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2
Departments of
*
Nutritional Sciences,
Animal Sciences and
**
Pediatrics, University of Wisconsin-Madison, WI 53706.
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSSample analysisRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • piglets • neonate • urea production • alanine • carbamoyl phosphate synthetase (CPS-1)
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSample analysisRESULTSDISCUSSIONREFERENCES |
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,
Morris 1992
). Flux through carbamoyl phosphate
synthetase-1
(CPS-1)3
is potentially a rate-limiting step in the formation of urea, and
its activity is related directly to the availability of
N-acetylglutamate (NAG), an essential cofactor
(Brusilow and Horwich 1989
, Meijer et al. 1985
). Therefore, the rate of urea synthesis is controlled by
substrate availability and the catalytic activities of the urea cycle
enzymes (Morris 1992
).
Measurement of the activity of CPS-1 in vitro is carried out under
controlled environmental conditions in which the pH and substrate
concentrations are adjusted to maximize activity (i.e.,
Vmax). In vitro enzyme activities have
been used routinely to study the development of the urea cycle
potential during gestation and after birth in rats (Kennan and Cohen 1959
, Miller and Chu 1970
, Raiha and Suihkonen 1968a
), piglets (Kennan and Cohen 1959
), and humans (Raiha and Suihkonen 1968b
).
In addition to being measured under nonphysiologic conditions, the
liver samples used to make homogenates in previous studies on human
fetuses were obtained from abortions, stillbirths, miscarriages and
unexpected infant deaths. These results may not be representative of
the capabilities of the healthy fetus in utero, or the newborn human
infant. Estimates of in vivo urea production in newborn infants are
difficult, given risk involved and the issue of parental consent. Thus,
it is not surprising that few such studies (Kalhan 1993
)
have been done.
In this experiment, newborn piglets were used to study urea cycle
capacity at birth. Our objective was to compare the in vivo urea
production potential obtained by providing a nitrogen load by an
intravenous infusion of alanine as was done previously (Davis et al. 2000
) and compare the in vivo production urea rate with a
calculated value by scaling the total liver activity
(Vmax) of CPS-1, the mitochondrial
enzyme thought to control urea cycle activity under nonsaturating
conditions (Meijer et al. 1985
). To assess CPS-1
capacity, it was measured in vitro and expressed in units of [mmol
citrulline/(h · g liver)], which could then be extrapolated to a
whole-animal "maximal urea production equivalents rate" [mmol
citrulline/(h · kg0.75)] based on liver weight
and body weight, assuming that urea is produced only in the liver
(Morris 1992
). The maximal urea production rate based on
the enzyme activity was then compared with the measured in vivo urea
production rate, as discussed previously (Davis et al. 2000
), in an attempt to assess whether the enzyme system was
saturated in vivo. This would be demonstrated by a measured in vivo
production rate similar to the whole-body "maximal urea
production equivalents rate," based on the activity of the presumed
first-limiting enzyme of the urea cycle (CPS-1), with both
expressed in similar units.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSample analysisRESULTSDISCUSSIONREFERENCES |
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Liquid phenol (90%), sulfuric acid (98%), ammonium chloride, perchloric acid and phosphoric acid were obtained from Fisher Scientific (Pittsburgh, PA). Urease (EC 3.5.1.5), ornithine transcarbamoylase (EC 2.1.3.3), ornithine, citrulline, 2,3-butanedione monoxime, NAG, ATP, tyrosine and hexadecyltrimethylammonium bromide (CTAB) were obtained from Sigma Chemical (St. Louis, MO). Alanine was obtained from U.S. Chemical (Cleveland, OH). Sodium hydroxide, sodium bicarbonate, glucose, sodium chloride, magnesium sulfate and 30% hydrogen peroxide were obtained from Mallinckrodt (Chesterfield, MO). Sodium nitroprusside was obtained from Eastman Organic Chemicals (Rochester, NY). Urea was obtained from Amend Drug & Chemical (Irvington, NJ).
Animals.
