Proline ameliorates arginine deficiency during enteral but not parenteral feeding in neonatal piglets

Janet A. Brunton1, Robert F. P. Bertolo2, Paul B. Pencharz1,2,3,4,5, and Ronald O. Ball1,2,3,5

1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5; 2 Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; and 3 The Research Institute, The Hospital for Sick Children, and Departments of 4 Paediatrics and 5 Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

The indispensability of arginine has not been conclusively established in newborns. Because parenteral feeding bypasses the gut (where de novo synthesis of arginine occurs from proline), a dietary supply of arginine that is sufficient to maintain urea cycle function may be of greater importance during intravenous compared with enteral feeding. Two-day-old piglets (n = 12) were fed nutritionally complete diets for 5 days via either a central vein catheter (IV pigs, n = 6) or a gastric catheter (IG pigs, n = 6). Subsequently, each piglet received three incomplete test diets [arginine free (-ARG/+PRO), proline free (-PRO/+ARG), or arginine and proline free (-ARG/-PRO)] in a randomized crossover design. Plasma ammonia was assayed every 30 min for 8 h or until hyperammonemia was observed. Ammonia increased rapidly in IV pigs receiving -ARG/+PRO and -ARG/-PRO (84 ± 36 and 74 ± 37 µmol · l-1 · h-1, respectively), requiring early diet cessation. A rapid increase was also exhibited by IG pigs receiving the -ARG/-PRO, but not the -ARG/+PRO diet (31 ± 15 vs. 11 ± 7 µmol · l-1 · h-1, respectively, P < 0.05). Plasma arginine and proline were indicative of deficiency (IG and IV groups) when deplete diets were infused. Arginine is indispensable in parenteral and enteral nutrition, independent of dietary proline.

hyperammonemia; total parenteral nutrition; newborn; urea cycle


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN VIVO EVIDENCE of net arginine release from the small intestine of young piglets (22, 27) and in vitro evidence from harvested enterocytes (5, 28) have demonstrated that the gut is an important site of endogenous arginine synthesis. Conditions of gut atrophy or dysfunction, such as malnutrition, intestinal disease, or parenteral feeding, will result in a lack of luminally derived metabolic precursors of arginine that could impair the biosynthetic capabilities of the neonate. This deficit in arginine synthesis must be met by higher dietary intake, but the quantity required is not known.

Endogenous arginine synthesis may be very important to the newborn, because arginine intake from milk is low relative to requirements for growth (26). The pathway for de novo synthesis of both arginine and proline is from glutamate via the intermediate conversion to L-Delta 1-pyrroline-5-carboxylate (P-5-C) by P-5-C synthase. Subsequently, P-5-C is converted into either proline by P-5-C reductase (EC 1.5.1.2) or ornithine by ornithine aminotransferase (OAT; EC 2.6.1.13), and then from ornithine to citrulline and arginine in the urea cycle. In the young animal, the conversion of glutamate to P-5-C is believed to be limited to the small intestine and thymus because P-5-C synthase is found exclusively in these tissues (13). Also, low P-5-C synthase activity during the suckling period (28) dictates that an alternate precursor for arginine synthesis must be responsible for meeting requirements.

Proline is one of the most abundant amino acids in porcine and human milk (7). Proline oxidase (no EC number assigned), responsible for the conversion of proline to P-5-C, and OAT have been identified in intestinal tissue and cultured enterocytes from suckling pigs (26). Conversion of gastrically infused [14C]proline to [14C]arginine has been reported in 10-day-old piglets fed a proline-deficient diet (16). Additional in vivo evidence is necessary to determine whether dietary proline contributes to whole body arginine requirements such that it could ameliorate dietary arginine deficiency.

A critical biological role of arginine is the disposal of ammonia via urea synthesis. Hyperammonemia was observed in term and preterm neonates fed early total parenteral nutrition (TPN) solutions with low (or zero) arginine concentrations (11, 20). The hyperammonemia was resolved by an infusion of L-arginine in most infants (11, 19), suggesting that arginine is indispensable. Preterm infants frequently demonstrate elevated plasma ammonia concentrations, perhaps due to immature hepatic amino acid metabolism or a subclinical arginine deficiency (2). As the precursor for nitric oxide, inadequate arginine availability has been implicated in the onset of, or delayed recovery from, neonatal diseases such as sepsis (23), persistent pulmonary hypertension (6, 24), and necrotizing enterocolitis (9). Improvement in our understanding of arginine metabolism during the neonatal period and of the enteral and parenteral dietary arginine requirements is critical for the optimal care of this compromised population.

