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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

First-Pass Metabolism Limits the Intestinal Absorption of Enteral -Ketoglutarate in Young Pigs¹

² U.S. Department of Agriculture/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, ³ Department of Internal and Occupational Diseases, Institute of Agricultural Medicine, Lublin, Poland, ⁴ Research Institute for the Biology of Farm Animals, Research Unit Nutritional Physiology "Oskar Kellner", D-18196 Dummerstorf, Germany, ⁵ Lund University, Department of Cell and Organism Biology, Lund, Sweden, and ⁶ Essentys AB, Lund, Sweden

^* To whom correspondence should be addressed. E-mail: dburrin{at}bcm.tmc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Our results in a previous study indicated that the portal absorption of intragastrically fed -ketoglutarate (AKG) was limited in young pigs. Our aim was to quantify the net portal absorption, first-pass metabolism, and whole-body flux of enterally infused AKG. In study 1, we quantified the net portal nutrient absorption in young pigs (n = 9) given an intraduodenal infusion of milk replacer [10 mL/(kg · h)] and either saline (control) or 930 µmol/(kg · h) AKG for 4 h. In study 2, we quantified the luminal disappearance of a duodenal AKG bolus in young pigs (n = 7). In study 3, we quantified the whole-body kinetics of ¹³C-AKG metabolism when infused either enterally (n = 9) or intravenously (n = 9) in young pigs. In study 1, when compared with the control group, enteral AKG infusion increased (P < 0.01) the arterial (13.8 ± 1.7 vs. 27.4 ± 3.6 µmol/L) and portal (22.0 ± 1.4 vs. 64.6 ± 5.9 µmol/L) AKG concentrations and the net portal absorption of AKG [19.7 ± 2.8 vs. 95.2 ± 12.0 µmol/(kg · h)]. The mean fractional portal appearance of enterally infused AKG was 10.23 ± 1.3%. In study 2, the luminal disappearance of AKG was 663 µmol/(kg · h), representing 63% of the intraduodenal dose. In study 3, the whole-body ¹³C-AKG flux [4685 ± 666 vs. 801 ± 67 µmol/(kg · h)] was higher (P < 0.05) when given enterally than intravenously, but ¹³CO₂ recovery was not different (37.3 ± 1.0 vs. 36.2 ± 0.7%dose). The first-pass splanchnic ¹³C-AKG utilization was 80%, of which 30% was oxidized to ¹³CO₂. We conclude that the intestinal absorption of AKG is limited in young pigs largely due to substantial first-pass gastrointestinal metabolism.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Previously, studies have shown that the portal appearance of intragastically infused AKG was limited in young pigs (9,10), representing only 10% of intake. In our first study, it was not possible to determine whether lack of intestinal transport or metabolism within intestinal tissue led to low portal appearances of AKG. Additionally, the intragastric infusion of AKG and a bolus feeding of a corn-based diet used in our previous study could have interfered with the continuous flow of AKG to the small intestine. However, subsequent studies have shown that there is excess capacity for sodium-dependent dicarboxylic acid transport (NaDC-1) in the small intestine and stomach to handle the AKG intake administered in our pig studies (11). Thus, it seemed apparent that the low rate of dietary AKG absorption is due to intestinal mucosal metabolism. To address this issue, we conducted studies in growing pigs to examine the metabolic fate of AKG when it is presented to the gut either intraduodenally or intragastrically and also in tracer form (¹³C-AKG) systemically or enterally. We hypothesized that the low rate of intestinal absorption of dietary AKG was due to extensive metabolism and oxidation by the gastrointestinal tract.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Studies 1 and 2 were approved by the Baylor College of Medicine Animal Protocol Review Committee. Housing and care of the animals conformed to USDA guidelines. Study 3 was approved by the Animal Care and Use Committee of the Ministry of Nutrition, Agriculture, Forestry, and Fishery, Schwerin, State Mecklenburg-Vorpommern, Germany.

