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-Ketoglutarate Is Limited in Young Pigs1 ,2
U.S. Department of Agriculture/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030;
*
Gramineer Int. AB, Ideon, SE-223 70 Lund, Sweden; and
Lund University, Department of Animal Physiology, SE-233 63 Lund, Sweden
3To whom correspondence and reprint requests should be addressed. E-mail: dburrin{at}bcm.tmc.edu.
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ABSTRACT |
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-ketoglutarate (AKG). Female pigs (n = 6; 21 d old) were implanted with arterial, venous, portal and gastric catheters and an ultrasonic portal flow probe and fed a corn and soybean meal–based diet. On the day of the experiment, the pigs received a 4-h intragastric infusion of sodium AKG at a rate equivalent to 0, 2.5, 5 or 10% of dietary intake. The net portal AKG balance of the control and 2.5% treatments did not differ and were not different from zero. However, the net portal AKG balance of both the 5 [163 µmol/(kg · h)] and 10% [159 µmol/(kg · h)] treatments were greater (P < 0.05) than the control. Despite significant net AKG absorption at the 5 and 10% levels, the net portal appearance represented only 10.8 and 6.7%, respectively, of the enteral input. The net portal appearances of glutamate, glutamine, ammonia and the branched-chain amino acids were not affected by dietary AKG level. We conclude that the absorption of dietary AKG is limited in young pigs and does not change the net portal balance of amino acids or ammonia.
KEY WORDS: • glutamate • ammonia • glutamate dehydrogenase • dicarboxylic acid transport • pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) were the first to demonstrate the quantitative importance of glutamine and glutamate as metabolic fuels for the small intestine. Their in situ rat studies with perfused jejunal segments showed that a large proportion of luminal glutamate (95%) and glutamine (70%) is taken up and metabolized by the mucosa. More recent in vivo studies have confirmed these findings in both piglets (2
) and humans (3
). Although glutamine has long been considered the key metabolic fuel for the gut, most of these previous studies have shown that glutamate is of equal importance. In vivo studies indicate that luminal glutamate is extensively oxidized and contributes the largest proportion of gut CO2 production in fed piglets (2
).
The first step in glutamate oxidation is either deamination via glutamate dehydrogenase (GDH)4
or transamination via branched-chain aminotransferase (BCAT). Both GDH and BCAT are expressed in gastrointestinal tissues (4
,5
). Deamination of glutamate via GDH produces ammonia and -ketoglutarate (AKG), whereas in the BCAT reaction, glutamate donates an amino moiety to a branched-chain -keto acid, forming AKG and the corresponding branched-chain amino acid (BCAA). The deamination of glutamate via GDH may contribute to the substantial amount of dietary nitrogen released from the gut as ammonia after a meal. On the other hand, the metabolic fate of AKG is entry into the tricarboxylic acid (TCA)-cycle and oxidation to CO2. Despite its key intracellular role as a metabolic intermediate in the TCA-cycle, however, there is limited information about the metabolic fate of extracellular AKG, especially within the gut. Interestingly, sodium/dicarboxylate cotransporters, which can transport AKG, have been identified on the brush border membrane of pig enterocytes and on the basolateral membrane of other species (6
). Thus, it is conceivable that AKG could serve as a metabolic fuel for the gut and spare other oxidative fuels, such as glutamate, glutamine and aspartate. Moreover, because the GDH reaction is reversible, AKG could serve to sequester ammonia produced within the gut as glutamate and thereby reduce urea production.
In recent years, some studies have reported beneficial metabolic effects of dietary supplementation with ornithine -ketoglutarate (OKG). OKG consists of two molecules of ornithine (ORN) and one molecule of AKG and has been shown to increase adaptive hypertrophy after intestinal resection (7
), to prevent decreases in muscle free glutamine after surgery (8
) and to prevent decreases in muscle protein synthesis after surgery (9
) and burns (10
). The beneficial effect of OKG on the gut may be due to the fact that ORN is a precursor for polyamines. Indeed, intragastric administration of OKG for 7 d has been shown to increase putrescine content and the amount of ornithine decarboxylase mRNA (7
). It is feasible that these effects are mediated by AKG, although the independent roles of AKG and ORN are unclear.
Thus, despite the central role of AKG in oxidative metabolism and the positive effects of OKG on intestinal function, the metabolic fate of dietary AKG in the gut remains obscure. Given the presence of transporters in the gut, we hypothesized that enterally fed AKG would be readily taken up and metabolized by the intestinal mucosa, much like glutamate. A second hypothesis was that increased mucosal uptake of AKG might shift the GDH reaction and serve to scavenge free ammonia and reduce its release into the portal circulation. To test these hypotheses, we quantified the net portal balance of AKG, ammonia and other amino acids in piglets enterally fed three different levels of AKG.
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MATERIALS AND METHODS |
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Animals and diets.