We conducted a study in two groups of newborn piglets, which were either 1) subjected to gut atrophy induced by parenteral feeding (using a previously established protocol) (3, 30) or 2) enterally fed via gastrostomy. The primary objectives were 1) to evaluate the indispensability of arginine during parenteral and enteral feeding, 2) to determine the impact of an atrophied gut on de novo arginine synthesis by feeding identical arginine-free diets parenterally or enterally, 3) to determine whether proline is a major precursor for arginine in piglets fed enterally or parenterally, and 4) to determine whether proline is indispensable in piglets fed enterally or parenterally.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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Animals and surgical procedures. Twelve intact male Yorkshire piglets were obtained from a specific pathogen-free herd (Arkell Swine Research Station, University of Guelph, Guelph, ON, Canada). Piglets were left with the sow for 2-4 days after birth and then were transferred to the laboratory for immediate surgical implantation of catheters under aseptic conditions. Anesthesia was induced with an intramuscular injection of acepromazine (0.5 mg/kg, Atravet; Ayerst Laboratories, Montreal, PQ, Canada) and ketamine hydrochloride (10 mg/kg, Rogarsetic; Rogar STB, Montreal, PQ, Canada) and was maintained during surgery with a mixture of oxygen and halothane (Fluothane, Ayerst Laboratories, Montreal, PQ, Canada) delivered by mask. Each piglet was implanted with a Silastic venous sampling catheter (Ed-Art, Toronto, ON, Canada), which was inserted into the left femoral vein and advanced to the inferior vena cava immediately caudal to the heart. Piglets in the intravenously fed (IV) group (n = 6) also received an infusion catheter that was inserted into the left external jugular vein and advanced to the superior vena cava immediately cranial to the heart. In the intragastrically fed (IG) group (n = 6), a Stamm gastrostomy was performed (16) to provide continuous enteral feeding.

After surgery, piglets were treated immediately with 0.15 mg/kg oxymorphone analgesic (Numorphone, Dupont Canada, Mississauga, ON, Canada) and 3.5 mg gentamicin sulfate (Garasol, Schering Canada, Pointe Claire, PQ, Canada) followed by 2.5 mg/day gentamicin sulfate for the next 3 days. Piglets were housed in individual circular metabolic cages that allowed visual and aural contact with other piglets; toys and blankets were also provided. The room was lit from 0800 to 2000 and was maintained at 28°C with supplemental heat provided by heat lamps. All piglets were weighed daily on an electronic balance (Sartorius, Germany), and sampling catheters were flushed with sterile heparinized saline to maintain patency. Piglets and cages were cleaned daily. All procedures were in accordance with the Canadian Council on Animal Care's Guide to the Care and Use of Animals (1993) and were approved by the local animal care committee.

Diets. The diets were based on an elemental parenteral TPN solution and were manufactured by the Parenteral Service Pharmacy (The Hospital for Sick Children, Toronto, ON, Canada) to the investigators' specifications (12). The TPN base solution was free of arginine, proline, and serine. For a complete amino acid diet, the three crystalline amino acids were dissolved in water, and the solution was injected into sterile bags of TPN through a 0.22-µm filter to provide final concentrations of 3.36 g arginine/liter, 4.58 g proline/liter, and 3.09 g serine/liter. For the three test diets, arginine (-ARG/+PRO), proline (-PRO/+ARG), or arginine and proline (-ARG/-PRO) were removed; serine was added to ensure that all diets were isonitrogenous. Just before feeding, vitamins (MVI Paediatric, Rhone-Poulenc Rorer, Montreal, PQ, Canada), minerals (Micro +6 Concentrate, Sabex, Boucherville, PQ, Canada), and lipid (Intralipid 20%, Pharmacia, Mississauga, ON, Canada) were added to the TPN solution. The diets were infused continuously (24 h) either intravenously or intragastrically, via pressure-sensitive peristaltic pumps, through a swivel-tether system (Alice King Chatham Medical Arts, Los Angeles, CA). The complete diet was designed to supply all nutrients required by piglets (30), including 15 g of amino acids · kg-1 · day-1 and 1.1 MJ metabolizable energy · kg-1 · day-1.