Study design

In studies 1 and 2, female piglets (n = 9) were purchased from the Texas Department of Criminal Justice, Huntsville, TX. Pigs (14 d of age) arrived at the Children's Nutrition Research Center and for a 7-d adjustment period were fed a liquid milk replacer diet (Litter Life, Merrick) at a rate of 50 g/(kg · h). The composition of the milk replacer (per kg dry matter) was 500 g lactose, 100 g fat, and 250 g protein. After 7 d, food was withdrawn from the piglets overnight and they were prepared for surgery as described previously (3). Briefly, under isoflurane anesthesia and aseptic conditions, the piglets were implanted with a polyethylene catheter (o.d., 1.27 mm, Becton Dickinson) in the common portal vein, and silastic catheters (o.d., 1.78 mm) in an external jugular vein and a carotid artery. An ultrasonic flow probe (8–10 mm i.d., Transonic) was placed around the portal vein. A silicone catheter (o.d., 2.17 mm, Baxter Healthcare) was implanted into the lumen of the duodenum. The catheters were filled with sterile saline containing heparin (25 kU/L), and exteriorized on either the left flank (portal and duodenal catheters, flow probe leads) or between the scapulae (jugular and carotid catheters). Immediately preoperatively, animals received an intramuscular injection of antibiotic (20 mg/kg enrofloxacin, Bayer) and an intramuscular injection of analgesic (0.1 mg/kg butorphenol tartrate, Fort Dodge Labs). Before enteral feeding was resumed postoperatively, pigs were maintained on total parenteral nutrition for 24 h at a rate of 5 mL/(kg · h). Pigs were allowed 7 d to recover from surgery. In all piglets, intakes and rates of weight gain had returned to preoperative levels.

    Study 1. Piglets were deprived of food for 15 h before initiation of the experiment. On the day of the experiment, at time –1 h, a primed (10 mL/kg; 25% w:w aqueous solution; oral), continuous duodenal infusion of milk replacer [10 mL/(kg · h), Litter Life, Merrick] was prepared as a 25% (w:w) aqueous solution that provided 920 kJ and 12.5 g protein/(kg · d). The treatments were dissolved in water and contained either NaCl (control; 930 mmol/L) or sodium-AKG (930 mmol/L; Sigma-Aldrich); the solutions were infused intraduodenally at 1 mL/(kg · h), such that the AKG infusion rate was 930 µmol/(kg · h). The level of AKG was chosen based on previous data from our laboratory, where intakes of >2.5% of diet dry matter was required to observe a detectable portal balance of AKG (10). Pigs also received an intravenous (200 µmol/kg), continuous, 6-h infusion of ¹⁵N₂-urea [20 µmol/(kg · h); 99%; Cambridge Isotope Laboratories]. At time 0 h, a primed (15 µmol/kg), continuous, 2-h infusion of NaH¹³CO₂ [15 µmol/(kg · h); 99%; Cambridge Isotope Laboratories] was initiated. Arterial samples were obtained at 0, 90, 105, and 120 min after initiation of NaH¹³CO₂ infusion to determine whole-body CO₂ production.

At time 2 h, the NaH¹³CO₂ infusion was terminated and a primed (40 µmol/kg), continuous, 4-h infusion of [1-¹³C]Leucine [40 µmol/(kg · h); 99%; Cambridge Isotope Laboratories] was initiated. Arterial and portal venous samples were obtained at time 4, 5, and 6 h for determination of leucine and urea kinetics as well as mass balance of ammonia, AKG, glucose, and amino acids. All pigs received both control and AKG treatments in a completely randomized design with at least 24 h between treatment periods.

    Study 2. After completion of study 1, the following day some of the pigs (n = 7) were given a duodenal bolus infusion [7.75 mL/kg; 25% (w:w) aqueous solution] of liquid milk replacer (Litter Life, Merrick) containing 25 g/L sodium AKG (1040 µmol/kg body weight). After 1 h, pigs were killed by i.v. injection of 50 mg/kg pentobarbitol. The small intestine was carefully clamped at the proximal duodenum and distal ileum, removed, and flushed with 2 x 50 mL of saline to wash the intestine. The washes were collected, pooled, and a 15 mL aliquot was flash frozen in liquid N₂ and stored at –80°C for later AKG analysis.