Female piglets (n = 6) were purchased from the Texas Department of Criminal Justice, Huntsville, TX. They were fed a mixed diet formulated to meet or exceed current NRC (11
) recommendations. The diet contained yellow corn (45.9%), lactose (17.15%), 47% soybean meal (12.5%), casein (9.75%), fish meal (7.5%), soy oil (5.0), 18.5% dicalcium phosphate (1.6%), Swine micro 4 (0.25%), NaCl (0.25%), L-threonine (0.07%), L-lysine · HCl (0.02%). Treatments consisted of control (saline alone), 2.5, 5 or 10% equivalent of dietary intake of sodium-AKG (Sigma-Aldrich, St. Louis, MO) infused into the gastric catheter [2 mL/(kg · h)] for 4 h. This is equivalent to 0, 1.25, 2.5 and 5 g AKG/(kg · d) (Na salt). To overcome differences in sodium content of the control and treatment infusions, sodium chloride (0.62 mol/L) was added to the control solution (water) to equal the sodium content of the 5% treatment.
Study design.
The piglets arrived at the Children’s Nutrition Research Center when they were 2 wk old. For the next 7 d, they were offered the mixed diet at a daily rate of 50 g/kg body wt. supplying 11.35 g protein/(kg · d) and 336.2 kJ of gross energy/(kg · d). After 7 d, food was withdrawn from the piglets overnight and they were prepared for surgery as described previously (2
). Briefly, under isoflurane anesthesia and aseptic conditions, the piglets were implanted with a polyethylene catheter (o.d., 1.27 mm, Becton Dickinson, Sparks, MD) 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, Ithaca, NY) was placed around the portal vein. A silicone catheter (o.d., 2.17 mm, Baxter Healthcare, McGaw Park, IL) was implanted into the stomach lumen, 
2 cm from the pyloric sphincter. The catheters were filled with sterile saline containing heparin (2.5 x 104 U/L) and exteriorized on either the left flank (portal and gastric catheters, flow probe leads) or between the scapulae (jugular and carotid catheters). Immediately preoperatively, piglets received an intramuscular injection of antibiotic (20 mg/kg enrofloxacin, Bayer, Shawnee Mission, KS). Immediately postoperatively, they received an intramuscular injection of analgesic (0.1 mg/kg butorphenol tartrate, Fort Dodge Labs, Fort Dodge, IA). Before enteral feeding was resumed postoperatively, pigs were maintained on total parenteral nutrition for 24 h at a rate of 5 mL/(kg · h).
In Experiment 1, the piglets (n = 6; 5.5 kg) were deprived of feed for 15 h before initiation of each treatment. At time 0, piglets were offered 50% of their daily food and a single priming dose of AKG (equal to 1 h of infusion) was administered via the gastric catheter. After the priming dose, piglets received a constant, 4-h infusion of AKG via the gastric catheter at 2 mL/(kg · h). Three replicate portal and arterial blood samples were obtained at time 120, 180 and 240 min after the start of AKG infusions.
Piglets were arranged in a completely randomized design in which each piglet received 3 of the 4 treatments during three separate 4-h periods. Piglets were allowed 24 h between treatment periods during which time they consumed the diet described above.
In Experiment 2, piglets (n = 6; 6.3 kg) were used to determine the plasma clearance rate of AKG [315 µmol/(kg · h)] infused into the jugular vein at 1 mL/(kg · h), and three of the pigs were used to determine the net extraction of AKG from the arterial supply by the portal drained viscera (PDV) during the same infusion. The piglets were deprived of food for 15 h before initiation of the experiment. At time 0, piglets were offered 33% of their daily food and a constant 2-h infusion of AKG into the jugular vein was initiated. To determine the net disappearance of AKG across the PDV, three arterial and portal samples were obtained from three of the piglets just before the end of the 2-h intravenous infusion of AKG. After 2 h of infusion, arterial samples were obtained from six piglets and the infusion was terminated (time 0). Arterial samples were obtained from each of the six piglets at 15, 30, 45, 60, 75, 90 and 105 min after stopping the infusion. The decrease in plasma AKG was used to assess the whole-body clearance rate of AKG.
Sample analysis.
Blood samples were immediately placed on ice and centrifuged. 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 cut-off filter. A 50-µL aliquot of the filtrate was dried and the amino acids were analyzed by reversed-phase HPLC of their phenylisothiocyanate derivatives (Pico Tag, Waters, Woburn, MA).
Plasma AKG was determined by the method of Bergmeyer and Bernt (12
) 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. After the initial absorbance reading, 6 U (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 (kit 171-C, Sigma-Aldrich). Plasma glucose was determined using a spectrophotometric assay kit (kit 315–100; Sigma-Aldrich).
Statistical analysis.
