Whole-body and splanchnic amino acid metabolism in sheep during an acute endotoxin challenge.

Supplemented protein or specific amino acids (AA) are proposed to help animals combat infection and inflammation. The current study investigates whole-body and splanchnic tissue metabolism in response to a lipopolysaccharide (LPS) challenge with or without a supplement of six AA (cysteine, glutamine, methionine, proline, serine and threonine). Eight sheep were surgically prepared with vascular catheters across the gut and liver. On two occasions, four sheep were infused through the jugular vein for 20 h with either saline or LPS from Escherichia coli (2 ng/kg body weight per min) in a random order, plus saline infused into the mesenteric vein; the other four sheep were treated with saline or LPS plus saline or six AA infused via the jugular vein into the mesenteric vein. Whole-body AA irreversible loss rate (ILR) and tissue protein metabolism were monitored by infusion of [ring-2H2]phenylalanine. LPS increased (P<0·001) ILR (+17 %), total plasma protein synthesis (+14 %) and lymphocyte protein synthesis (+386 %) but decreased albumin synthesis (-53 %, P=0·001), with no effect of AA infusion. Absorption of dietary AA was not reduced by LPS, except for glutamine. LPS increased the hepatic removal of leucine, lysine, glutamine and proline. Absolute hepatic extraction of supplemented AA increased, but, except for glutamine, this was less than the amount infused. This increased net appearance across the splanchnic bed restored arterial concentrations of five AA to, or above, values for the saline-infused period. Infusion of key AA does not appear to alter the acute period of endotoxaemic response, but it may have benefits for the chronic or recovery phases.

Responses in whole-body amino acid kinetics to an acute, sub-clinical endotoxin challenge in lambs.

Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations.

Response in hepatic removal of amino acids by the sheep to short-term infusions of varied amounts of an amino acid mixture into the mesenteric vein.

Under conditions of chronic supply the liver removes most amino acids (AA) in excess of net anabolic needs. Little information is available, however, on how acute alterations in AA supply (as might occur with once-daily feeding regimens) are controlled by the liver. Are these also extracted completely in a 'first-pass' manner or are there limitations to hepatic uptake? Furthermore, is the rate of removal 'saturable' (by Michaelis-Menten kinetics) over the range of supply experienced under normal feeding conditions? These questions have been addressed in a study that involved acute (4.5 h) increases in AA supply. Four sheep were prepared with trans-hepatic vascular catheters and were offered a basal diet (equivalent to 1.6xenergy maintenance) throughout. On four occasions, at 7 d intervals, they were infused with various amounts of an AA mixture into the mesenteric vein over a 4.5 h period. The mixture contained fourteen AA in the proportions present in rumen microbial protein. The amounts infused were calculated to provide an additional one, two, three and four times that absorbed from the basal diet. Continuous blood collections were removed over 2 h intervals before (basal diet only) and at 0.5-2.5 and 2.5-4.5 h of AA infusion. Transfers of AA, from the digestive tract and to the liver, were calculated for both plasma and total blood. The recovery of the infused AA across the portal-drained viscera (PDV) was quantitative (100%) only for histidine and proline, the remaining AA were recovered at 56-83 %. These losses correlated with the arterial concentrations and were probably due to removal of AA from the systemic circulation by the tissues of the digestive tract. Despite the wide range of net PDV appearances (i.e. absorbed plus infused), the percentage of most AA removed by the liver remained constant, but the percentage varied with AA (from 34 for proline to 78 for tryptophan for blood transfers). Thus, even when supply was increased 5-fold over baseline there was no indication that the transport into the liver declined, indeed the absolute removals continued to increase. In contrast, the branched-chain AA (isoleucine, leucine and valine) did not exhibit constant percentage extractions. Their percentage extractions were always the lowest (16, 10 and 25 respectively) and tended to decline at the highest infusion rates, indicative of saturation in hepatic transport and/or metabolism. The arterial concentrations of all infused AA increased with rate of infusion, again indicative that the liver did not extract all the net AA available across the PDV. Absolute amounts removed were similar between plasma and blood, indicating that most of the hepatic transfers occurred from plasma. The fractional rates of transfer from total inflow to the liver (i.e. with re-circulated AA included) were 3- to 4-fold lower than rates based on the amounts absorbed plus infused. The highest percentage extraction for total blood inflows was for serine (27), but most were between 6 and 16, except for the branched-chain AA, which were all <1. Use of percentage extractions based on total inflows are probably more appropriate for development of mathematical models of liver metabolism, and the current data suggest that constant values may be applied. The needs of the liver for specific mechanisms involving phenylalanine and histidine (plasma protein synthesis), glycine (detoxification of xenobiotics) and alanine (gluconeogenesis) probably also require to be included in such models.