Postsurgically, piglets in the IV group were immediately infused with complete TPN at one-half of the maximal rate for 12 h, at an increase to 75% for 12 h, and then at full rate. Piglets in the IG group also received intravenous infusions of complete TPN for 12 h at 50% to allow healing of the stomach. Subsequently, the TPN solution was infused intragastrically but was diluted with water at a volume ratio of 1:2 (TPN-water) for the first 12 h, then 1:1 for the next 12 h; at full rate, IG diets included the infusion of water (2:1, TPN-water) to lower the osmolarity. All piglets were maintained on full, nutritionally complete feeding until the morning of day 5, at which time all piglets were fasted for 12 h, followed by 12 h of feeding a complete diet.

Experimental design. Beginning on the morning of day 6, each piglet was subjected to the three dietary treatments on three consecutive days in a randomized, crossover design. Immediately after blood sampling for baseline data (time 0), the test diet was initiated. Venous blood was sampled every 30 min for 9 h, with a final sample taken at 10 h.

A preliminary study to test the response of piglets (n = 2) to arginine-free TPN demonstrated that the onset of hyperammonemia was much more rapid and extreme than anticipated; plasma ammonia concentration exceeded 1,000 µmol/l during an overnight infusion, and symptoms of ammonia toxicity were observed. For this reason, during the main experiment, the test diets were ceased if hyperammonemia was detected in excess of the predetermined critical value (CrV) of 270 µmol/l. This CrV was established in our laboratory by sampling plasma from eight healthy sow-fed piglets; it represents the group mean ± SD (58 ± 32 µmol/l) times 3. Diets were infused for a maximum of 8 h if the CrV was not exceeded.

At diet cessation, all piglets were "rehabilitated" with a bolus of amino acid(s) equal to the deficiency accrued during the test period. The amino acids were infused in a saline solution for 1 h into either the femoral (IV piglets) or gastric (IG piglets) catheters.

Plasma ammonia concentration was determined every 30 min during treatment infusion by a colorimetric assay based on the amination of 2-oxoglutarate to glutamate, with simultaneous oxidation of NADPH (procedure no. 171-UV, Sigma Diagnostics, St. Louis, MO). Whole blood pH, bicarbonate, electrolytes, CO2 and O2 pressures (PCO2, PO2, respectively), and osmolality were determined every 90 min (Nova Statprofile 9+, Nova Biomedical, Waltham, MA). Plasma urea was determined in hourly samples by use of a colorimetric assay in which ammonia was liberated from urea by enzymatic hydrolysis. Subsequently, alpha -ketoglutarate was aminated to glutamate with concurrent oxidation of NADH (procedure no. 66-UV, Sigma Diagnostics). Amino acid concentrations were determined by reverse-phase HPLC. For plasma free amino acids, 100 µl of plasma were mixed with 20 µl of 2.5 mmol/l norleucine (internal standard) and 1 ml of protein precipitant (0.5% trifluoroacetic acid in methanol), vortexed, and centrifuged at 3,000 g for 5 min to remove proteins. Preparation of phenylisothiocyanate derivatives for reverse-phase HPLC was then performed as previously described (4).

Statistics. The slope of the change in plasma ammonia concentration from 2 h after initiation of the test diet until cessation was calculated for each piglet and then compared by analysis of covariance, with diet order and previous treatment included as covariates (BMDP Student Version, BMDP Statistical Software, Los Angeles, CA). Only slopes that were significantly different from zero (P < 0.05) were included. Two hours was chosen as the first point for the regression analysis, because after the treatments were started, plasma ammonia initially declined in all piglets. Changes in plasma urea were similarly compared by use of values from baseline to cessation. Growth performance and ammonia concentration, plasma amino acids, bicarbonate, and pH at diet cessation (8 h or CrV) were compared between IG and IV groups by one-way ANOVA (Minitab Statistical Software, Minitab, State College, PA).


    RESULTS
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INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

The IG- and IV-fed piglets were similar in age and body weight at the time of surgery. Rate of weight gain after adaptation (day 2 to day 5) to the nutritionally complete diet also was not different between IG and IV piglets (Table 1) and was similar to that of sow-fed piglets (30). During the 3-day period (day 6 to day 9) when IG and IV piglets received the same test diets, the IG piglets gained weight at a greater rate than IV piglets (Table 1), indicating that the effect of the deficient diets was more severe in the IV piglets.