    Study 3. Portions of the experimental design, diets, and protocol have been described previously (12). Eighteen castrated male pigs (German Landrace) with initial body weight of 15 kg and 8 wk of age were housed in individual metabolic cages. Pigs were surgically implanted with a 3 catheters into the jugular vein, the stomach/duodenum for infusion of nutrients and AKG, and into the carotid artery for blood sampling. The catheters were filled with sterile saline containing heparin. Immediately after surgery, pigs received an intramuscular injection of 4 mL antibiotic (gentamycin, Vepha-Gent, Veyx-Pharma GmbH) and 1 mL Rimadyl (carprofen, Pfizer Animal Health) i.m. After surgery the pigs were fed intraduodenally an elemental diet composed of free amino acids, dextrose, Intralipid, electrolytes, and vitamins for 7 d. The diet provided the following approximate daily nutrient intakes per kg body weight: 4 g protein, 12 g glucose, and 5.5 g lipid. Three days postsurgery, pigs began receiving a continuous infusion of AKG [5.95 mmoL sodium AKG/(kg · d), equivalent to 1 g/(kg · d)] either intraduodenally or intragastrically. At 7 d postsurgery, pigs received a continuous infusion of 15 mg/kg [1-¹³C]AKG (Chemotrade) dissolved in 50 mL isotonic saline solution for 3 h. The AKG was infused either intravenously (jugular vein) or via the enteral catheters (intraduodenal or intragastric). The number of pigs studied in each group was as follows: intraduodenal/¹³C-AKG intraduodenal (ID-fed, n = 4), diet intraduodenal/¹³C-AKG intravenous (ID-fed, n = 5), diet intragastric/¹³C-AKG intragastric (IG-fed, n = 4), diet intragastric/¹³C-AKG intravenous (IG-fed, n = 5). Blood samples were taken 1-h before (–1) and 1, 1.5, 2, 2.5, and 3-h after the start of [1-¹³C]AKG infusion.

Sample analysis

Blood samples were immediately placed on ice and centrifuged at 3000 x g for 10 min. Plasma was collected, immediately frozen in liquid N₂, and stored at –80°C until analysis. For plasma amino acid analysis, a 0.2 mL aliquot of plasma was mixed with an equal volume of an aqueous solution of methionine sulfone (4 mmol/L) and centrifuged at 10,000 x g for 120 min through a 10-kDa cutoff filter. A 50 µL aliquot of the filtrate was dried and the amino acids were analyzed by reverse-phase HPLC of their phenylisothiocyanate derivatives (Pico Tag, Waters).

Plasma AKG was determined by the method of Bergmeyer and Bernt (8) with minor modifications. The assay was carried out in 0.5 mL of working solution consisting of 100 mmol/L phosphate buffer (pH 7.6), 4 mmol/L ammonium chloride, and 50 µmol/L NADH. To the working solution, an appropriate amount of plasma containing 1–10 nmol of AKG was added. An initial absorbance reading was obtained at 340 nm. Following the initial absorbance reading, 6 units (in a volume of 10 µL) of bovine GDH (G2501; Sigma-Aldrich) was added to each tube. After a 10-min incubation, a second absorbance reading was taken at 340 nm. The amount of AKG in the sample is directly proportional to the decrease in absorbance between the first and second reading. The AKG concentration was calculated by the use of a standard curve. Plasma ammonia was determined using a spectrophotometric assay kit (171-C, Sigma-Aldrich). Plasma glucose was determined using a spectrophotometric assay kit (315–100; Sigma-Aldrich).

