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J. Anim. Sci. 2004. 82:2033-2042
© 2004 American Society of Animal Science

ANIMAL NUTRITION

Splanchnic metabolism of volatile fatty acids absorbed from the washed reticulorumen of steers1

N. B. Kristensen*,2 and D. L. Harmon{dagger}

* Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Denmark and and {dagger} Department of Animal Sciences, University of Kentucky, Lexington 40546


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
Six steers fitted with a ruminal cannula and chronic indwelling catheters in the mesenteric artery, mesenteric vein, hepatic portal vein, hepatic vein, as well as in the right ruminal vein were used to study metabolism of VFA absorbed from buffers in the emptied and washed reticulorumen. [2-13C]Acetate was infused into a jugular vein to study portal-drained visceral (PDV) uptake of arterial acetate, hepatic unidirectional uptake of acetate, and whole-body irreversible loss rate (ILR). Isobutyrate was infused into the right ruminal vein to calibrate VFA fluxes measured in the portal vein. On sampling days, the rumen was emptied and incubated in sequence with a 0-buffer (bicarbonate buffer without VFA), a VFA-buffer plus continuous intraruminal infusion of VFA, and finally another 0-buffer. Ruminal VFA absorption was determined as VFA uptake from the VFA-buffer and metabolic effects determined as the difference between metabolite fluxes with VFA-buffer and 0-buffers. Steady absorption rates of VFA were maintained during VFA-buffer incubations (4 h; 592 ± 16, 257 ± 5, 127 ± 2, 17 ± <1, 20 ± <1 mmol/h, respectively, of acetate, propionate, butyrate, isovalerate, and valerate). The portal flux of acetate corrected for PDV uptake of arterial acetate accounted for 105 ± 3% of the acetate absorption from the rumen, and the net portal flux of propionate accounted for 91 ± 2% of propionate absorption. Considerably less butyrate (27 ± 3%) and valerate (30 ± 3%) could be accounted for in the portal vein. The sum of portal VFA and 3-hydroxybutyrate as well as lactate represented 99 ± 3% of total VFA acetyl units and 103 ± 2% of VFA propionyl units. Estimates are maximum because no accounting was made for lactate derived from glycolysis in the PDV. The net splanchnic flux of VFA, lactate, 3-hydroxybutyrate, and glucose accounted for 64 ± 2% of VFA acetyl units and 34 ± 5% of VFA propionyl units. Results indicate that there is a low "first-pass" uptake of acetate and propionate in the ruminal epithelium of cattle, whereas butyrate and valerate are extensively metabolized, though seemingly not oxidized to carbon dioxide in the epithelium but repackaged into acetate, 3-hydroxybutyrate, and perhaps other metabolites. When PDV "second-pass" uptake of arterial nutrients is accounted for, PDV fluxes of VFA, lactate, and 3-hydroxybutyrate represent VFA production in the gastrointestinal tract and thereby VFA availability to the ruminant animal.

Key Words: Blood Flow • Cattle • Energy Metabolism • Volatile Fatty Acids


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
Volatile fatty acids are important energy substrates for ruminants (Sutton, 1985Go); however, knowledge of VFA metabolism has hitherto been used sparingly in practice probably owing to uncertainty about their metabolism by gut microbes and splanchnic tissues. Until recently, the ruminal epithelium has been considered to oxidize a large fraction of VFA absorbed from the gastrointestinal tract (Bergman and Wolff, 1971Go). Recent findings showing that rumen epithelial uptake of acetate and propionate was considerably less in sheep under washed rumen conditions (Kristensen et al., 2000aGo) compared with fed sheep (Bergman and Wolff, 1971Go) and the finding that ruminal microbes seem to metabolize acetate extensively (Kristensen, 2001Go) challenge the view that an extensive oxidation of VFA takes place in the ruminal epithelium.

