Arginine, ornithine, and proline interconversion is dependent
on small intestinal metabolism in neonatal pigs
Robert F. P.
Bertolo1,
Janet A.
Brunton1,
Paul B.
Pencharz1,2,3,4, and
Ronald O.
Ball1,2,4
1 Department of Agricultural, Food and Nutritional
Science, University of Alberta, Edmonton, Alberta T6G 2P5; and
2 The Research Institute, The Hospital for Sick
Children, and the Departments of 3 Paediatrics and
4 Nutritional Sciences, University of Toronto, Toronto,
Ontario, Canada M5G 1X8
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ABSTRACT |
We have previously shown that
arginine deficiency is exacerbated by the removal of dietary proline in
orally, but not parenterally, fed piglets. Therefore, we hypothesized
that the net interconversions of proline, ornithine, and arginine
primarily occur in the small intestine of neonatal piglets. Ten
intragastrically fed piglets received either intraportal (IP) or
intragastric (IG) primed, constant infusions of
[guanido-14C]arginine and
[U-14C]ornithine + [2,3-3H]proline. By
infusing amino acid isotopes via the stomach compared with the portal
vein, we isolated small intestinal first-pass metabolism in vivo.
During IP infusion, fractional net conversions (%) from proline to
ornithine (0), ornithine to arginine (11 ± 6), and
ornithine to proline (5 ± 1) were lower (P < 0.05) than during IG infusion (39 ± 8, 18 ± 6, and 42 ± 12, respectively); we speculate that these data are due to the
localization of ornithine aminotransferase to the gut. The balance of
these conversions indicated a large synthesis of arginine (70.0 µmol · kg
1 · h
1)
by the gut, with a corresponding degradation of ornithine (70.8 µmol · kg
1 · h
1)
and no change in proline balance. Gut synthesis of arginine from
proline (48.1 µmol · kg
1 · h
1)
was 50% of its requirement, whereas proline synthesis from arginine (33.0 µmol · kg
1 · h
1)
amounted to 10% of its requirement. Overall, arginine synthesis is
more dependent on the gut than proline synthesis. In situations in
which gut metabolism is compromised, such as during parenteral nutrition or gastrointestinal disease, arginine and proline are individually indispensable because their biosyntheses are negligible.
biosynthesis; intraportal infusion; ornithine aminotransferase; amino acid kinetics; first-pass metabolism
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INTRODUCTION |
UNLIKE IN THE
ADULT, ARGININE SYNTHESIS occurs primarily in the neonatal small
intestine; indeed, the net synthesis of arginine by the small intestine
of the piglet has been demonstrated using arteriovenous differences
across the gut (18, 23). Because of low
pyrroline-5-carboxylate (P-5-C, EC no. not yet assigned) synthase
activity in the neonatal small intestine, glutamate and glutamine are
not significant precursors for arginine synthesis in the neonate
(14, 22). As a result, proline is believed to be the
primary dietary precursor for de novo synthesis of arginine by the gut.
Indeed, in a previous experiment employing arginine deficiency-induced
hyperammonemia as the primary outcome, we demonstrated that arginine
and proline are coindispensable in intragastrically fed piglets
(7). In that study, we concluded that 1) the
neonatal piglet cannot synthesize sufficient arginine to maintain the
urea cycle (and dispose of ammonia); 2) the piglet cannot
synthesize sufficient proline to maintain plasma concentrations; and
3) arginine synthesis from proline is dependent on gut
metabolism, which is diminished as a result of gut atrophy during
parenteral feeding. Although these observations were convincing, they
were indirect.
In another study (5), we demonstrated that the free pools
of the P-5-C amino acids (arginine, ornithine, citrulline, proline, glutamate, and glutamine) were dramatically altered when identical diets were fed to piglets via the stomach, central vein, or portal vein. In particular, we observed dramatic changes in the ornithine pools in liver, small intestinal mucosa, kidney, and plasma. We hypothesized that gut atrophy due to parenteral feeding caused lower
nitrogen retention because of lowered conditionally indispensable amino
acid (i.e., arginine and proline) synthesis by the atrophied gut
(4). Taken together, these results led to the present
multi-isotope experiment, which was designed to demonstrate that
proline and arginine interconversion via ornithine is dependent on
first-pass metabolism by the gastrointestinal tract.
