Splanchnic net balance of oxygen and metabolites in response to mesenteric vein infusion of ammonia in sheep

In order to simulate daily episodes of high ammonia (NH4 +) absorption associated with the intake of diets with high content of non-protein N or rapidly rumen degradable protein, four wethers (42±3.4 kg BW), surgically fi tted with indwelling catheters in the posterior aorta and splanchnic veins, were infused with 340 μmol NH4 HCO3 -/min into the mesenteric vein for 3 h, over 7 consecutive days. On the 7th day, net mass transfers of NH4 +, urea, glucose, lactate, ß-OH-butyrate and oxygen were measured across portal-drained viscera (PDV), liver and splanchnic tissues during the last 90 min of the NH4 + infusion (NH4 + treatment, AT). Measurements were repeated on the following day, after withdrawal of the NH4 + infusion (Control treatment, CT). NH4 + infusion increased liver NH4 + uptake (+396 μmol/min; sed, 72; P=0.04) and urea production (+152 μmol/min; sed, 55; P=0.14), and oxygen consumption by the liver (+151 μmol/min; sed, 6; P=0.002), the PDV (+224 μmol/min; sed, 56; P=0.03) and the splanchnic tissues (+352 μmol/min; sed, 57; P=0.009). Net mass transfers of glucose, lactate and ß-OH-butyrate across the PDV and the liver, and the acid-base status of the animals were unchanged.


INTRODUCTION
In ruminants, a high proportion of the dietary N is absorbed as NH 4 + .The liver removes all the NH 4 + absorbed and then converts it to urea.NH 4 + metabolism could alter both liver energy expenditure and glucose production, but there is a controversy about the intensity and the sign (in the latter case) of these effects.In addition, it is unknown whether liver metabolism of other energy metabolites could also be altered.The objective of the present experiment was to examine the response of hepatic, portal-drained viscera (PDV) and splanchnic net mass transfers of NH 4 + , glucose, urea, lactate, ß-OH-butyrate and oxygen to a 3-h NH 4 + infusion into the mesenteric vein, administered during 7 d in order to simulate daily episodes of high NH 4 + absorption associated with the intake of diets with high concentration of non-protein N or rapidly rumen degradable protein.

Animals, diet and design
Four Corriedale wethers (42±3.4kg BW), surgically prepared with indwelling catheters in the posterior aorta, portal, hepatic and mesenteric veins, were housed in individual pens (2.6 m 2 ) and fed 450 g/d of lucerne hay plus 360 g/d of cracked maize (8.1 MJ ME/d; 16.0 g N/d) in 4 equal portions, at 0:30, 6:30, 12:30 and 18:00 h, during 4 weeks, using automatic feeders.Water was offered ad libitum.Throughout the last week, at 8:30 h, a solution of 900 mM NH 4 + HCO 3 -was infused into the mesenteric vein for 3 h (340 µmol NH 4 + /min; 8.1 µmol NH 4 + /kgBW/min).On the 7 th day, a solution containing 0.1M Na p-aminohippuric acid (pAH), 0.05 M Na phosphate buffer (pH 7.4) and 460 UI heparin/g (APH solution) was also infused into the mesenteric vein (0.36 g/min) during the last 135 min of the NH 4 + HCO 3 infusion, in order to determine the portal and hepatic blood fl ow (NH 4 + treatment, AT).On the next day, the NH 4 + HCO 3 -infusion was withdrawn and only the APH solution was infused at identical times (Control treatment, CT).

Samples and analysis
In both treatments, 45 min after the start of the infusion of the APH solution, two simultaneous blood samples were continuously withdrawn from the aorta and the portal and hepatic veins over 90 min (0.17 ml/min).The collection lines were allowed to pass through ice-cold water and the blood samples were collected directly into 10 ml syringes stored in ice-cold water.Samples were carefully mixed and analysed for blood pO 2 , pCO 2 and pH immediately after collection, using a Blood Gas Analyser.The packed cell volume, blood haemoglobin and pAH concentration were analysed as described by Milano et al. (2000).A portion of blood was centrifuged (1000 g; 4ºC), and the plasma was processed for determination of NH 4 + , urea and glucose (Milano et al., 2000), lactate (Lactate, Randox ® ) and ß-OH-butyrate (Ranbut, Randox ® ).Infusions and blood collections were performed with peristaltic pumps.

Calculations and statistics
Blood fl ow and net mass transfers of metabolites and oxygen across the liver, the PDV and the splanchnic tissues were calculated as described by Milano et al. (2000).The data were analysed by ANOVA, with animals and treatment as main factors.

RESULTS AND DISCUSSION
The results are shown in Table 1.PDV and liver data represent the average of 3 data points per treatment because the portal vein catheter lost patency in one of the animals.

Glucose
There was a trend for lower absorption or higher utilization of glucose by the PDV in AT (130 µmol/min; P=0.11).Hepatic glucose production was not signifi cantly different between treatments.Blood BE and pH were not altered by NH 4 + HCO 3 -infusion in the current experiment, suggesting that the enhanced glucose production observed in previous experiments with sheep during NH 4 + Cl - infusion into the mesenteric vein (30 µmol NH 4 + Cl -/kg BW/min; Barej et al., RECAVARREN M.I. ET AL. 1987;6.7 µmol NH 4 + Cl -/kgBW/min; Milano et al., 2001), might have been linked to a NH 4 + Cl --induced metabolic acidosis.

Urea, lactate and ß-OH-butyrate
The differences between treatments in mass transfers of urea, lactate and ß-OH-butyrate across the PDV were not signifi cant.There was a trend for a higher hepatic urea production in AT (+303 µmol urea-N/min; P = 0.14).This increase represents a recovery of 77% of the N infused.There were no differences between treatments in liver lactate uptake or ß-OH-butyrate production.

Oxygen
PDV, liver and splanchnic oxygen consumption was signifi cantly higher in AT (224, 151 and 352 µmol/min, respectively).Liver oxygen consumption increased by 0.39 mol/mol NH 4 + removed or 0.49 mol/mol urea-N produced, these ratios being slightly higher than the stoichiometric ratios accepted for urea synthesis (0.33 mol/ urea-N; Stryer, 1988).Milano et al. (2000) found that the liver oxygen consumption increased by 0.69 mol/mol NH 4 + removed or 0.61 mol/mol urea-N produced (P=0.13)during continuous infusion of 400 µmol/min of NH 4 + HCO 3 -into the mesenteric vein of wethers for 4 days.When extrapolated to the 180 min of the NH 4 + infusion, the increment in splanchnic oxygen consumption observed in the current experiment amounts to 63.4 mmoles or 29 KJ (0.352 mmol/min × 180 min × 460 KJ/mmol O 2 ), i.e. the equivalent of 1% of the daily ME intake destined to deposition (based on a daily EM intake of 8.2 MJ and 5.3 MJ ME/d of maintenance energy requirements).

CONCLUSIONS
The 3 h infusion of NH 4 + HCO 3 -for 7 d, increased NH 4 + liver uptake, with a consequently higher urea production, and oxygen consumption by the liver, the PDV and the splanchnic tissues.No changes were observed in hepatic net mass transfers of glucose, lactate and ß-OH-butyrate or in the acid-base status of the animals.

Table 1 .
Mass transfers of NH 4