A total of twenty-four piglets (0.839–2.078 kg), six at a time, were brought into the laboratory within ~3–8 h of birth, with at least 2 litters represented in each group. The piglets were paired, the smaller piglets of each group with the larger of the group, and pairs were assigned randomly to one of four treatment groups as follows: baseline control (n = 8), 18 h of alanine intravenously (IV) at 50% of resting energy expenditure (REE) (n = 4), 36 h of alanine IV at 50% REE (n = 6) or 36 h of glucose IV at 50% REE (n = 6). The order in which the 12 pairs of piglets were studied was completely randomized. Care and handling of piglets were reviewed and approved by the College of Agricultural and Life Sciences Research Animal Resource Committee.
Animal preparation.
Animals underwent surgical procedures for insertion of catheters as
previously described (Davis et al. 2000
).
Experimental protocol.
After recovery from anesthesia, each piglet was infused with water
intragastrically (IG) via the Foley catheter until well hydrated (based
on the urine excretion rate). The experiment began once piglets were
hydrated, usually a few hours after the start of IG water infusion
(~12 h of age). All piglets were infused with water IG at 8–10 mL/h
for a 6-h baseline period (Table 1
). At the end of the baseline period, the piglets designated as controls
were anesthetized with 3% halothane in oxygen, the entire livers were
removed and the piglets were killed by exsanguination. The livers were
immediately blotted, weighed, minced and placed on ice until analysis
of the activity of the mitochondrial enzyme, CPS-I. The piglets
assigned to be infused with alanine for 18 or 36 h and those to be
infused with glucose for 36 h were infused with their respective
solutions containing 50% REE as alanine or glucose, provided IV at a
rate of 8–10 mL per h.
|
). The infusion levels of alanine or glucose were based on
the previous stair-stepped infusions of alanine or glucose at 25,
50 and 75% REE (Davis et al. 2000
). The intent was to
provide an amount of nitrogen that would challenge the piglet to make
urea, but not result in metabolic complications due to hyperammonemia. After the infusions of alanine or glucose, piglets were anesthetized for removal of the livers, and were killed by exsanguination. Livers were blotted, weighed, minced and analyzed for the activity of CPS-1 as described below. All livers were kept on ice until the time of analysis, which was completed within 60–120 min of liver removal.
Sample collection.
One-hour urine aliquots were continuously collected throughout the
experiment from the urachal catheter, directly into tubes containing
100 µL of 6 mol/L HCl (urine pH<2.0). Blood (0.25 mL)
was collected every 3 h throughout the experiment, deproteinized
by the method of Somogyi (1930)
and the supernatants
stored at -10°C until the time of analysis.
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Sample analysis |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSample analysisRESULTSDISCUSSIONREFERENCES |
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Acidified urine samples were diluted 10- to 40-fold with 0.1–0.4 mol/L
phosphate buffer (pH 7.0) and analyzed for urea plus ammonium nitrogen
or only ammonium nitrogen colorimetrically, as previously described
(Davis et al. 2000
). Recovery of urea and ammonium
sulfate standards added to samples were 103.6 and 101.4% on average,
respectively. Urinary urea nitrogen was calculated by difference. Urine
samples were composited over 3-h periods and analyzed for total
nitrogen by micro-Kjeldahl digestion (Mann 1967
)
followed by colorimetric determination of ammonium in the digested
samples by the Berthelot reaction. Recovery of ammonium sulfate-N
added to samples in the colorimetric assay was 98.4% on average.
Recovery of tyrosine-N through the Kjeldahl procedure was 102.3%.
Blood urea nitrogen (BUN).
BUN was determined in deproteinized blood samples by incubation of the sample with urease, followed by colorimetric analysis of ammonium, a modification of the Berthelot reaction. Recovery of urea added to samples was 99.8% on average.
Carbamoyl phosphate synthetase assay.
The activity of CPS-1, which catalyzes the reaction of ammonium and
bicarbonate to form carbamoyl phosphate, was assayed by a modification
of the method of Brown and Cohen (1959)
. Due to the
instability of carbamoyl phosphate (Meijer et al. 1985
),
the reaction was coupled with ornithine transcarbamoylase (OTC) to form
citrulline (cit), which was measured colorimetrically (Archibald 1944
) to determine the enzyme activity
(Vmax) of CPS-1 [mmol cit/(h · g wet
liver)].