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METHODS |
Animals and surgical procedures.
Intact male Yorkshire piglets were obtained from the University of
Alberta's swine unit at 1-3 days of age and 1.4-1.8 kg body
wt. Piglets were removed from the sow and transported to the
laboratory, where the piglets immediately underwent surgery to implant
catheters. All procedures used in this study had been approved by the
Animal Care Committee of the University of Alberta.
By use of a method modified from Wykes et al. (26) and
Rombeau et al. (16), custom-made Silastic catheters
(Ed-Art, Don Mills, ON, Canada) were installed using aseptic
techniques. For all piglets, feeding catheters were installed in the
stomach by Stamm gastrostomy with a Silastic tube (16).
Tracer infusion catheters were implanted in the umbilical vein for
intraportally infused (IP) piglets; the stomach catheter was used for
both diet and tracer infusion in intragastrically infused (IG) piglets. All pigs were fitted with a femoral vein catheter for blood sampling. The umbilical catheter was introduced transperitoneally into the umbilical vein and advanced to the portal-hepatic junction. The femoral
catheter was introduced into the left femoral vein and advanced to the
inferior vena cava caudal to the heart. All catheters were tunneled
under the skin from the point of exit on the left side of the chest to
the points of entry into the blood vessels or stomach. Piglets were
housed individually in circular metabolic cages that allowed visual and
audio contact with other piglets. Toys and blankets were provided for
environmental enrichment. The room was lighted from 0800 to 2000 and
was maintained at 28°C with supplemental heat provided by heat lamps.
Piglets and cages were cleaned daily.
An elemental and complete diet (3) was fed via the gastric
catheter continuously for 6 days after surgery. Diets were premixed and
administered through a tether-swivel system (Alice King Chatham Medical
Arts, Los Angeles, CA) with pressure-sensitive infusion pumps. The
infusion regimen was designed to supply all nutrients required by
piglets (26); targeted intakes were 15 g amino
acids · kg
1 · day
1
and 1.1 MJ metabolizable
energy · kg
1 · day
1,
with glucose and lipid (Intralipid 20%, Fresenius Kabi, Uppsala, Sweden) each supplying 50% of nonprotein energy. The amino acid pattern was similar to that of a commercial total parenteral nutrition (TPN) solution, which is based on human milk protein (Vaminolact, Fresenius Kabi) and consisted of (mg/g total L-amino acids)
105 alanine, 60 arginine, 60 aspartic acid, 14 cysteine, 104 glutamic acid, 31 glycine, 30 histidine, 45 isoleucine, 103 leucine, 102 lysine,
19 methionine, 40 phenylalanine, 82 proline, 55 serine, 4 taurine, 52 threonine, 21 tryptophan, 21 tyrosine (supplied as the soluble
dipeptide glycyl-L-tyrosine), and 52 valine. The sterile
TPN solutions were manufactured in our laboratory, as previously
described (26). TPN was stored in the dark at 4°C until
used; immediately before usage, vitamins (MVI Paediatric, Rhone-Poulenc
Rorer Canada, Montreal, PQ) and minerals were added.
After surgery, all piglets received intravenously infused diet at 50%
targeted intake until the following morning to prevent dehydration and
provide nutrients. Pigs then received diet via the gastric catheter at
50% of the full rate for 12 h, at 75% of the full rate for
12 h, and then at full rate (324 ml · kg
1 · day
1)
for 4.5 days; distilled water was simultaneously infused so that total
diet volume infused was ~324
ml · kg
1 · day
1
throughout adaptation. Piglets were weighed each morning, and infusion
rates were adjusted accordingly.
Constant tracer infusions.
Ten piglets were blocked by body weight between the two treatments: IP-
or IG-infused piglets. On the morning of day 5, arginine kinetics were determined by a primed [111 kBq (3 µCi)/kg], constant infusion [185 kBq (5 µCi) · kg
1 · h
1]
of L-[guanido-14C]arginine. The urea product
pool was also primed [463 kBq (12.5 µCi)/kg] with
[14C]urea via the femoral catheter (n = 4); as a result of preliminary evidence of overpriming, this prime was
reduced to 370 kBq (10 µCi)/kg for the remaining pigs
(n = 6). The constant infusion continued for 6 h,
with blood sampled every 30 min. On the morning of day 6,
ornithine kinetics were determined by a primed [481 kBq (13 µCi)/kg], constant infusion [370 kBq (10 µCi) · kg
1 · h
1]
of L-[U-14C]ornithine. Simultaneously,
proline kinetics were determined by a primed [370 kBq (10 µCi)kg],
constant infusion [370 kBq (10 µCi) · kg
1 · h
1]
of L-[2,3-3H]proline. All priming doses were
determined in a pilot experiment in similar pigs; specific
radioactivity data after bolus isotope infusions were fitted to a
monoexponential function, and turnover time (i.e., prime-to-constant
ratio) was calculated (21).