To estimate the enrichment of blood bicarbonate, an aliquot of whole blood (1.0 mL) was placed in a 10-mL vacutainer (Becton Dickinson), and 0.5 mL of perchloric acid (10% w:w) was added. Room air (10 mL) filtered through soda lime (Sodasorb; Grace Container Products) was injected into the vacutainer, removed into a gas-tight syringe, and transferred to a second vacutainer. The isotopic enrichment of the carbon dioxide in the gas sample was measured on a continuous flow gas isotope ratio mass spectrometer (Thermo Finnigan Gasbench-II coupled with Delta^plusXL Isotope Ratio MS).

Plasma ketoisocaproic acid (KIC) was isolated by cation exchange chromatography (AG-50V resin, Bio-Rad). Eluants were treated with sodium hydroxide (100 µL; 10 mol/L) and hydroxylamine HCl (200 µL; 0.36 mol/L) and heated (60°C; 30 min). After cooling, the pH of the samples was adjusted to <2. The keto acids were extracted in 5 mL of ethylacetate and dried under nitrogen at room temperature. Derivatization of KIC was accomplished by adding 50 µL of N-methyl-N-t-butyl-dimethylsilys-trifluoroacetamide plus 1% t-butyl-dimethylchlorosilane. The isotopic enrichment of KIC was determined by EI GC-MS (GC-MS model HP-6890/5973 MSD, Hewlett Packard) by monitoring ions at 316 m/z and 317 m/z.

Plasma urea isotopic enrichments were determined by EI GC-MS analysis. Proteins were precipitated from 50 µL of plasma with 200 µL of ice-cold acetone. After vortexing, the protein was separated by centrifugation (3000 x g for 20 min), and the supernatant was removed and dried under nitrogen. To the dried supernatant, 250 µL of a 1:20 dilution of malonaldehyde bid (dimethyl acetal) and concentrated HCl (30 w %) was added, the sample was incubated at room temperature for 2 h, and then completely evaporated (Speedvac, Savant Instruments, Forma Scientific). The urea was derivitized with 50 µL of N-methyl-N-t-butyl-dimethylsilys-trifluoroacetamide + 1% t-butyl-dimethylchlorosilane and the isotopic enrichment in plasma was determined using EI GC-MS analysis by monitoring ions at 153 to 155 m/z.

Plasma [1-¹³C]AKG isotopic enrichments were determined by EI GC-MS analysis. AKG was eluted from 200 µL of acidified plasma after application to cation exchange column (AG-50W resin, Bio Rad). The cation exchange effluent was alkalized with 100 µL of 10 mol/L NaOH, combined with 200 µL of 0.36 mol/L hydroxylamine (Sigma Chemical), sonicated for 1 min at a room temperature, and heated at 60°C for 30 min. Samples were then cooled to room temperature and acidified with 200 µL HCL (Fisher Scientific) to pH <2, and extracted 3 times with 5 mL of ethylacetate (Fisher Scientific). The organic extracts were completely evaporated under N₂ in room temperature. The residue was reacted with 100 µL MTBSTFA + 1% TBDMCS (Regis Chemical) for at least 24 h. A 10 µL aliquot was injected into a GC-MS (GC-MS model HP-6890/5973 MSD, Hewlett Packard) equipped with RESTEK RTX-1 column. The mass spectrometer was operated in the Electron Impact (EI) mode and ions monitored were at m/z 446 for -KG and at m/z 447 for [1-¹³C] -KG.

Calculations

Net portal balance of metabolites [µmol/(kg · h)] was calculated as follows:

(Eq. 1)

when Conc. is the concentration in the blood (µmol/L), PORT and ART refer to portal and arterial blood and PBF is portal blood flow [L/(kg · h)].

Whole-body leucine flux [Q; µmol/(kg · h)], was calculated as follows:

(Eq.2)

where R is the tracer infusion rate [µmol/(kg · h)] and IE_infusate and IE_plasma are the isotopic enrichments (expressed as mol%) of the infused tracer and plasma KIC, respectively. Whole-body AKG and urea flux were calculated using Eq. 2 based on the steady-state plasma IE of [¹³C]AKG and [¹⁵N]urea, respectively.