                              
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Table 1.   Characteristics and growth performance of piglets

The initiation of the -ARG/+PRO and -ARG/-PRO diets for both the IV and IG piglets resulted in a significant decline in plasma ammonia compared with baseline during the 1st h of treatment (P < 0.05; Fig. 1, A and B). Subsequently, the IV piglets fed -ARG/+PRO or -ARG/-PRO had a rapid rise in plasma ammonia (Table 2), with all but one IV piglet reaching the CrV before 8 h for both treatments (Fig. 1, A and B). Time to CrV and peak plasma ammonia concentrations were not different in IV piglets fed -ARG/+PRO compared with -ARG/-PRO diets (Table 2). A significant difference was observed in the peak ammonia concentrations of IV vs. IG piglets fed either -ARG/+PRO or -ARG/-PRO diets (Table 2); IV-fed piglets demonstrated a more rapid rise and a greater peak concentration at time of CrV or diet cessation (Table 2). During the -PRO/+ARG diet infusions to both IG and IV groups, plasma ammonia declined during the 1st h, with no subsequent significant changes (Fig. 1C).


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Fig. 1.   Plasma ammonia concentrations of individual piglets receiving intravenous (IV) or intragastric (IG) infusions of an arginine-free diet (-ARG/+PRO; A), an arginine- and proline-free diet (-ARG/-PRO; B), or a proline-free diet (-PRO/+ARG; C). Horizontal dotted line, the critical value (CrV) for ammonia (270 µmol/l); cessation of test diet occurred when piglets exceeded this concentration.


                              
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Table 2.   Time from initiation to cessation of treatments and change in plasma ammonia and urea concentrations during IG and IV diet treatments

The inclusion of proline in the IG-fed -ARG/+PRO diet prevented hyperammonemia. None of the piglets attained the CrV during the IG -ARG/+PRO feeding (Fig. 1A). The plasma ammonia concentration at diet cessation for IG piglets was significantly higher after the -ARG/-PRO treatment than after the -ARG/+PRO diet (Table 2). The exclusion of proline (-PRO/+ARG) from the IV and IG diets containing arginine did not result in a rise in plasma ammonia (Table 2).

The bolus infusion of arginine or arginine and proline at cessation of the test diets resulted in an immediate decline in plasma ammonia concentration in hyperammonemic piglets. Most piglets returned to baseline levels within 2 h of being fed a replete diet (Fig. 2).


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Fig. 2.   Plasma ammonia concentrations of individual piglets after cessation of IV and IG test diet infusions, when diets without arginine (A and C) or arginine and proline (B and D) were reinitiated. Horizontal dashed and dotted lines, baseline ammonia concentration before initiation of test diets (means ± SD, respectively).

Plasma urea concentration declined significantly (P < 0.05) during all diet treatments, with no significant difference in rates of decline between IG and IV pigs (Table 2). Blood pH was similar between IV and IG pigs before the initiation of the test diets (all piglets: 7.428, pooled SD: 0.033), and pH did not change during any of the treatments (data not shown). Blood bicarbonate concentration was significantly less in the IV compared with the IG pigs at cessation of the -ARG/+PRO (27.2 vs. 32.2 mmol/l, pooled SD: 3.7, P < 0.05) and -ARG/-PRO (26.3 vs. 33.0 mmol/l, pooled SD: 2.2, P < 0.001) treatments. At diet cessation, the bicarbonate concentration as a percentage of baseline was higher in the IG vs. IV pigs (-ARG/+PRO, 106 vs. 93%, pooled SD: 9%, P < 0.05; -ARG/-PRO, 96 vs. 109%, pooled SD: 10%, P < 0.05).