For all statistical tests, a P-value of 0.05 was considered to represent statistical significance. In Experiment 1, the effects of AKG on the arterial, portal and net portal appearance of individual amino acids, AKG, glucose and ammonia were analyzed using the Mixed procedure of SAS (SAS Institute, Cary, NC). The model contained the effects of level of AKG supplementation, and pig was included as a random variable. Treatment means were computed using the LSMEANS option. Linear and quadratic trends were estimated using orthogonal contrasts for unequally spaced treatments. The effect of sample time (120, 180 or 240 min) was tested using the Mixed procedure of SAS. The effects of pig, treatment and time were included in the model and pig was included as a random variable. The effect of AKG level on portal blood flow was tested using 10-min interval means. The effect of level of AKG was included in the model and pig was included as a random variable.
In Experiment 2, the PDV extraction of arterial AKG was analyzed using the Mixed procedure of SAS. The model contained the effects of sample site (arterial or portal), and pig was included as a random variable. Treatment means were computed using the LSMEANS option.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Portal blood flow (Table 1
; Fig. 1
) was not affected by AKG infusion. Arterial and portal AKG concentrations (Table 1)
were increased (P < 0.01) by AKG infusion. Furthermore, the rates of net portal AKG appearance within a treatment were not different across the three sampling times, suggesting that the rate of AKG absorption had attained a quasi-steady state. Therefore, the mean AKG net portal appearance was used in all calculations. Arterial AKG concentration was increased above control only at the 10% infusion rate but the increase was linear (P < 0.01) as tested by orthogonal contrasts. Portal AKG was increased above the control at the 5% infusion rate, and the 10% infusion rate was greater than the other treatments. The mean net portal balance of AKG when piglets were administered the control treatment was 47 µmol/(kg · h), but this did not differ from 0 (P = 0.44). The net portal appearance of AKG for the 2.5% treatment was 112.7 µmol/(kg · h) and was not different (P = 0.14) from control, the absolute value only tended to differ from 0 (P = 0.08). Both the 5 and 10% treatments resulted in greater (P < 0.05) net portal balances of AKG than did the control treatment. AKG net portal balance increased linearly (P = 0.01) and tended to increase quadratically (P = 0.08) with increasing dietary AKG. From the net portal balance data, we calculated the amount of infused AKG that appeared in the portal vein (Table 1)
. The proportion of infused AKG appearing in the portal plasma was low for all treatments (Table 1)
. For the 2.5 and 5% treatments, the proportion of infused AKG appearing in portal plasma was 11%. The mean fractional portal appearance of AKG for the 10% treatment was 6.7%. The net portal appearances of glutamate, glutamine, ammonia and the BCAA were not affected by AKG infusion.
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There was no net utilization of arterial AKG across the PDV (P = 0.83). Mean arterial and portal AKG concentrations were 137 and 138 (SEM = 16.9) µmol/L, respectively. Intravenous AKG was cleared rapidly. The concentration of AKG in plasma at time 0 (before termination of infusion) was 145 ± 11 µmol/L. At 15 min after the termination of infusion, the plasma concentration had dropped to 2.13 ± 0.27 µmol/L. All subsequent sample times yielded concentrations of <1.0 µmol/L.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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At all treatment levels, no more than 11% of dietary AKG appeared in the portal drainage. The observation that such low proportions of infused AKG appear in the portal plasma raises several possibilities. First, it is possible that the infused AKG was not transported across the brush border membrane and remained in the lumen of the gastrointestinal tract intact, or it was metabolized by the microbial population of the gastrointestinal tract. Because sodium/dicarboxylate cotransporters, which can transport AKG, exist on pig brush border membranes (6
), it seems unlikely that AKG would not be taken up by the enterocytes. However, the abundance and capacity of these sodium/dicarboxylate transporters in the pig intestine has not been established and thus, uptake of AKG may have been limiting. If transport of AKG was not limiting, the portal appearance of only 11% suggests that AKG is highly metabolized and/or oxidized by first-pass metabolism because the net portal extraction of systemic AKG was not significant. The net portal appearance of glutamate, glutamine and ammonia was also not affected by AKG infusion. Thus, it does not appear that providing a relatively high level of AKG affected the sequestration of ammonia via GDH activity. It might be expected that the release of glutamate and glutamine would not be increased by AKG, given that very little dietary glutamate or glutamine is released by the PDV under normal feeding conditions (1
,2
). AKG is involved in other transamination reactions within the body. BCAA transaminase catalyzes the reaction between AKG and BCCA (leucine, isoleucine and valine). The BCAA is transaminated, forming glutamate from AKG and the respective keto-acid from each of the BCAA. It is conceivable that supplemental AKG might decrease the net release of BCAA from the PDV by stimulating the transamination of BCAA to form glutamate. Of the BCAA, only the net portal balance of leucine tended to be decreased (P = 0.07); however, this decrease and those of other BCAA were not significant. Thus, our results indicate that dietary AKG supplementation did not alter the net release of other amino acids from the PDV.