It is necessary to consider two sources of substrate supply to accurately describe splanchnic metabolism. Only tissues actually involved in nutrient absorption (epithelia) will be able to extract nutrients in their "first pass" (i.e., following absorption from the lumen). Most tissues, even those in the portal-drained viscera (PDV), will have to take up nutrients from the blood (i.e., "second pass"; Reynolds, 2002Go). Use of the washed rumen technique and systemic infusion of [2-13C]acetate in multicatheterized steers will minimize ruminal microbial VFA metabolism and allow estimation of the PDV uptake of arterial acetate. Our hypothesis was that steers will also metabolize small quantities of acetate and propionate during the first pass, through the ruminal epithelium.

The current study was undertaken to investigate the first-pass uptake and metabolism of VFA absorbed from the washed reticulorumen in steers using systemic infusion of [2-13C]acetate to quantify PDV second-pass acetate uptake and to investigate the effects of the intraruminal VFA load on PDV and liver net metabolism of VFA, glucose, lactate, 3-hydroxybutyrate, glutamine, and glutamate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Implications
 Literature Cited
 
All procedures involving animals were conducted at the UKARC Beef Unit and approved by the University of Kentucky Animal Care and Use Committee (Protocol No. 00387A2002).

Animals and Feeding
Six Holstein bull calves were fitted with a ruminal cannula (#2C rumen cannula, Bar Diamond Inc., Parma, ID), castrated, and fitted with permanent indwelling catheters in the mesenteric artery, mesenteric vein, hepatic portal vein, and the hepatic vein (Huntington et al., 1989Go) as well as in the right ruminal vein (silicone tubing 1.02 mm i.d., 2.16 mm o.d.; Helix Medical Inc., Carpinteria, CA). Catheter patency was maintained by weekly aspiration of catheter fluid, flushing with saline, and filling with saline containing 100 U/mL of heparin and 0.1% (vol/vol) benzyl alcohol.

Steers were fed 2.1 kg of DM/d of mixed grass hay and 3.3 kg of DM/d of corn-based concentrate (composition of dietary DM, %: OM = 94; ether extract = 4; CP = 13; and NDF = 34). The steers had access to a mineral mix (composition of DM: Ca =13 to 15%; P = 6%; NaCl = 17 to 20%; Mg = 3%; S = 1%; K = 1%; Zn = 2,300 ppm; Mn = 2,200 ppm; Cu = 1,050 ppm; I = 45 ppm; Co = 15 ppm; and Se = 29 ppm). The mineral mix contained vitamin A (66,000 IU/100 g) and vitamin E (28 IU/100 g). The steers were housed individually in 3- x 3-m pens, with the temperature at 18 to 24°C and lit from 0530 to 2130. The steers had free access to water except during samplings. The steers were fed their whole ration in the morning (between 0730 and 0830) except on sampling days, when steers were fed after the experimental procedure was completed.

The steers were sampled 21 and 35 ± 2 d after surgery, with an average BW of 251 and 260 ± 6 kg at the two samplings, respectively.

Intravenous Infusion
One steer was sampled per sampling day. The steer was weighed, and a temporary jugular catheter (16G Delmed I catheter; Delmed Inc., Canton, MA) was introduced into the right jugular vein. Continuous infusion of [2-13C]acetate (1.96 ± 0.02 mmol/h, sodium acetate [2-13C], 99%; Cambridge Isotope Laboratory, Andover, MA) into the jugular vein, p-aminohippuric acid (pAH, 16.7 ± 0.2 mmol/h, Sigma-Aldrich Corp., St. Louis, MO) into the mesenteric vein, and saline (Butler Co., Columbus, OH) into the ruminal vein was initiated. After two to three sets of blood samples (see below), the ruminal vein infusion was changed from saline to isobutyrate (16.1 ± 0.2 mmol/h; isobutyric acid 99+%; Acros Organics, Geel, Belgium). The infusates were prepared the day before sampling by dissolving [2-13C]acetate (30 mmol/kg infusate) in sterile saline (Butler Co.) and pAH (250 mmol/kg infusate) as well as isobutyric acid (220 mmol/kg infusate) in sterile water (Butler Co.). The pH of the pAH and isobutyrate infusates was adjusted to 7.4. The infusates were transferred to autoclaved bottles by sterile filtration (0.2-µm Nalgene sterile filter; Nalgene Int., Rochester, NY). All infusion rates were determined gravimetrically, and infusion solutions were weighed multiple times during infusions to check that the infusion rates were constant throughout the sampling day.