Plasma concentrations of arginine and ornithine at baseline were significantly higher in the IG compared with the IV piglets (Table 3). During the infusions of -ARG/+PRO and -ARG/-PRO diets, plasma arginine fell to >2 SD below a reference mean (31) within 1 h of initiation in the IV piglets, and within 2 h in the IG piglets; subsequently, arginine remained low in all animals until cessation of treatment (Fig. 3, A and B). The -PRO/+ARG diet induced a fall in plasma proline in both IV and IG piglets, also to levels indicative of a deficiency state (Fig. 3C). At cessation of the -ARG/+PRO and -ARG/-PRO diets, IG piglets had higher concentrations of arginine and ornithine compared with IV piglets (Table 3). IG piglets fed -PRO/+ARG also maintained greater plasma proline and ornithine concentrations than IV piglets receiving identical intakes (Table 3). IV vs. IG feeding resulted in higher plasma concentrations of glutamine, glutamate, and aspartate at the cessation of the -ARG/+PRO and -ARG/-PRO diets (Table 3).

                              
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Table 3.   Plasma amino acid concentrations at initiation (baseline) and at cessation of IG and IV diet treatments



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Fig. 3.   Plasma arginine or proline concentrations of individual piglets receiving IV or IG infusions of an arginine-free diet (-ARG/+PRO; A), an arginine- and proline-free diet (-ARG/-PRO; B), or a proline-free diet (-PRO/+ARG; C). Horizontal solid and dotted lines, mean concentration and 2 SD, respectively, from sow-fed reference data (31).

Mode of feeding also resulted in the IV piglets having significantly greater plasma concentrations of the branched-chain amino acids, histidine, phenylalanine, and lysine in response to the -ARG/+PRO and -ARG/-PRO diets (P < 0.05, data not shown). No differences in branched-chain amino acid concentrations were observed between IG- and IV-fed piglets receiving the -PRO/+ARG diet.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, severe hyperammonemia developed within hours of feeding arginine-free diets to intravenously fed neonatal piglets, clearly demonstrating the indispensability of arginine during parenteral nutrition. In enterally fed piglets, moderate hyperammonemia developed after 8 h of being fed arginine-free diets; combined with lower plasma concentrations of arginine and urea, this indicates that even orally fed piglets could not synthesize sufficient arginine to maintain urea synthesis. By employing identical diets and using plasma ammonia as an indicator of arginine deficiency, we also conclude that the arginine requirement is clearly higher for neonatal piglets fed parenterally vs. enterally.

Parenteral feeding involves a lower metabolic capacity due to gut atrophy and a nutrient supply that is exclusively systemic. Because the neonatal small intestine is an important site for arginine synthesis (5, 22), these factors could explain the present observations that, when both IV and IG groups were provided with identical arginine-free (-ARG/+PRO) diets, the rapid development of hyperammonemia was observed only in intravenously fed piglets. Alternately, there are complex metabolic changes that occur with parenteral (vs. enteral) feeding, such as the hormonal responses evoked by intraluminal nutrients (1), which could cause alterations in arginine synthesis or catabolism. We previously showed no conversion of intravenously infused [14C]proline or [14C]glutamate into ornithine, citrulline, or arginine in piglets fed orally, although these conversion products were detected when the tracers were infused intragastrically (16). Because those piglets were all orally fed and had healthy intestines, our results (16) and those of others (27) have demonstrated that arterial proline and glutamate were minimally available to the small intestine. Thus luminally derived precursors and/or a healthy small intestine may be necessary for de novo arginine synthesis in the neonate. Our current study confirmed that plasma proline was not available to the enterocyte for arginine synthesis, because hyperammonemia occurred equally as quickly in the IV piglets fed -ARG/+PRO and -ARG/-PRO.

Glutamine is also a potential precursor for arginine via the same pathways as glutamate (27). In vitro research suggested that enterocytes require a source of glutamine for arginine synthesis from proline (26). During the catabolism of glutamine, an ammonia group is donated to the intramitochondrial synthesis of carbamoyl phosphate, which is necessary for the conversion of proline-derived ornithine to citrulline in the urea cycle (26). Our diets did not contain glutamine; however, in vivo research shows that glutamine is readily taken up by the enterocyte across the basolateral membrane (22, 25). During the IV infusions of -ARG/+PRO and -ARG/-PRO treatments, plasma glutamine increased at a rate of ~200 µmol · l-1 · h-1 (Table 3). Therefore, glutamine was available as a nitrogen donor and as a potential precursor and was not likely a limiting factor in the synthesis of arginine in IV-fed piglets. Regardless of the mechanisms, during parenteral nutrition, endogenous arginine synthesis from potential precursors cannot meet even the basic arginine requirement for urea synthesis, let alone for growth.