A 10% liver homogenate was prepared by mincing the liver in 1:10 (wt/v) of 0.1% CTAB (wt/v) in 0.15 mol/L KCl (wt/v; 1 g liver to 9 mL CTAB) in a Potter Elvjehem tube kept in ice. The tissue was then homogenized with three passes of a tight-fitting pestle and an aliquot was preincubated at 37°C for the assay.
The assay medium consisted of the following: 50 µmol ammonium chloride prepared in 0.05 mol/L phosphate buffer (pH 7.3), 50 µmol sodium bicarbonate prepared in 0.05 mol/L phosphate buffer (pH 7.3) and gassed with 95% O2/5% CO2, 24 µmol magnesium sulfate prepared in water, 5 µmol NAG prepared in 0.05 mol/L phosphate buffer (pH 7.3), 20 µmol ATP prepared in water, adjusted to pH 7.3 with NaOH, 5 µmol L-ornithine prepared in 0.05 mol/L phosphate buffer (pH 7.3), 3–4 U OTC diluted in 0.05 mol/L phosphate buffer (pH 7.3) and 0.1 mL of 0.4 mol/L phosphate buffer (pH 7.3) plus 0.05–0.2 mL homogenate in a final volume of 1.0 mL.
Thirty-minute incubations were carried out at 37°C in 15-mL glass test tubes in duplicate or triplicate, unstoppered, without shaking. After a 10-min preincubation, the reaction was started with the addition of 0.05 to 0.2 mL homogenate and stopped with 3 mL of 0.5 mol/L perchloric acid. The mixture was vortexed and immediately transferred into a 12-mL thick-walled centrifuge tube and centrifuged for 10 min at 4000 x g to remove the protein precipitate. The blank used was a tissue blank to which the perchloric acid was added immediately before the homogenate at time zero.
When assays were incubated for 30 min at 37°C, the production of citrulline as a function of volume of a 10% newborn piglet liver homogenate added to the assay system was linear up to 0.5 mL homogenate (y = 3.236x + 0.0739; R2 = 0.9884, where x = volume of 10% homogenate in mL).
Citrulline.
The activity of CPS-1 was based on the colorimetric determination of
citrulline, which was carried out on 1 mL of supernatant in duplicate
by the method of Archibald (1944)
. Optical densities
were read on a Gilford 200 spectrophotometer in a 1-cm glass
cuvette at wavelength 490 nm. The mean recovery of citrulline added to
supernatant was 102.1%.
Recovery of citrulline in the enzyme assay system was determined by addition of known amounts of citrulline to the system, with and without incubation. Recovery of citrulline after 30 min of incubation with homogenate, compared with time zero, was 101.5% on average, confirming that measurements of activity were based on production of citrulline within the enzyme system.
Calculations.
The equation used for the calculation of the rate of urea production is
the same as that used previously (Davis et al. 2000
) and
is very similar to that used by Mitton et al. (1991)
:
 |
where Up is urea produced,
Ue is equivalent to the urea excreted
in the urine over a given period of time, and
Ub1 and
Ub2 correspond to the estimated total
urea in the water space of the piglet at the beginning and the end of
the timed urine collection. To estimate the body urea content, it is
assumed that the piglet is 80% water on the basis of our own data
(Mickelson, unpublished data from our laboratory, comparable piglets
12 h old, n = 16, 81% water; piglets infused IV
at 50% REE and 72 h old, n = 7, 82.5% water) and
that urea is distributed uniformly in the water space of the piglet
(Mitchell and Steele 1987
).
Statistics.
An initial analysis was performed to determine whether the initial weights of the piglets (0.839 to 2.078 kg) affected the following: 1) the change from baseline in the rate of urea or total nitrogen excretion, 2) the change from baseline in BUN concentration, 3) the change from baseline in the estimated rate of urea production, or 4) the final Vmax of CPS-1 for each piglet. A multiple regression analysis allowing for different slopes for the four distinct treatment groups was performed and initial weight had no significant effect for any of the treatments for any of the response variables. Therefore, initial piglet weight could be ignored in future analyses.