To collect 14CO2 during
L-[U-14C]ornithine infusions, piglets were
placed in a covered Plexiglas box (60 × 40 × 40 cm) and
acclimatized for 30 min. Air was drawn through the box at 25 l/min
through three gas-washing bottles in series containing a total of 400 ml of CO2 absorber composed of ethanolamine and
2-methoxyethanol (Fisher Scientific, Nepean, ON) (1:2, vol/vol). After
an initial 45-min collection, fresh absorber was exchanged every 30 min, volumes in each bottle were recorded, and aliquots were collected for scintillation counting. The constant infusions continued for 6.5 h, with blood sampling and 14CO2
collection conducted every 30 min.
Analytical procedures.
Plasma amino acids and the specific radioactivity (SRA) of plasma
glutamate, hydroxyproline, glutamine, citrulline, arginine, proline,
and ornithine were measured by reverse-phase HPLC using phenylisothiocyanate derivatives as previously described
(10). A 400-µl plasma sample was mixed with two internal
standards: norleucine and
L-[U-14C]phenylalanine (NEN Life Science
Products, Boston, MA). Radioactive derivatives were collected
postcolumn in 1-ml fractions; 5 ml of scintillant were added, and
fractions were counted on a liquid scintillation counter by use of the
dual-isotope counting protocol for 3H and 14C.
Plasma urea concentrations were determined using a spectrophotometric assay kit (Sigma Chemical, St. Louis, MO); radioactivity associated with urea was determined by collecting the underivatized urea fraction
by HPLC. The rate of 14CO2 expiration was
determined for each collection period by quantifying the total
radioactivity collected in the 14CO2 absorber
with liquid scintillation counting of a 1-ml aliquot of absorber mixed
with 5 ml of scintillant (Atomlight; Dupont Canada, Mississauga, ON).
Calculations.
The following calculations were used for both plasma amino acids and
urea. SRA was calculated as
SRA for plasma amino acids for each time point during the
infusion study were plotted. Plateau values were calculated as the
means of the plasma SRA at the time points within the plateau. Plateaus
were visually determined and then verified using regression analysis by
determining that the slope was not different from zero. All plateaus
included
4 time points.
Fluxes of infused amino acids (arginine, ornithine, and proline)
were calculated as
The first-pass small intestinal "extraction" was determined
from the fluxes during IP and IG tracer infusions for the respective amino acids (calculated for each amino acid in each pig). This method
is similar to that used previously for determination of "splanchnic
disappearance," which used IG and intravenous tracer infusions
(2)
The rate of 14CO2 expiration
(
14CO2,
Bq · kg
1 · h
1)
was plotted for each collection period, and plateaus were determined as
for plasma SRA. The isotopic steady state was determined, and the
14CO2 was corrected for bicarbonate
retention (0.93) as described by Wykes et al. (26). The
percentage of [14C]ornithine dose oxidized was determined
by
To compare the role of first-pass small intestinal metabolism on
net amino acid interconversions, we used SRA ratios to represent "fractional net conversion": fractional net conversion (% of
product flux) = (SRAplasma product at
plateau/SRAplasma precursor) × 100. The fractional
net conversion of arginine to ornithine was equivalent to that of
arginine to urea, because equimolar amounts of ornithine and urea are
produced from arginine breakdown. Similar to the approach of Beaumier
et al. (2), molar rates of conversion of plasma labeled
amino acid precursor to labeled product (
precursor to
product) were calculated for those conversions where product
flux was determined (i.e., proline, ornithine, arginine).
precursor to product calculations used mean IP
flux data, which reflect a more accurate estimate of flux, because the
isotope is infused into the sampled pool (2)
To determine the proportion of precursor flux converted to
product,
precursor to product was divided
by fluxamino acid precursor (Table 3). Molar
conversion of proline to arginine (
proline to
arginine) is calculated from the molar conversion of proline to
ornithine multiplied by the proportion of ornithine flux converted to
arginine (and similarly for the flux of arginine to proline)
Statistical analyses.