Synthesis and release of significant amounts of arginine by the intestine for use by the liver and periphery appear to occur only in the young animal; mature animals appear to synthesize arginine in the kidney from intestinally derived citrulline that bypasses the liver (8, 27). Low arginase (EC 3.5.3.1) activity has been demonstrated in enterocytes cultured from preweaning pigs (5), which would account for the release of arginine as opposed to ornithine or citrulline. These important differences in arginine metabolism between young and mature animals make it essential that arginine requirements be quantified for the neonate as opposed to being extrapolated from adult requirements.

The intragastrically infused -ARG/+PRO and -ARG/-PRO diets evoked very different metabolic responses, in contrast to the IV infusions. The inclusion of proline in the diet of the IG-fed piglets sustained sufficient urea cycle function to avoid hyperammonemia. The IG piglets fed -ARG/-PRO experienced a rise in plasma ammonia that was rapid compared with the IG -ARG/+PRO treatment. Interestingly, the rise in plasma ammonia during the -ARG/-PRO infusions was not as severe in the IG compared with the IV-fed piglets. Because there was no dietary proline available, it is likely that some conversion of dietary glutamate to arginine was occurring in the healthy gut. Although excess glutamate was provided in all of our diets, our observation of a slow rise in plasma ammonia during the IG -ARG/-PRO diet infusion demonstrates that its use as a precursor for arginine is limited, likely due to low P-5-C synthase activity in the neonate (29). Murphy et al. (16) demonstrated in young pigs fed orally that a greater amount of arginine was synthesized from labeled proline than could be detected from a similar intragastric infusion of labeled glutamate. It is important to note that the increases in plasma ammonia in IG piglets fed arginine-free diets were significant (11 and 31 µmol · l-1 · h-1 for -ARG/+PRO and -ARG/-PRO diets, respectively), although not as rapid as in the IV-fed piglets (Table 2). Even in orally fed pigs, the removal of dietary arginine would eventually have resulted in critical hyperammonemia. Thus proline and arginine should be considered "coindispensable"; however, proline can only ameliorate arginine deficiency during enteral feeding.

The indispensability of arginine was also demonstrated by plasma arginine concentrations, based on levels indicative of a deficiency state during the infusion of either of the arginine-free test diets in both IV and IG piglets (Fig. 3). In IG piglets fed -ARG/+PRO, a portion of the dietary proline must have been converted to arginine to avert hyperammonemia; however, plasma proline concentration did not decline. Therefore, the decline in plasma arginine in the IG piglets was not due to inadequate dietary proline but rather to insufficient de novo synthetic capabilities that could not fulfill whole body arginine requirements. The indispensability of arginine was further demonstrated by the rapid recovery from hyperammonemia with the addition of arginine to the diet. All piglets recovered within 2 h of repletion, as determined by biochemical indexes such as plasma ammonia, bicarbonate, urea, and amino acids.

Plasma proline concentrations during the -PRO/+ARG diets were also indicative of deficiency for both IG- and IV-fed piglets. Proline concentrations were <20% of the baseline values after 8 h of infusion of the proline-free diet. Final plasma proline concentration was twofold greater in the IG- compared with the IV-fed piglets, but both groups were >2 SD below the mean reference value for sow-fed piglets (31). The difference may be attributed to the conversion of intragastrically, but not intravenously, infused glutamate as a precursor for proline (16). Arginine can also act as a precursor for proline synthesis when provided intravenously or enterally. Approximately 40% of the proline deposited in proteins during growth in neonatal pigs adapted to a low-proline diet could have been synthesized from arginine (15). The piglets fed -PRO/+ARG in this study were likely not receiving enough dietary arginine to support adequate proline synthesis. Furthermore, we observed a decline in plasma urea concentration over time in both IG- and IV-fed piglets receiving the -PRO/+ARG diets, which suggests that at our concentration of dietary arginine, ureagenesis was impeded. Further quantification of the coindispensability of arginine and proline is essential to establish the safe, minimal dietary requirement for both amino acids.