Differences in the Vmax of CPS-1 between
treatment groups were detected by one-way ANOVA with means
comparisons by Least Significant Differences (LSD). The calculated rate
of urea production for the last 6 h of the experiment as a
function of the Vmax of CPS-1 for each pig
was analyzed by a multiple regression that allowed for different slopes
and intercepts for the four distinct treatment groups (Fig. 6)
. The
changes from baseline in the rate of urinary urea-N excretion,
blood urea nitrogen concentration and the rate of urea production, were
analyzed by paired t test between the baseline mean (6
h) and the final mean (last 6 h of infusion) for piglets infused
with alanine or glucose.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSample analysisRESULTSDISCUSSIONREFERENCES |
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Urinary nitrogen.
Urine composited over 6-h periods was analyzed for total (Kjeldahl)-N,
urea-N and ammonium-N (Fig. 1
). Total-N excretion [mg N/(6h · kg0.75)]
increased in piglets infused with alanine for 36 h from 89.6
± 42.0 (baseline), to 345.1 ± 75.7 (P < 0.05) and decreased in piglets infused with glucose for 36 h from
89.6 ± 42.0 to 38.8 ± 10.3 (P < 0.05). The
decrease in N-excretion of the piglets infused with glucose
suggests that body fuel and thus protein was being spared. Although one
cannot measure this in piglets infused with alanine, it may also have
been the case for those piglets because they were infused with an equal
amount of energy, on the basis of ATP.
|
). Excretion of UUN [mg N/(h · kg0.75)]
increased from 10.6 ± 5.9 to 53.2 ± 11.1 in piglets infused
with alanine (P < 0.002) and decreased in piglets
infused with glucose from 10.6 ± 5.9 to 3.7 ± 1.3
(P < 0.002). Urinary urea plus ammonium-N made up
79% of the total (Kjeldahl)-N excreted throughout the experiment on
average (range 65–95%; Fig. 2
).
|
BUN concentrations (mmol urea N/L; Fig. 3
) increased nearly fivefold in piglets infused with alanine for 36 h from 9.1 ± 3.0 to 52.0 ± 7.0 (P < 0.002)
and decreased in piglets infused with glucose for 36 h from 9.1
± 3.0 to 5.4 ± 2.3 (P < 0.01).
|
The calculation of Up [mmol urea/(h ·
kg0.75)] is based on the urea excretion and the
changes in the body urea pool (see calculations section). Therefore,
the pattern of the calculated rate of urea production is very similar
to patterns followed by UUN excretion and BUN concentrations over time
(Fig. 4
). The rate of Up [mmol urea/(h ·
kg0.75)] increased sixfold from 0.34 ± 0.21 to 2.39 ± 0.53 (P < 0.002) in piglets
infused with alanine for 36 h, and decreased from 0.34 ± 0.21 to 0.16 ± 0.11 in piglets infused with glucose (P
< 0.02).
|
The Vmax of CPS-1 [mmol citrulline/(h
· kg0.75)] for piglets infused with alanine
for 36 h (4.4 ± 1.5) was increased significantly
(P < 0.05) compared with all other treatment groups.
The Vmax of CPS-1 for all other
treatments were not different from each other (P > 0.05; Fig. 5
).
|
The best-fit model (R2 = 91.8%)
explaining the final rate of urea production [mmol urea/(h 8729·
kg0.75)] at baseline, 18 or 36 h after the
start of alanine or glucose infusion, as a function of the
Vmax of CPS-1 [mmol cit/(h
8729 · kg0.75), Fig. 6
] at the time the piglet was killed revealed a positive relationship
(slope = 0.263; P = 0.002). The
y-intercepts for the 18-h alanine group (0.77) and the 36-h
alanine group (1.24) were significantly different (P < 0.001) from the y-intercept of the baseline controls and the
36-h glucose controls (-0.21), which was not significantly different
from zero (P = 0.20), suggesting that for a given level
of enzyme activity, the infusion of alanine resulted in an increase in
the urea production rate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSample analysisRESULTSDISCUSSIONREFERENCES |
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CPS-1 is first limiting for urea production under normal physiologic
conditions (Meijer et al. 1985
) and was therefore the
enzyme chosen for analysis in this experiment. On the basis of in vitro
Vmax comparisons [mmol product/(h ·
kg0.75)], urea cycle flux in rat pups, adult
rats, and human infants is limited by argininosuccinate synthetase (AS)
(Miller and Chu 1970
, Raiha and Suihkonen 1968a
and 1968b
, Table 2
). It is interesting that the calculated urea production rate of piglets
at baseline [Fig. 4
; 0.34 ± 0.21 mmol urea/(h ·
kg0.75)] closely resembles the
Vmax of AS of humans and rats at birth
(see Table 2
); however, it would be incorrect to assume, therefore,
that AS is rate limiting in vivo under these nonsaturating conditions
because single in vitro Vmax
conditions are not at all representative of in vivo conditions, in
which substrate concentrations and amount of enzyme activity vary.