In study 1, whole-body CO₂ production was calculated as follows:

(Eq.3)

where IE infusate is the enrichment of in the infusate (mol % excess), IE arterial bicarbonate is the enrichment in arterial blood (mol % excess), and tracer infusion rate [µmol/(kg · h)] during the i.v. bicarbonate infusion that preceded each treatment period. The entire equation was divided by 0.81 to correct for recovery of infused labeled carbon in bicarbonate (13).

Whole-body leucine oxidation [µmol/(kg · h)] was calculated as follows:

(Eq. 4)

where IE_CO2 is the isotopic enrichment of bicarbonate during the [1-¹³C]leucine infusion and IE_LEU is the isotopic enrichment of 1-¹³C-KIC during the [1-¹³C]leucine infusion.

Whole-body nonoxidative leucine disposal (NOLD) is an estimate of leucine incorporation into muscle. NOLD [µmol/(kg · h)] was calculated by the following equation:

(Eq. 5)

Whole-body endogenous leucine appearance rate (Ra) [µmol/(kg · h)] is an estimate of leucine released into the plasma pool by tissue proteolysis and was calculated as:

The fractional first-pass splanchnic extraction of enterally infused [¹³C]AKG was calculated as follows:

where PE represents the steady-state plasma enrichments of [¹³C]AKG during enteral and intravenous infusion. The fraction of enteral [¹³C]AKG that was oxidized on first pass by the splanchnic tissues was calculated as follows:

where fox represents the fractional oxidation of [¹³C]AKG given either enterally or intravenously as described previously.

Statistical analysis

For all statistical tests, a P-value of 0.05 was considered to be significant. In study 1, the effects of AKG on the arterial, portal, and net portal appearance of individual amino acids, AKG, glucose, ammonia, and leucine kinetics were analyzed using the General Linear Model procedure (Minitab). The model contained the effects of AKG supplementation and pig. Pig was included as a random variable. In study 3, whole-body kinetic data were analyzed using a 2-way ANOVA with diet (ID vs. IG fed) and tracer infusion route (IV vs. Enteral) as main effects. A 1-way Student's t test was used to test whether AKG net portal balance was significantly greater than zero during control treatments. Values in the text are means ± SEM.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Expt. 1. The body weights of pigs in the control (7.45 ± 0.3) and AKG- infused group (7.29 ± 0.4) did not differ. AKG infusion increased (P < 0.01) arterial and portal AKG concentration and the net portal balance of AKG (Table 1). Even when no AKG was infused into the duodenum, the net portal absorption of AKG was significantly greater than zero. However, the net portal absorption of AKG was increased (P < 0.001) with AKG treatment compared with control. The net portal balance of AKG represented only 10.23% of the amount infused. The net portal balance of 10.23% was actually a slight overestimate of infused AKG absorption, because when only saline was infused, there was a significant absorption of AKG. When we corrected the data for absorption of AKG from the control diet, the proportion of infused AKG appearing in the portal venous drainage was 8.12%.

    Expt. 3. Plasma isotopic enrichments of [¹³C]AKG in 18 pigs infused with [¹³C]AKG either enterally or intravenously for 3 h and fed an elemental diet either intraduodenally or intragastrically are presented in Figure 1. Body weights did not differ among the 4 groups studied (overall mean 17.2 ± 0.3 kg). The [¹³C]AKG enrichment (MPE) was significantly higher in pigs after intravenous compared with enteral [¹³C]AKG infusion (Fig. 1). The whole-body [¹³C]AKG flux of was higher in pigs after enteral than intravenous [¹³C]AKG infusion (tracer infusion route effect, P < 0.05) (Table 3). However, the whole-body flux of [¹³C]AKG was also higher after in the IG compared with ID fed groups (diet effect, P < 0.05). There was a significant (P < 0.05) diet x tracer infusion route interaction such that the increase in whole-body [¹³C]AKG flux associated with the IG diet was greater during the enteral tracer infusion mode. CO₂ recovery was not affected by either diet (ID vs. IG) or tracer infusion route (IV vs. ENT ¹³C-AKG infusion) and was 37% of the dose administered. The calculated rates of first-pass splanchnic [¹³C]AKG utilization and oxidation to ¹³CO₂ were 80 and 30% among the 2 modes of enteral AKG infusion. First-pass splanchnic oxidation of [¹³C]AKG represented 80% of the whole-body [¹³C]AKG rate. The pigs used in study 3 (9 wk of age) were older than those used in Studies 1 and 2 (4 wk of age) and thus may have experienced some developmental changes in gut metabolism. This may explain why the splanchnic first-pass [¹³C-AKG] utilization was only 80% in study 3, yet the net intestinal AKG utilization determined in study 1 was 90%.