The fact that the net portal balance of glutamate and other amino acids was not increased by AKG infusion leads us to question the fate of enterally fed AKG. Another possible fate of AKG within the enterocyte is oxidation within the TCA cycle. If, indeed, all of the carbon infused as AKG was oxidized to CO2, we would expect this to increase CO2 output, yet the net portal appearance of CO2 did not increase with AKG infusion. However, it is also possible that AKG was oxidized preferentially and therefore spared other compounds, whereas the total oxidation in the PDV remained constant. Another possibility is that the AKG was converted to glutamate or glutamine and incorporated into protein. When piglets were administered all treatments, portal blood flow increased markedly (+50% above fasting baseline) after the bolus meal and as expected, tended to decline over the 4-h period. Yet, portal blood flow did not differ among the treatments over the 4-h period. Despite this lack of significant differences, the portal blood flow after the 10% treatment appeared to decrease more rapidly between 75 and 120 min after feeding than after the other treatments. However, we have no explanation for this observation.
In conclusion, only a small proportion (7–11%) of dietary AKG appeared in portal plasma. The net portal appearances of glutamate, glutamine, ammonia and the BCAA also were not significantly affected. We conclude that the net portal absorption of AKG is limited in young pigs, suggesting that either AKG transport capacity is limiting or that the intestinal mucosa or luminal microbes extensively metabolize AKG. Therefore, to fully understand whether enteral AKG is absorbed and metabolized by the PDV and the whole body, further study that utilizes detailed kinetics analysis with isotopic tracers is warranted.
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FOOTNOTES |
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2 Supported by a grant from Gramineer International, Inc., and by federal funds from the U.S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number 58–6250-6–001. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
4 Abbreviations used: AKG, -ketoglutarate; BCAA, branched-chain amino acid; BCAT, branched-chain aminotransferase; GDH, glutamate dehydrogenase; OKG, ornithine -ketoglutarate; ORN, ornithine; PDV, portal drained viscera; TCA, tricarboxylic acid.
Manuscript received 4 June 2002. Initial review completed 17 July 2002. Revision accepted 28 August 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Windmueller, H. G. & Spaeth, A. E. (1975) Intestinal metabolism of glutamine and glutamate from the lumen as compared to glutamine from blood. Arch. Biochem. Biophys. 171:662-672.[Medline]
2. Stoll, B., Burrin, D. G., Henry, J., Hung, Y., Jahoor, F. & Reeds, P. J. (1999) Substrate oxidation by the portal drained viscera of fed piglets. Am. J. Physiol. 277:E168-E175.
3. Matthews, D. E., Marano, M. A. & Campbell, R. G. (1993) Splanchnic bed utilization of glutamine and glutamic acid in humans. Am. J. Physiol. 264:E848-E854.
4. Madej, M., Lundh, T. & Lindberg, J. E. (1999) Activities of enzymes involved in glutamine metabolism in connection with energy production in the gastrointestinal tract epithelium of newborn, suckling and weaned piglets. Biol. Neonate 75:250-258.[Medline]
5. Suryawan, A., Hawes, J. W., Harris, R. A., Shimomura, Y., Jenkins, A. E. & Hutsun, S. M. (1998) A molecular model of human branched-chain amino acid metabolism. Am. J. Clin. Nutr. 68:72-81.[Abstract]
6. Pajor, A. M. (1999) Sodium-coupled transporters for Krebs cycle intermediates. Annu. Rev. Physiol. 61:663-682.[Medline]
7. Czernichow, B., Nsi-Emvo, E., Galluser, M., Gossé, F. & Raul, F. (1997) Enteral supplementation with ornithine ketoglutarate improves the early adaptive response to resection. Gut 40:67-72.
8. Blomqvist, B. I., Harrarqvist, F., Von der Decken, A. & Wernerman, J. (1995) Glutamine and -ketoglutarate prevent the decrease in muscle free glutamine concentration and influence protein synthesis after total hip replacement. Metabolism 44:1215-1222.[Medline]
9. Wernerman, J., Hammarqvist, F. & Vinnars, E. (1990) -ketoglutarate and postoperative muscle metabolism. Lancet 335:701-703.[Medline]
10. Cynober, L. (1991) Ornithine -ketoglutarate in nutritional support. Nutrition 7:313-322.[Medline]
11. National Research Council (1998) Nutrient Requirement of Swine 10th ed. 1998 National Academy Press Washington, DC. .
12. Bergmeyer, H. U. & Bernt, E. (1974) 2-Oxoglutarate. UV spectrophotometric determination. Bergmeyer, H. U. eds. Methods of Enzymatic Analysis 2nd ed. 1974 Academic Press New York, NY. .
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