Washed Rumen Procedure
Immediately after the intravenous infusions were begun, the rumen was emptied and washed with approximately 3 x 5 L of warm tap water and 10 L of saline. The ruminal contents were stored in a plastic barrel and warmed before reintroduction to the steer at the end of the day. Following washing, 15 kg of bicarbonate buffer without VFA (0-buffer; Table 1Go) was added to the rumen. All buffers and saline added to the rumen were heated to 40°C over night in a forced-air oven. An infusion and gassing device described previously (Kristensen et al., 2002Go) was placed in the buffer in the rumen and the ruminal cannula was closed. The buffer in the rumen was continuously gassed with a mixture of 75% CO2:25% N2. The 0-buffer was pumped out of the rumen after 45 min and 15 kg of VFA-buffer (exact weight recorded) was added to the rumen (Table 1Go). The ruminal cannula was closed and intraruminal infusion of VFA-infusate (1,008 ± 8 g/h; Table 1Go) was initiated. After 245 min with VFA buffer and VFA infusion, the rumen was emptied and washed twice with 7.5 kg of saline. The weight of buffer and saline from the rumen was recorded. Another 15 kg of 0-buffer (Table 1Go) was added and maintained in the rumen for 45 min following the same procedure as described for the first 0-buffer incubation. Following buffer incubations and sampling, ruminal contents, warmed in a water bath, were placed into the rumen and the steer was fed.


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  		Table 1. Composition of ruminal buffers and ruminal infusate (mmol/kg of buffer or infusate)
 
Blood and Ruminal Sampling
Blood samples were simultaneously drawn from the artery, portal vein, and hepatic vein catheters by slowly drawing blood into 20-mL syringes flushed with a heparin solution (10,000 IU heparin/mL). Blood was sampled after 40 min of each 0-buffer incubation and after 10, 30, 60, 120, 180, and 240 min of the VFA-buffer incubation. Blood samples were placed on ice immediately after collection. Packed cell volume was determined by centrifugation of capillary tubes (3 min, Autocrit Ultra 3, Becton Dickinson, Franklin Lakes, NJ). Blood plasma was separated by centrifugation (2,500 x g, 4°C, 20 min), stored below –20°C, and kept on dry ice when transported between laboratories.

Ruminal buffer samples were obtained after 10 min of each 0-buffer incubation and after 10, 30, 60, 180, and 240 min of the VFA-buffer incubation. Samples were obtained from VFA-buffer and saline pumped out of the rumen after the VFA-buffer incubation. Buffer pH was measured immediately, and the buffer samples were stored and transported as described for blood plasma.

Analytical Procedures
Plasma concentrations of L-lactate (L-lactate oxidase), D-glucose (D-glucose oxidase), L-glutamate (L-glutamate oxidase), and L-glutamine (glutaminase plus L-glutamate oxidase) were determined by membrane-immobilized enzymes (YSI Inc., Yellow Springs, OH). Membrane linearity was checked daily according to the manufacturer’s recommendations. Plasma concentrations of D-3-hydroxybutyrate were determined using a kit based on D-3-hydroxybutyrate dehydrogenase (Stanbio Laboratory, Boerne, TX). Plasma pAH concentrations were determined colorimetrically as previously described (Harvey and Brothers, 1962Go). Volatile fatty acid concentrations in infusates, buffers, and plasma, as well as determination of carbon-13 abundance in acetate by GC-IRMS (Finnigan MAT, Bremen, Germany) were done as previously described (Kristensen, 2000Go), except that samples for carbon-13 abundance were pooled within 0-buffer and VFA-buffer samples and acetate in the samples was concentrated by ultrafiltration (5000 Da, Centrisart I; Sartorius, Goettingen, Germany).