View larger version (16K): [in this window] [in a new window]   Figure 1  Plasma isotopic enrichments (MPE) of 13C-AKG in young pigs infused with 13C-AKG either enterally or intravenously for 3 h and fed an elemental diet either intraduodenally (ID) or intragastrically (IG). Values are means ± SEM, n = 4–5.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

The observation that only 10% of infused AKG appeared in the portal plasma raises several possibilities as to the fate of luminal AKG. One possible explanation for the low AKG portal appearance is that luminal AKG transport is limited. Sodium-dicarboxylate cotransporter, NaDC-1, which transports AKG, is present in the stomach, intestine, and colon (11,14). Moreover, kinetic estimates of tissue ¹⁴C-AKG uptake predict an excess capacity for mucosal AKG uptake, so it seems unlikely that AKG transport is limiting. To test this, we infused a single duodenal bolus of 1040 µmol/kg and found that over 660 µmol/kg (64%) disappeared from the small intestine of piglets in 1 h (study 2). This data, taken together with the portal mass balance of AKG during a similar AKG duodenal infusion (study 1), indicates that substantial metabolism of AKG occurs in either the lumen of the intestine via microbial metabolism, or in the intestinal enterocyte. However, it is impossible from our results to distinguish between enteroctye vs. microbial metabolism of AKG. We are not aware of any studies that have characterized the metabolism of AKG by mixed cultures of commensal pig microbes, yet is seems highly likely that some degree of microbial metabolism occurs in the intestinal lumen. What is even more intriguing from the available evidence is the fact that specific transporters for AKG (i.e., NaDC-1) exist on the intestinal apical mucosa despite the fact that AKG is not a major constituent of most mammalian diets. This evidence implies a functional need to transport AKG from the intestinal lumen, which may arise from gut microbial production. This possibility is supported by the presence of a low rate of portal AKG absorption in control pigs that were not given supplemental AKG.

The net portal appearance of glutamate and glutamine were also not affected by AKG infusion as observed previously (10). It seems likely that AKG was converted to glutamate via either abundant amino-transaminases (alanine and aspartate) or glutamate dehydrogenase, all of which are present in the intestine (8). If the absorbed AKG was converted to glutamate via transamination, it could either be released into the portal blood or converted to other amino acids. However, it might be expected that the release of glutamate would not be increased by AKG even if substantial conversion to these amino acids occurred, given that very little dietary glutamate is released by the PDV under normal feeding conditions (3). Alternatively, proline can be synthesized from enteral glutamate by the intestinal mucosal tissue (15,16). Moreover, we found that net portal proline balance was 60% higher in pigs supplemented with AKG. Given that the net increase in proline balance was 138.1 µmol/(kg · h) in AKG treated pigs, and that over 800 µmol/(kg · h) of AKG was unaccounted for in the portal balance, it is possible that the increase in proline release can be completely accounted for by conversion from AKG. Such a large conversion of AKG to proline in the enterocyte could have led to a decrease in portal ammonia balance, but portal ammonia balance remained unchanged. The lack of effect on portal ammonia balance was also reflected in similar rates of whole-body urea synthesis in the 2 groups.

