. J., Wang, Y., Campbell, C. E., De Boeck, G. and Wood, C. M. (2001). Patterns of nitrogenous waste excretion and gill urea transporter mRNA expression in several species of marine fish. Mar. Biol. 139,839 -844.[CrossRef]
Wekell, J. C. and Barnett, H. (1991). New method for analysis of trimethylamine oxide using ferrous sulfate and EDTA. J. Food Sci. 56,132 -138.
Wiggs, A. J., Henderson, E. B., Saunders, R. L. and Kutty, M. N. (1989). Activity, respiration, and excretion of ammonia by Atlantic salmon (Salmo salar) smolt and postmolt. Can. J. Fish. Aquat. Sci. 46,790 -795.
Wood, C. M. (1993). Ammonia and urea metabolism and excretion. In The Physiology of Fishes (ed. D. Evans), pp. 379-425. Boca Raton: CRC Press.
Wood, C. M. (1995). Excretion. In Physiological Ecology of the Pacific Salmon (ed. C. Groot, L. Margolis and W.C. Clarke), pp. 381-438. Vancouver: Government of Canada Special Publications Branch; UBC Press.
Wood, C. M. (2001). The influence of feeding, exercise, and temperature on nitrogen metabolism and excretion. In Fish Physiology, vol. 20 (ed. P. A. Anderson and P. A. Wright), pp. 201-238. Orlando: Academic Press.
Wood, C. M., Milligan, C. L. and Walsh, P. J. (1999). Renal responses of trout to chronic respiratory and metabolic acidoses and metabolic alkalosis. Am. J. Physiol. 277,R482 -R492.[Medline]
Wood, J. D. (1958). Nitrogen excretion in some marine teleosts. Can. J. Biochem. Physiol. 36,1237 -1242.
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