Calculations and Statistical Procedures
Data on lactate, glucose, glutamine, glutamate, and 3-hydroxybutyrate are given as plasma concentrations and plasma fluxes. Blood flow rates and fluxes of VFA are given as whole-blood values. Blood concentrations of VFA were obtained from plasma concentrations assuming a 45% dilution space for VFA in erythrocytes (Kristensen, 2000Go). The net portal fluxijk was calculated as follows: portal blood or plasma flowijk x (portal concentrationijk – arterial concentrationijk), using either whole-blood or plasma values as indicated: i = Steer 1 to 6, j = Repetition 1 to 2, and k = Sample 1 to 8. The net hepatic fluxijk was calculated as follows: hepatic blood or plasma flowijk x hepatic concentrationijk – {(portal blood or plasma flowijk x portal concentrationijk) + [(hepatic blood or plasma flowijk – portal blood or plasma flowijk) x arterial concentrationijk]}. The hepatic extraction ratioijk was calculated as follows: –net hepatic fluxijk / {(portal blood or plasma flowijk x portal concentrationijk) + [(hepatic blood or plasma flowijk – portal blood or plasma flowijk) x arterial concentrationijk]}. The flux at each sampling time was weighted by time. The time represented by each sampling was calculated as the time halfway between the foregoing (or initiation of buffer incubation) and the actual sampling to the time halfway between the actual and the following sample (or end of buffer incubation). Calculations on [2-13C]acetate abundance and fluxes were described previously (Kristensen et al., 1996bGo). Ruminal VFA absorption was calculated as follows: VFA added with ruminal buffer + VFA infused during VFA-buffer incubation – VFA in ruminal buffer and wash water after incubation. Isobutyrate-corrected recovery of ruminally absorbed VFA was calculated as [(portal appearance with VFA-bufferij – portal appearance with 0-bufferij) / VFA absorptionij] / portal recovery of isobutyrate infused into the ruminal veinij, where i = Steer 1 to 6 and j = Repetition 1 to 2. The portal recovery of isobutyrate was calculated from the difference in net portal flux between the period with saline and isobutyrate infusion into the ruminal vein. The following weights were assigned in calculation of acetyl unit balance: acetate = 1, butyrate = 2, isovalerate = 3, valerate = 1, and 3-hydroxybutyrate = 2. The following weights were assigned in calculation of propionyl unit balance: propionate = 1, valerate = 1, lactate = 1, and glucose = 2. Isovalerate was assigned to three acetyl units due to its carboxylation during metabolism, yielding a purely ketogenic C6 intermediate (3-methyl-glutaconyl-CoA; Bender, 1985Go).

Differences in the amount of ruminal buffer and ruminal pool sizes at initiation of VFA buffer incubations compared with values after 240 min of incubation were evaluated by paired t-test using the means procedure of SAS (SAS Inst. Inc., Cary, NC). Effects of buffer were analyzed by ANOVA using the GLM procedure of SAS. The model included buffer and steer. Significance was declared for P < 0.05. Means in the text are means of six steers ± SEM when not stated otherwise.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Implications
 Literature Cited
 
Rumen
The VFA concentrations in the rumen were relatively stable during the VFA buffer incubation (Figure 1Go). The mean ruminal concentrations of acetate, propionate, butyrate, isovalerate, and valerate were 64 ± 3, 25 ± 1, 11 ± 0.2, 1.7 ± 0.02, and 1.2 ± 0.02 mmol/kg buffer, respectively. The mean concentrations of all VFA were numerically lower in the rumen compared with the concentrations in the VFA-buffer. However, the ruminal liquid pool increased (P < 0.01; 2.9 ± 0.4 kg) during incubation and only the ruminal valerate pool was found to decrease (P = 0.01; –12 ± 4%) during incubation. The steers absorbed 68 to 80% of the VFA added to the rumen by VFA-buffer and VFA infusion (Table 2Go), and normal physiological absorption rates were obtained compared with fed growing steers (Harmon et al., 1985Go; Krehbiel et al., 1992Go). The mean ruminal pH with the VFA buffer incubation was 6.5 ± 0.1. The washed rumen procedure allowed a precisely defined time for onset and end of VFA absorption from the rumen. The VFA content of the second portion of wash water was only 0.2% of the ruminal VFA pool with the VFA-buffer incubation. The fractional absorption rate of VFA (absorption rate divided by ruminal pool size) in the ruminal buffer was lowest for acetate (0.56 ± 0.05 h–1) and highest for valerate (1.03 ± 0.09 h–1). Dijkstra et al. (1993)Go reported fractional absorption rates in lactating dairy cows that generally were lower than those of the current study (0.21 to 0.85); however, the Dijkstra et al. (1993)Go data were corrected for liquid passage from the rumen and were obtained under experimental conditions with increasing pH during incubations, and the buffers were not agitated by gassing as the buffer was in the current study.