Effects of diet quality on urea fates in sheep as assessed by refined, non-invasive [15N15N]urea kinetics.

The effect of diet quality on urea production, entry into the gastrointestinal tract (GIT) and subsequent diversion to anabolic or catabolic fates was examined in four sheep (mean live weight 49.5 kg). The animals received, in a crossover design, each of two rations, hay-grass pellets (1:1 HG) and a mixed concentrate-forage (CF). Measurements were made of N balance and urea kinetics based on a 4 d continuous intravascular infusion of [15N15N]urea. Enrichments of [15N15N]- and [14N15N]urea in the urine, and faecal 15N content were determined each day. After 24 h of infusion, urinary [15N15N]urea enrichments reached constant enrichment but a further 24 h was required before [14N15N]urea enrichment was at plateau. The latter is derived from hydrolysis of urea to 15NH3 in the digestive tract with subsequent absorption and reconversion to urea. The diets were not isonitrogenous (14.3 v. 17.1 g N supplied daily for HG and CF respectively) but showed no difference in N balance. Urea-N production was much greater (16.3 v. 11.1 g/d; P = 0.011) for CF compared with HG and more urea-N entered the GIT (9.9 v. 7.7; P = 0.07). A larger proportion of GIT entry was returned to ureagenesis (51 v. 42%; P = 0.047) for the CF diet but a smaller fraction was lost in the faeces (3.3% v. 7.1%; P = 0.013). In consequence, most of the additional urea-N which entered the GIT on the CF diet was returned to the ornithine cycle (probably as NH3) and the absolute amount available for anabolic purposes was similar between the rations (3.9 v. 4.5 g N/d).

Transfers of N metabolites across the ovine liver in response to short-term infusions of an amino acid mixture into the mesenteric vein.

The effect of acute (4.5 h) infusions into the mesenteric vein of an amino acid (AA) mixture, which simulated the composition of rumen microbial protein, on net transfers of NH3, urea and total AA across the portal-drained viscera (PDV) and liver in the ovine has been examined. Four wether sheep were surgically prepared with vascular catheters across the PDV and liver (Lobley et al. 1995) and were offered a basal diet of 1000 g grass pellets/d (approximately 1.4 x energy maintenance). Each animal was infused at weekly intervals with one of four dilutions of the AA mixture. These dilutions provided 0.44, 0.88, 1.32 and 1.84 mmol AA-N/min infused, the lowest of which approximately doubled the net absorption of AA-N from the basal diet. Animals were treated with heparin to allow continuous collection of blood by peristaltic pump for 2 h preceding, and between 0.5-2.5 and 2.5-4.5 h after, the start of the AA infusions. Blood flow in the hepatic artery increased (100 v. 208 g/min; P = 0.002) in response to AA infusion, while hepatic portal venous flow decreased (2090 v. 1854 g/min; P = 0.006). The AA infusion also stimulated O2 uptake by the PDV (P < 0.001) and liver (P = 0.016). Absorption across the PDV and hepatic removal of NH3 were unchanged between basal and amino acid infusion conditions. Urea-N removal across the PDV was unaltered, but hepatic production increased (P < 0.001) with level of AA infusion. During infusions, net appearance of AA across the PDV was below the theoretical level. This may have been due to inhibition of AA uptake from the small intestine, and/or increased removal by the digestive tract of AA from the systemic circulation associated with greater arterial concentrations. Hepatic extraction of AA increased with level of infusion, both for total AA and those included in the infusate. Total hepatic urea-N production tended towards a maximum (estimated as 2 mumol N/g liver wet weight per min). The AA removed by the liver and not used for ureagenesis remained similar (170 mumol AA-N/min) between basal and AA infusions. This was presumed available for anabolic purposes (mainly synthesis of export proteins). The proportion of net AA-N appearance (absorption plus infused) across the PDV removed by the liver declined from 0.71 to 0.53 between basal and AA infusions. In contrast to findings from cattle (Wray-Cahen et al. 1997), increased AA infusion did not alter the net removal of glutamine across the liver. This may reflect differences between the studies in NH3: AA-N absorbed. Further differences between the cattle study and the current findings may relate to the different physiological state (pregnancy v. growth), which may alter the partition of AA between anabolic and catabolic fates.