|
]. The results are consistent
with the idea that higher activity of CPS-1 (and probably other urea
cycle enzyme activities) can result in a significantly higher urea
production rate (slope = 0.263; P < 0.002). It
would be interesting to know whether this slope persists in groups of
animals infused with alanine for longer periods of time. One may
hypothesize that, at a constant rate of infusion, substrate
concentration and enzyme activity may come into some "balance." If
this happens, an increase in the activity of the enzyme may not result
in an increase in the rate of urea production because of a constant
substrate infusion. It is also significant that infusion of alanine for
18 and 36 h resulted in increases in the rate of urea production
for a given level of CPS-1 activity (P < 0.001).
Having discussed the statistical model selected, it should be noted
that, due to the limited range of the CPS-1
Vmax within a given treatment group
(Fig. 6)
, the ability to select alternative models to describe the data
set is limited.
The increase in CPS-1 activity (P < 0.05) in response
to alanine infusion (Fig. 5)
is likely the result of short-term
urea cycle regulation, specifically, an increase in the concentration
of intramitochondrial NAG, a required cofactor of CPS-1
(Tatibana and Shigesada 1976
). Meijer and Hensgens (1982)
suggested that under conditions of increased
ammonium concentrations, such as after an increase in the supply of
amino acids to the liver, in order to buffer the intracellular ammonium
concentration short term, there is an increase in the concentration of
NAG in mitochondria, which results in an increase in the activity of
CPS-1.
Schimke (1962)
demonstrated long-term regulation of
the urea cycle. In these experiments, increases in the activities of
all urea cycle enzymes were observed in livers of rats weighing 50–60
g at the start of the experiment. Rats were fed a low protein (15%
casein) or high protein (60% casein) diet for 14 d. The results
indicated a two- to threefold increase in the enzyme activities of all
urea cycle enzymes in response to the high protein diet. This response
was thought to be a result of an increase in the amount of total enzyme
protein present, based on purification of arginase and OTC, an example
of long-term urea cycle regulation that may require up to 6 d
to attain a new steady state.
The results of this experiment are supported by the work of
Snodgrass and Lin (1981)
who showed that the activities
of all five urea cycle enzymes in rat liver [µmol/(h
·100g rat)] increased 30 to 90+% either by IG
infusion of casein hydrolysate [2 g N/(d · kg)] or by alanine,
glycine or methionine alone [2 g N/(d · kg)] for 2 d. The same
increase in urea cycle enzyme activities does not occur as a result of
infusion of ammonium nitrogen (Hutchinson et al. 1964
)
or any of the other 21 amino acids (Snodgrass and Lin 1981
). From these studies it was concluded that excess waste
nitrogen does not necessarily lead to increases in enzyme activity in
rat liver.
On the basis of the ability of piglets in these experiments to increase
urea production sevenfold and to increase the activity of CPS-1 by more
than twofold after infusion of alanine at 50% of REE for 36 h,
piglets are not compromised in their ability to manage a nitrogen load,
given as alanine or possibly other amino acids, at birth and in the
first 2 d of life. One wonders whether the situation is similar in
human infants because the developmental pattern of enzyme activities,
based on Vmax comparisons, of piglets
in utero and after birth (Kennan and Cohen 1959
) is very
similar to that seen in humans (Raiha and Suihkonen 1968b
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AS, argininosuccinate synthetase; BUN, blood urea nitrogen; cit, citrulline; CPS-1, carbamoyl phosphate synthetase; CTAB, hexadecyltrimethylammonium bromide; IG, intragastric; IV, intravenous; NAG, N-acetylglutamate; OTC, ornithine transcarbamoylase; REE, resting energy expenditure; UUN, urinary urea nitrogen.
Manuscript received October 25, 1999. Initial review completed January 19, 2000. Revision accepted March 24, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSample analysisRESULTSDISCUSSIONREFERENCES |
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