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  		Figure 1. Ruminal buffer concentration of acetate (circles) and propionate (squares) in steers incubated in sequence with a 0-buffer (bicarbonate buffer without VFA, first sample), a VFA buffer plus continuous intraruminal infusion of VFA, and finally, another 0-buffer (last sample) in the washed rumen. Each point is the mean of six steers ± SEM.

 

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  		Table 2. Ruminal input and absorption of VFA in steers incubated with bicarbonate buffers in the washed rumen
 
Arterial Concentrations
The packed cell volume as well as the concentration of valerate and pAH were not affected by buffer; however, the concentration of all other metabolites determined in plasma increased (P < 0.05) with the VFA-buffer incubation compared with the 0-buffer incubation (Table 3Go). All time plots (not shown) for metabolites indicated stable experimental conditions; the mean arterial and portal concentration of acetate is shown as an example in Figure 2Go. The blood VFA concentrations observed with the 0-buffer incubation were similar to the levels observed in sheep incubated with 0-buffer (Kristensen et al., 2000aGo) and the blood VFA concentrations observed with the VFA-buffer were similar to values in fed growing steers (Harmon et al., 1985Go; Krehbiel et al., 1992Go).


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  		Table 3. Packed cell volume as well as arterial concentrations of VFA, p-aminohippurate, glucose, lactate, 3-hydroxybutyrate, glutamate, and glutamine in steers incubated with bicarbonate buffers in the washed rumen
 


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  		Figure 2. Arterial (circles) and portal (squares) blood concentration of acetate in steers incubated in sequence with a 0-buffer (bicarbonate buffer without VFA; first sample), a VFA buffer plus continuous intraruminal infusion of VFA, and finally, another 0-buffer (last sample) in the washed rumen. Each point is the mean of six steers ± SEM.

 
Blood Flow
Portal as well as splanchnic blood flow increased (P < 0.05) with the VFA-buffer incubation compared with the 0-buffer incubation (Table 4Go). The increase was about 17% for the portal blood flow and 9% for splanchnic blood flow. The increase in portal blood flow agrees with observations from sheep, wherein portal blood flow increased approximately 10% with incubation of VFA-buffer compared with 0-buffer (Kristensen et al., 2000aGo). Sellers et al. (1964)Go reported rumen arterial blood flows of 36 to 176 L/h (calculated as two times the flow of the right ruminal vein) in dairy cows. These authors observed that the rumen arterial blood flow responded to the presence of VFA and it seems likely that at least part of the observed increase in portal blood flow in the current study could be due to a direct effect of VFA on rumen epithelial blood flow.


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  		Table 4. Portal and splanchnic blood flow, net portal flux as well as net hepatic flux of VFA, glucose, 3-hydroxybutyrate, lactate, glutamate, and glutamine in steers incubated with bicarbonate buffers in the washed rumena
 