IG and IP treatments (means ± SD) were compared using a paired
sample t-test (two-tailed) (version 6.02, Corel Quattro Pro, Ottawa, ON, Canada) and were considered significantly different if
P < 0.05.
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RESULTS |
Piglet performance.
The piglets remained healthy and were interested in the environment
during the course of all protocols. The weight upon arrival (1.57 kg,
pooled SD: 0.15), final weight (2.52 kg, pooled SD: 0.22), and rate of
body weight gain (178 g/day, pooled SD: 25) was not significantly
different between IG and IP treatments.
Fate of constant radiotracer infusions.
In one of the pigs in the IP infusion group, the femoral sampling
catheter was not patent during the [guanido-14C]arginine
infusion protocol, and blood could not be collected; thus the IP group
consists of four pigs for the arginine infusion data. However, the
catheter was patent during the [14C]ornithine and
[3H]proline infusion protocol, and these data included
five pigs for both groups.
It is important to note that the validity of all kinetic equations
employed was verified by plasma amino acid concentrations that were not
different between IG and IP groups during any of the infusion protocols
(P > 0.05) and that were not different within pigs
over time during infusion protocols (P > 0.05).
SRA plateaus for each infusate were reached before 3.5 h in all IG pigs for [14C]ornithine (mean: 2.2 h),
[3H]proline (mean: 2.5 h),
[guanido-14C]arginine (mean: 2.2 h), and
[14C]urea (mean 2.1 h). In all IP pigs, SRA
plateaus were reached before 3 h for [14C]ornithine
(mean: 1.5 h), [3H]proline (mean: 1.4 h),
[guanido-14C]arginine (mean: 2.0 h), and
[14C]urea (mean 3.1 h). Typical SRA curves for each
of the infused tracers are presented in Fig.
1 (A: ornithine and proline,
B: arginine and urea). Plateaus for the SRA of all
[14C]ornithine, [3H]proline, and
[guanido-14C]arginine products (including infusate and
breath 14CO2) included
4 time points and are
summarized in Table 1. Coefficients of
variation (CV) for the plateaus of each product were determined within
pigs and averaged for each group (Table 1). The percentage of ornithine
oxidized and fractional net conversions to products during
[14C]ornithine infusion are presented in Table
2. A greater percentage of the flux was
oxidized to CO2 during IP infusion (P = 0.006). Fractional net conversions of ornithine to citrulline,
arginine, proline, or hydroxyproline were all lower when
[14C]ornithine was infused IP (P < 0.02). Fractional net conversions of ornithine to urea
(P = 0.22), glutamate (P = 0.14), or
glutamine (P = 0.08) were not significantly different
between infusion routes. The fractional net conversions to products for
[3H]proline and [guanido-14C]arginine are
presented in Table 2. Only the fractional net conversion of proline to
ornithine was significantly lower during IP [3H]proline
infusion (P < 0.01); these conversion data (1 ± 1) were not different from zero. Fractional net conversions of proline to glutamate or hydroxyproline were not significantly different between
infusion routes (P > 0.05); conversions to other
products (i.e., arginine, citrulline, and glutamine) were not
calculable due to negligible radioactivity in plasma. Fractional net
conversion of arginine to urea was not different between infusion
routes (P > 0.05).

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Fig. 1.
Typical specific radioactivity (SRA) plateau curves in
intragastrically fed piglets after (A) primed, constant
intragastric infusions of [14C]ornithine and
[3H]proline or (B) a primed, constant
intragastric infusion of [guanido-14C]arginine, including
an overpriming intravenous dose of [14C]urea;
[14C]urea overprime was resolved in remaining pigs
by reducing bolus prime dose by 20% (not shown).
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Table 1.
Specific radioactivity at plateau and CV of average plateau for labeled
products from intragastric or intraportal infusions of
[14C]ornithine, [3H]proline, and
[guanido-14C]arginine in intragastrically fed piglets
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Table 2.