P-5-C is central to the interconversion of glutamate to urea cycle metabolites (ornithine, citrulline, and arginine) or to proline. P-5-C reductase (P-5-C to proline) is present in most tissues, but P-5-C synthase (glutamate to P-5-C) is found only in the intestine and thymus (13). Neonates with reduced enteric mucosal mass (secondary to TPN feeding) would therefore have limited conversion of glutamine and glutamate to proline or ornithine during TPN feeding and during reinitiation of oral feeding. In vivo evidence of limited conversion of glutamate to proline was recently reported in premature infants receiving TPN (14). The label recovered in proline from an infusion of [13C]glucose was only ~10% of that in glutamate (14). Enrichment of urea cycle metabolites was not reported. Therefore, neonates have greater dietary requirements for both proline and arginine during TPN feeding. Additional arginine and proline should also be supplied during the reintroduction of enteral feeding after parenteral feeding due to limited de novo synthetic capacity.

The plasma concentrations of ornithine at baseline differed between IV- and IG-fed piglets, demonstrating that the mode of feeding significantly affects the metabolism of this amino acid as well. The difference was not due to a dilution effect, because baseline blood osmolarity was not lower in the parenterally fed piglets. Plasma ornithine concentration declined in the IG pigs during the -PRO/+ARG infusion; in the IV pigs, plasma ornithine was significantly lower than in IG pigs at baseline and remained constant. This decline in the IG piglets may reflect the conversion of ornithine to proline by OAT and P-5-C reductase in the liver or gastrointestinal tract. This mechanism appears to be limited in its capability to maintain plasma proline concentration. The P-5-C reductase activity in tissues of piglets adapted for 11 days to a low-proline diet was similar to that in control piglets (18). Therefore, upregulation of endogenous synthesis of proline from ornithine and arginine does not appear to be a mechanism that can compensate for a dietary deficiency.

Glutamine synthesis is considered to be an important pathway for nitrogen disposal in response to ammonia toxicity secondary to arginine deficiency. This mechanism was not clearly exhibited in 20- to 50-kg pigs fed arginine-deficient diets, even when challenged with 60-min infusion of ammonium into the portal vein to induce hyperammonemia (17). Prior and Gross (17) speculated that glutamine synthesis may not be as important for ammonia disposal in pigs as previously demonstrated in rats. The rapid rise in plasma glutamine and glutamate concentrations in the IV-fed piglets in this study indicates that endogenous glutamine synthesis is an important mechanism for nitrogen disposal; the discrepancy in results between our study and that of Prior and Gross could be due to differences in amino acid metabolism in neonatal pigs vs. older pigs (20-50 kg).

There was an effect of diet order on the rate of rise in ammonia concentration in the IV-fed piglets that was not seen in IG-fed piglets. The randomized crossover design allowed for unbiased analyses, which eliminated effects that might have been introduced by diet order. However, the significance of the diet order covariate indicates that the IV piglets did not fully recover, during the 16 h between studies, from the metabolic aberrations induced by the treatments. Although none of the other biochemical parameters that we measured at baseline indicated that diet order was a confounding variable, the IV-fed piglets did not grow as rapidly as IG-fed piglets during the 3 days of test diets.

By employing piglet models fed identical diets either intravenously or intragastrically, we have clearly demonstrated that arginine and proline are indispensable amino acids for both parenterally and enterally fed neonates. Furthermore, we established that the gut acts as an important mediator of whole body arginine supply by using proline as a major substrate for arginine synthesis; however, dietary proline or glutamate cannot fully spare the whole body requirement for arginine. Hence, dietary requirements for arginine and proline appear to be "codependent" and are altered by the mode of feeding. Clearly, the dietary arginine requirement is greater for parenterally than for enterally fed neonates. Low plasma arginine concentrations have been observed in preterm infants on TPN, particularly during stress or surgical trauma, suggesting that the commercial formulas currently available do not contain enough arginine.


    ACKNOWLEDGEMENTS

The amino acids were donated by Pharmacia-Upjohn, Stockholm.


    FOOTNOTES

This study was supported by grants from the Natural Sciences and Engineering Council of Canada, The Alberta Pork Producers Development Corporation, and Alberta Agricultural Research Institute.

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. §1734 solely to indicate this fact.

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).

Received 5 October 1998; accepted in final form 15 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alverdy, J. C. Amino acids to support gut function and morphology. In: Amino Acid Metabolism and Therapy in Health and Nutritional Disease, edited by L. A. Cynober. New York: CRC, 1995, p. 435-440.

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Am J Physiol Endocrinol Metab 277(2):E223-E231
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