Branched-chain amino acid (BCAA) aminotransferase (BCAT) catalyzes the reaction between AKG and BCAA (leucine, isoleucine, and valine). The presence of BCAT in the intestine has been shown in pigs and other species based on enzyme activity, immunohistochemistry, and in vivo metabolism (17–20). The BCAA is transaminated, forming glutamate from AKG and the respective keto-acid from each of the BCAA. We had hypothesized that supplemental AKG would lead to a decrease in the net release of BCAA from the PDV by stimulating the transamination of BCAA to form glutamate. In contrast, however, the portal release of leucine and isoleucine was increased by AKG, yet this did not affect whole-body leucine kinetics. The net portal balance of lysine was also increased with AKG. Because the net portal balance of many amino acids was near 100% with the AKG treatment, it is not clear if AKG spared the amino acids or increased amino acid release due to proteolysis within the portal-drained viscera.

An additional probable fate of AKG within the enterocyte is oxidation via the TCA cycle. If indeed all of the carbon infused as AKG was oxidized to CO₂, an increase CO₂ output from the PDV would be expected, yet the production of CO₂ in the whole body did not increase with AKG infusion. Interestingly, the net portal balance of glucose was decreased with AKG treatment, yet the reason for this decrease is not clear. However, the results from the ¹³C-AKG tracer study suggest that first-pass splanchnic utilization of dietary AKG was substantial, representing 80% of the dietary intake. The finding of 80% first-pass splanchnic metabolism in study 3 was slightly less than the estimate of 90% gut metabolism observed in study 1. This may reflect differences in the age and diets used in these 2 studies, insofar as some of the enzymes involved in AKG metabolism increase with age (8). Moreover, the AKG intake was >5-fold higher in study 3 and study 1, which may have resulted in a lower fractional rate of gut metabolism. The oxidation rate of ¹³C-AKG based on ¹³CO₂ recovery was relatively high (35% dose), yet there was no difference in the fractional recovery between intravenous and enterally administered tracer modes. This translated into a calculated first-pass AKG oxidation rate of 30%, which is lower than the gut oxidation rate we have observed previously for dietary glutamate (e.g., 50%)(3–5,21). Based on our current findings, it would appear that the substantial first-pass intestinal utilization of dietary AKG occurs and that a majority of the AKG is converted via nonoxidative metabolic end products, such as proline, and yet oxidation to CO₂ is quantitatively important. Furthermore, the splanchnic tissues, mainly gut, are the major site of AKG oxidation, representing 80% of whole-body AKG oxidation.

In conclusion, our results indicate that a substantial amount of dietary AKG disappeared from the lumen of the small intestine, but is not absorbed into the portal blood stream. It is likely that some of the AKG in the diet was metabolized to proline, accounting for the 60% increase net portal proline appearance. In vivo tracer results suggested that large amounts of AKG are metabolized in first-pass by the splanchnic tissues (80% intake), however, nonoxidative pathways predominate over complete metabolism to CO₂. We found no effect of dietary AKG on either whole-body urea or leucine fluxes. These results generally agree with previous data where AKG was provided intragastrically and suggest that gastric metabolism of dietary AKG is not significant. We conclude that the net intestinal absorption of AKG is limited in young pigs due to extensive metabolism by mucosal epithelial cells or by bacteria in the intestinal lumen. However, the fractional rate of AKG oxidation to CO₂ in the gut is less than other major gut fuels, namely, glutamate and glutamine.

	FOOTNOTES

 
¹ This research was supported by USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX. Financial support was received from Essentys AB and federal funds from the USDA, Agricultural Research Service under cooperative agreement 58-6250-6-001 and by NIH grant HD33920 (D.G.B.). The content of this publication does not necessarily reflect the views or policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

⁷ Abbreviations used: AKG, -ketoglutarate; BCAT, branched chain amino transferase; NOLD, nonoxidative leucine disposal; PDV, portal drained viscera.

Manuscript received 16 May 2006. Initial review completed 22 June 2006. Revision accepted 18 August 2006.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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