Fractional net conversions to products and ornithine oxidation from
intragastric or intraportal infusion of [14C]ornithine,
[3H]proline and [guanido-14C]arginine in
intragastrically fed piglets
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Fluxes during IP infusion were significantly lower than those during IG
infusion for ornithine (P = 0.007), proline
(P = 0.005), and arginine (P < 0.002)
(Table 3). Plasma ornithine fluxes were lower than those for arginine and proline, which was not surprising given that arginine and proline have inputs from both the diet and
protein breakdown, unlike ornithine. The small intestinal extractions
for ornithine and arginine (51 and 40% of dose, respectively) were
greater than the extraction for proline (14% of dose; Table 3).
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Table 3.
Small intestinal first-pass extraction of ornithine, proline, and
arginine from intragastric and intraportal infusions of
[14C]ornithine, [3H]proline, and
[guanido-14C]arginine in intragastrically fed
piglets
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DISCUSSION |
In our previous study, the IP infusion of diet was developed as a
new and unique way to isolate small intestinal metabolism by
maintaining hepatic first-pass metabolism (4). It is
important to note that the plexus of anastomotic channels, which
function as a ductus venosus in the fetal piglet, closes before birth
(8), so that portal infusions via the umbilical vein are
completely metabolized by the liver on first pass. To our knowledge,
this method of in vivo separation of small intestinal metabolism has not previously been used to study amino acid kinetics. Thus, in the
present study, we infused amino acid tracers into the portal vein or
stomach, thus separating the respective absence or presence of
intestinal first-pass metabolism. Unlike previous studies in which
"splanchnic metabolism" was estimated by tracer infusion into a
central vein rather than the stomach (2, 14), the present
experiment reduced splanchnic metabolism to "gut
metabolism," thereby excluding the substantial effects of hepatic
metabolism. This more focused model is critical in establishing the
role of the small intestine in P-5-C amino acid metabolism, because
very different metabolic activities occur in the small intestine and the liver (11, 13). Therefore, using an in vivo model in
intragastrically fed piglets, we have elucidated the role of small
intestinal first-pass metabolism on P-5-C amino acid kinetics,
including the net interconversions among proline, ornithine, and arginine.
This study has clearly demonstrated that small intestinal metabolism is
necessary for the net conversion of proline to arginine. Indeed, no net
conversion was evident when gut first-pass metabolism was bypassed,
whereas 16% of arginine flux [42% (proline to ornithine) × 39%
(ornithine to arginine), Table 2] or 48 µmol
proline · kg
1 · h
1
(Table 4) were converted when gut
first-pass metabolism was maintained. The net conversion of proline to
arginine appears to occur during first-pass metabolism by the gut,
because our models do not preclude the arterial extraction by the gut
of precursors (i.e., proline) for the synthesis of arginine. Neonatal
mammals are in a continuously fed state due to suckling, and so their dependence on first-pass metabolism of dietary precursors by the gut
would not be a limiting scenario for the synthesis of conditionally indispensable amino acids.
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Table 4.
Rates of conversion (Qprecursor to product) of proline,
ornithine and arginine from intragastric or intraportal infusions
of [14C]ornithine, [3H]proline and
[guanido-14C]arginine in intragastrically fed
piglets
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Proline synthesis from arginine was also dependent on small intestinal
metabolism, as indicated by the fractional net conversion differences
for the pathway from ornithine to proline (Table 2). The fractional net
conversion of arginine to ornithine (via arginase; EC 3.5.3.1),
measured as arginine to urea as in Table 2, was not different between
IP and IG infusions, probably because arginase activity is negligible
in the neonatal piglet small intestine (6, 22) and is
exceedingly abundant in the neonatal liver (12). However,
the conversion of ornithine to proline appeared to be localized to the
gut, because a 75% lower fractional net conversion was observed during
IP infusion (Table 2). Indeed, the molar rate of conversion to proline
was 41.5 µmol
arginine · kg
1 · h
1
in IG infused pigs, which was reduced to 8.5 µmol
arginine · kg
1 · h
1
when gut first-pass metabolism was bypassed (Table 4). The importance of proline in the rapidly growing piglet was demonstrated by the fractional net conversions to hydroxyproline. Although proline net
conversion to hydroxyproline was not affected by route of isotope
infusion, the finding that hydroxyproline SRA reached plateau rapidly
during either [3H]proline or [14C]ornithine
infusions indicates a very rapid turnover of collagen in the neonatal
piglet. Because hydroxyproline synthesis is posttranslational and
irreversible, this rapid collagen turnover must be a considerable proline "sink" in the neonatal pig; indeed, proline has been shown to be indispensable in the piglet (1). Furthermore, this
proline loss may be extreme in situations that lead to increased
collagen turnover, such as during wound or burn healing; such
situations would increase proline requirements and should be quantified.
