Changes in net hepatic flux of nutrients by deacetylation of p-aminohippuric acid in dairy cows.

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1 Changes in net hepatic flux of nutrients by of p-aminohippuric acid in dairy cows. Rodríguez-López, J.M., Cantalapiedra-Hijar, G., Durand, D., Thomas, A., and Ortigues-Marty, I. INRA, UR 1213, Unité Mixte de Recherches sur les Herbivores, F Saint-Genès-Champanelle, France. Work associated to the REDNEX European project

2 Introduction Study of the in vivo metabolism of nutrients in the liver Arterio-venous difference method INPUT OUTPUT Portal vein Hepatic artery O 2 O 2 O 2 O2 O 2 O 2 O 2 O 2 O 2 O 2 O 2 O 2 Hepatic vein [nutrient] blood flow rate [nutrient] blood flow rate Net uptake or release = output input

3 Introduction Dilution method with p-aminohippuric acid (PAH) Ideal marker: homogenization in blood, no loss and an easy determination. (Eisemann et al., 1987) PAH hepatic acetylation: Sheep (Katz and Bergman, 1969) O Cows (Kristensen et al., 2009) H 2 N PAH infusion N H O OH O CH 3 Acetyl group 16% 50% Solution: Inclusion of a step Liver H 2 N O N H O O Acetylated PAH O CH 3

4 Material and methods 1 st objective: PAH validation process The accuracy profile (NF V03-110, 2010) Accuracy Trueness Closeness of agreement between the accepted reference value and each value measured Closeness of agreement between the accepted reference value and an average value from several repetitions Precision: Repeatability Intermediate precision Same analytical procedure, same operator and equipment during a short interval of time Same analytical procedure, different operator or equipment during a more prolonged interval of time

5 Material and methods 1 st objective: PAH validation process The accuracy profile (NF V03-110, 2010) PAH concentration levels: 0, 2.5, 5, 10, 15, 20, 25 and 30 mg/l 3 repetitions per level for 5 days With and Without the PAH step

6 Material and methods 2 nd objective: effect of the PAH on the net hepatic flux PAH infused into ruminal and mesenteric veins: 38 mmol/h, 6h 4 Blood collected from portal and hepatic vein and mesenteric artery (hourly samples over the last 5 hours). Plasma PAH was measured by spectrophotometer (Hamburger et al., 1948) With Without the PAH step Heating cycle with HCl 5N at 90ºC for 60 min (Isserty et al., 1998) Plasma flow x Nutrient concentrations = Net hepatic flux of nutrient Statistical analyses Paired-t test

7 Bias (%) Bias (%) Results 1 st objective: PAH validation process With PAH Without PAH 130% 130% 120% 120% 110% 110% 100% 90% 100% 90% % 80% 70% PAH concentration level /mg/l) 70% PAH concentration level /mg/l) Trueness: 99.9% SD Repeatability: 0.12 SD Intermediate precision: 0.33 Trueness: 99.2% SD Repeatability: 0.16 SD Intermediate precision: 0.16

8 Absorbance 540 nm Results 1 st objective: PAH validation process Effect of the inclusion of the PAH step on the standard curve y = 0.041x R² = 1 y = x R² = Concentration (mg/l) Concentration (mg/l) Without PAH With PAH Slopes and y-intercepts were significantly different between methods.

9 PAH Plasma concentration Flow Rate (L/min) (mg/l) Results 2 nd objective: effect of the PAH on the plasma PAH concentration = P-value < 0.05 With PAH 0 Mesenteric artery Portal vein Hepatic vein Without PAH All PAH concentrations in plasma were significantly higher (10-15%) when the PAH step was included in the analysis.

10 Plasma flow fate (L/min) Results 2 nd objective: effect of the PAH on the plasma flow N. S Portal vein Hepatic vein = P-value < 0.05 With PAH Without PAH Method affected hepatic plasma flow rate (more than 10%) with no significant influence on portal vein plasma flow rate.

11 Plasma flow (L/min) Hepatic artery contribution (%) Results 2 nd objective: effect of the PAH on the plasma flow Hepatic artery % 19.8% Hepatic artery/ hepatic vein = P-value < 0.05 With PAH Without PAH Hepatic artery plasma flow rate and its contribution to total hepatic flow rate changed by more than 50% between methods.

12 Results 2 nd objective: effect of the PAH on the net hepatic flux Change (%) P-value Net hepatic flux (mmol/min) Acetate 70.6 β-hydroxybutyrate 20.0 Oxygen 21.3 Total amino acids 40.4 Butyrate 5.0 Glucose 8.9 Propionate 2.9 Ammonia 1.8 N.S. Urea 7.3 N.S. Lactate 0.7 N.S. = P-value < 0.05 Modified 0-20% Modified >20% The net hepatic flux of nutrients was affected by the PAH. The extent of this effect may be related to the A-V concentration differences

13 Conclusions This work confirms the results obtained by Kristensen et al. (2009) that cattle liver acetylates PAH and quantifies the changes on the net hepatic nutrient fluxes when the laboratory procedure includes the PAH step. 15% of PAH in hepatic vein is acetylated With the PAH step, portal blood flow was not modified, while hepatic venous and arterial blood flows were reduced by 10 and 57%, respectively. The PAH analysis method affected the net hepatic flux of nutrients, with changes greater than 20%.

14 Thank you very much for your attention