Several data in this study strongly support the conclusion that the
synthesis of arginine from proline is more dependent on first-pass
metabolism than is the synthesis of proline from arginine. In
particular, the contribution of the gut to the fractional net conversion of ornithine to proline (75%, Table 2) was not as profound
as that of proline to ornithine (100%, Table 2). In addition, the
overall molar flux from proline to arginine (48.1 µmol
proline · kg
1 · h
1,
Table 4) was entirely dependent on the gut, whereas the
arginine-to-proline molar conversion attributable to the gut amounted
to 33.0 µmol arginine · kg
1 · h
1.
More revealing are the arginine balance data (to and from ornithine), which indicate a large net synthesis of arginine (i.e., 40.9 µmol · kg
1 · h
1)
when gut metabolism is included, and a large net catabolism of arginine
(i.e.,
29.1
µmol · kg
1 · h
1)
when gut metabolism is bypassed. These data suggest a net synthesis of
70.0 µmol
arginine · kg
1 · h
1
from ornithine during first-pass gut metabolism and a net degradation of arginine by the rest of the body. These data are in contrast to
those for proline balance, when equal amounts of proline were synthesized from ornithine regardless of gut metabolism; indeed, the
large degradation of ornithine by the gut (70.8 µmol · kg
1 · h
1)
was entirely accounted for by the higher synthesis of arginine during
gut metabolism. Despite the greater dependence of arginine synthesis on
gut metabolism, it is obvious from the combined data that proline
synthesis also relies on gut metabolism and that ornithine is central
to these conversions.
One of the goals of our multiple tracer infusion protocol was to
identify the limiting step in the pathway that results in arginine
synthesis being dependent on gut metabolism. As shown in Fig.
2, P-5-C is the central metabolite for
the interconversions among ornithine, proline, and glutamate. The
pathway from ornithine to proline involves the two enzymes, ornithine
aminotransferase (OAT, ornithine to P-5-C) and P-5-C reductase (EC
1.5.1.2, P-5-C to proline); the reverse pathway from proline to
ornithine involves proline dehydrogenase (EC 1.5.99.8, proline to
P-5-C) and OAT (P-5-C to ornithine). The limiting step in the pathway
for proline synthesis from arginine was the net conversion from
ornithine to proline (4.5 vs. 18%). Furthermore, the limiting step for
arginine synthesis from proline was the net conversion of proline to
ornithine (0 vs. 42%). The interconversion between ornithine and
proline appears to be the pathway that is localized to the small
intestine. Therefore, the gut-localized enzymes are either OAT or P-5-C
reductase and proline dehydrogenase. Proline dehydrogenase is also the
first enzyme in the pathway from proline to glutamate; however, despite the relatively high concentrations of proline dehydrogenase in the
neonatal pig small intestine (17, 24), this net conversion was not dependent on the gut, because IP and IG infusions gave similar
rates (22 vs. 18%, respectively, P = 0.20). However,
OAT is also involved in the conversion from ornithine to glutamate, and
this net conversion was lower when gut metabolism was bypassed (4.2 vs.
6.7%, P = 0.04). Consequently, we conclude that, in
the neonatal pig, OAT must be predominantly localized to the small intestine, and this localization therefore explains the negligible net
interconversion between arginine and proline when gut metabolism is
bypassed.

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Fig. 2.
Pathways demonstrating the localization of ornithine
aminotransferase to the gut. Fractional net conversions from
[3H]proline to [3H]ornithine and
[3H]glutamate (solid arrows and boxes) and from
[14C]ornithine to [14C]proline and
[14C]glutamate (dashed arrows and boxes) with respective
enzymes for each conversion pathway. P5C, pyrroline-5-carboxylate. Data
(mean ± SD) were from intragastric (IG) or intraportal (IP)
infusions of [3H]proline and [14C]ornithine
in intragastrically fed piglets. Underlined values were significantly
greater (P < 0.05).
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It is important to note that this extrapolation of plasma amino acid
kinetic data to intracellular enzyme activity assumes that
equilibration is achieved among the various pools. Although true enzyme
activity cannot be determined from such data, the net in vivo
consequence of these activities can be inferred, because all amino acid
precursors and products reached kinetic equilibrium in the plasma.
Nevertheless, the present data add to previous studies by measuring the
net interconversions among the amino acids associated with OAT activity
with and without small intestinal first-pass metabolism. Indeed,
although all of the enzymes involved in arginine and proline
interconversion have been shown to be present in the small intestine of
neonatal pigs (17, 22), OAT activity is present
predominantly in the small intestine (24). The critical
role of intestinal OAT for arginine synthesis has also been
demonstrated in mice and humans with intestinal OAT deficiency; this
deficiency results in hypoornithinemia, hypocitrullinemia, hypoargininemia, hyperammonemia, and even death during the neonatal period (20). Furthermore, gabaculine (an inhibitor of OAT)
results in decreased plasma concentrations of ornithine, citrulline,
and arginine by 59, 52, and 76%, respectively, in 4-day-old suckling piglets (9). The present interpretation of plasma kinetic
data, suggesting the localization of enzyme activity to the gut, is supported by these other studies and provides important in vivo verification of in vitro evidence.
The relative differences in fractional net conversions when the gut is
or is not bypassed clearly indicate that interconversions among
arginine, ornithine, and proline are dependent on first-pass gut
metabolism. The quantitative importance of these conversions on whole
body metabolism can be extrapolated from the flux data. On first pass,
the small intestine extracted 40% of dietary arginine and 15% of
dietary proline; although ornithine was not provided in the diet, 51%
of the oral dose was also extracted on first pass (Table 3). The fate
of extracted arginine and proline within the small intestine is
oxidation, protein synthesis, and conversion to other metabolites. For
extracted ornithine, which is not a component of protein, its fate is
oxidation and conversion. Ornithine oxidation was significantly higher
during IP infusion, suggesting that ornithine oxidation occurs
primarily in the liver. However, during IG infusion of ornithine
isotope, 49% of the ornithine dose reaches the liver (i.e., 51%
extraction), and 35.7% of that dose is oxidized (i.e., IP infusion
oxidation rate). This calculation predicts an ornithine oxidation rate
of 17.5% during IG infusion, which is lower than the rate observed
(i.e., 25.1%). Therefore, ~7.6% of the ornithine dose must be
oxidized within the small intestine. Of the ornithine extracted by the
small intestine, ~15% was oxidized, and the remaining 85% must have
been converted. The flux data also indicate that 40% of dietary
arginine was extracted by the gut on first pass. Because
arginine-to-ornithine conversion was not dependent on the gut,
intestinal arginine oxidation via ornithine was negligible. Therefore,
the majority of this extracted arginine must have been either
converted or incorporated into protein. Stoll et al. (18)
estimated that threonine was incorporated into piglet mucosa at a rate
of 2.2 µmol · kg
1 · h
1.
With a relative proportion of 3.4% of protein, this threonine incorporation involves ~4.4 µmol
arginine · kg
1 · h
1
and 5.3 µmol
proline · kg
1 · h
1,
with the assumption that piglet protein includes 6.8% arginine and
8.2% proline (25). Thus protein synthesis accounts for
6% of the 78 µmol
arginine · kg
1 · h
1
and 9% of the 57 µmol
proline · kg
1 · h
1
extracted by the gut on first pass. Therefore, the primary fate of the
substantial amounts of arginine, proline, and ornithine extracted by
the gut is conversion to other amino acids, as well as to other minor
products such as nitric oxide, polyamines, and creatine.
In combination with the fractional conversion differences between
routes of infusion, we conclude that interconversion among the P-5-C
amino acids is dependent on first-pass metabolism by the piglet small
intestine and amounts to a significant proportion of whole body fluxes
of these amino acids. Indeed, of the 57 µmol proline · kg
1 · h
1
extracted by the gut from the diet, an estimated 84% (i.e., 48.1 µmol · kg
1 · h
1,
Table 4) is converted to arginine; similarly, ~42% (i.e., 33.0 µmol · kg
1 · h
1,
Table 4) of the extracted 78 µmol
arginine · kg
1 · h
1
is converted to proline. In total, the gut releases 48.1 µmol of
synthesized arginine and 117 µmol · kg
1 · h
1
of dietary arginine, which is in excess of the recommended arginine requirement for piglets of 96 µmol · kg
1 · h
1
(i.e., 2.3 mmol · kg
1 · day
1)
(15). As a comparison, the gut releases 33 and 348 µmol · kg
1 · h
1
of synthesized and dietary proline, respectively, compared with an
estimated requirement of 329 µmol · kg
1 · h
1
(i.e., 7.9 mmol · kg
1 · day
1)
(1). In other words, the gut synthesizes 50% of the
arginine required by the piglet but only 10% of the proline
requirement. Therefore, the de novo synthesis of arginine from proline
is not only more dependent on the gut, it is also more important in
relation to whole body supply and requirement.
Because ornithine is not a component of protein or the diet, the only
input to ornithine flux is de novo synthesis. However, there appears to
be a substantial amount of ornithine synthesis (i.e., flux) that is not
accounted for by molar inputs from arginine and proline (i.e., 149 µmol · kg
1 · h
1
in IG and 76 µmol · kg
1 · h
1
in IP). It is possible that some ornithine is synthesized from glutamate and glutamine via P-5-C synthase and OAT. However, in vitro
data suggest that P-5-C synthase activity is negligible in intestinal
tissues (14, 22) and thus probably does not account for
the 55% of ornithine synthesis that is missing. Alternatively, it is
probable that ornithine plasma flux estimates do not reflect all
aspects of ornithine metabolism (2) and can be
overestimated if corrections for recycling are not employed
(19). For example, the conversion of ornithine to
citrulline was over 100% of citrulline flux (Table 2), which suggests
either tracer recycling, possibly via nitric oxide synthase, which
bypasses ornithine, or more likely, preferential conversion to
citrulline during first-pass intestinal metabolism (2).
These data suggest that absolute flux rates and associated stochastic
calculations need to be used with caution, given the inappropriateness
of an imposed single-pool model to complicated amino acid metabolism.
In the present study, the relative conversions, which are independent
of flux data, provide the primary basis for our conclusions.
In conclusion, the data in this study support our previous findings
that arginine and proline are coindispensable in gastrically fed
piglets (7). In situations in which first-pass gut
metabolism is bypassed or compromised (i.e., TPN, gut resection,
intestinal disease, or weaning-induced gut stress), arginine and
proline are individually indispensable because their biosyntheses are negligible. Therefore, the requirements for arginine and proline need
to be established for parenterally fed or gut-stressed neonates. Because the net synthesis of arginine from proline was more dependent on small intestinal metabolism than the reverse net conversion, we
speculate that the increase in arginine requirement in such neonates
will be greater than the increase in proline requirement. There is a
need to quantify the synthetic capacity of the piglet as well as the
minimum dietary requirement. Because of their coindispensability, the
arginine and proline requirements need to be determined together; then
the maximum ratio of arginine to proline (and vice versa) should be
determined, similar to the way requirements are expressed for
phenylalanine/tyrosine or methionine/cysteine. Because of the role of
the gut in arginine and proline metabolism, in situations of gut stress
or bypass (i.e., parenteral nutrition), the arginine and proline
"requirement ratio" will be very different from that in healthy
orally fed neonates and should be determined separately.
 |
ACKNOWLEDGEMENTS |
The amino acids were generously donated by Biokyowa/Nutriquest, St.
Louis, MO.
 |
FOOTNOTES |
This study was supported by grants from the Natural Sciences and
Engineering Research Council of Canada, the Alberta Pork Producers
Development Corporation, and the Alberta Agricultural Research Institute.
Present address for R. F. P. Bertolo and J. A. Brunton:
Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada A1B 3X9.
Address for reprint requests and other correspondence:
R. O. Ball, Dept. of Agricultural, Food, and Nutritional
Science, Univ. of Alberta, Edmonton, AB, Canada T6G 2P5 (E-mail:
rball{at}afns.ualberta.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 14, 2003;10.1152/ajpendo.00269.2002
Received 19 June 2002; accepted in final form 7 January 2003.
 |
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