Net Portal and Net Hepatic Fluxes
The net portal flux of VFA, lactate, and 3-hydroxybutyrate were strongly affected (P < 0.01) by buffer treatment. The net portal flux of glucose, glutamate, and glutamine were not affected by buffer treatment (Table 4Go). Net hepatic flux of glutamate and glutamine were positive with both buffer treatments and increased (P < 0.05) with VFA-buffer compared with 0-buffer incubation. Net hepatic flux of acetate and 3-hydroxybutyrate were positive but were not affected by buffer incubation (Table 4Go). Net hepatic flux of propionate, butyrate, isovalerate, and valerate were negative with both buffer treatments, and the hepatic uptake of these compounds increased (P < 0.01) with the VFA-buffer compared with the 0-buffer incubation. The hepatic net flux of lactate was negative with both treatments, however, the hepatic uptake of lactate decreased (P < 0.01) with VFA buffer incubation (Table 4Go). The hepatic lactate uptake accounted for 23% and 15% of the net hepatic glucose flux with 0-buffer and VFA-buffer incubation, respectively. The hepatic net flux of glucose did not change (P = 0.45) with buffer and the hepatic uptake of propionate could account for approximately 11% of glucose-C with the 0-buffer; however, with the VFA-buffer, 90% of the glucose-C could be accounted for by propionate on a net basis. Even with a large deficit in propionate-C for gluconeogenesis, the liver released glutamate and glutamine with the 0-buffer. Glucose irreversible loss rate (ILR), measured by infusion of [U-13C]glucose in a jugular vein, was also unaffected by the presence or absence of propionate in the washed rumen of lactating cows (Kristensen et al., 2002Go). Although lactate accounted for a small part of the gap in glucose precursors with the 0-buffer, the limited range of metabolites analyzed in the present experiment does not allow us to evaluate whether glycogen or amino acids other than glutamate and glutamine were the main source of the glucose released with low propionate supply.

Hepatic Extraction Ratio
The hepatic extraction ratio (fraction of afferent hepatic flux taken up by the liver) of propionate and butyrate decreased (P < 0.05) with the VFA-buffer compared with 0-buffer (data not shown); however, the hepatic extraction ratio of isobutyrate, isovalerate, and valerate all increased (P < 0.01) with VFA-buffer compared with 0-buffer. This opposite direction of the change in hepatic extraction of butyrate and valerate is noteworthy compared with the metabolism of these VFA in the ruminal epithelium. It was observed in sheep that an increased intraruminal infusion of butyrate was followed by a decrease in the epithelial uptake of both butyrate and valerate (Kristensen et al., 2000bGo). In the current study, the hepatic extraction ratio of butyrate and valerate were similar with the 0-buffer incubation (76 and 74%, respectively); however, with VFA-buffer, the hepatic extraction ratio decreased to 65% for butyrate and increased to 92% for valerate. These changes would support the hypothesis that there is a different acyl-CoA synthetase for valerate in the liver compared with ruminal epithelium and this enzyme is activated by VFA (isobutyrate, isovalerate, or valerate) or a metabolic intermediate of these VFA.

Isobutyrate Calibration
The arterial concentration, the net portal flux, and the net hepatic flux of isobutyrate increased (P < 0.01) following isobutyrate infusion into the ruminal vein (Table 5Go). The increase in net portal flux of isobutyrate could account for 90 ± 4% of the infused isobutyrate. Isobutyrate infusion was done parallel to pAH infusion to validate the VFA flux measurements. Mixing of blood in the portal vein was assumed to be the primary constraint to obtaining a precise and accurate measurement of blood flow and nutrient fluxes. The estimate on isobutyrate recovery is, however, sensitive to a potential uptake of arterial isobutyrate by the PDV. If it is assumed that 50% of the arterial isobutyrate is removed by the PDV, the estimate on portal recovery of isobutyrate would be 94% instead of 90 and the values on portal recovery of ruminal VFA a little lower than those presented below.


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  		Table 5. Arterial concentration, net portal flux, net hepatic flux, and portal recovery of isobutyrate infused into the ruminal vein of steers
 
Given that the potential loss of pAH from arterial blood entering the PDV is limited to lymphatic drainage and the net absorption of water is minute compared with blood flow, potential errors in blood flow measurements are primarily the result of sampling and analytical errors. Sampling errors could arise from obtaining samples that do not represent the portal blood relative to nonsampling times. If blood from the cranial mesenteric vein and vena lienalis is not completely mixed, blood flow measured from pAH infusion into the mesenteric vein will not correspond to the portal arterial concentration difference of isobutyrate infused into the right ruminal vein (entering the portal system via the vena lienalis). Because different analytical methods were used to measure pAH and isobutyrate concentrations, it is not possible to differentiate strictly between the mixing problem of blood and analytical errors. Nevertheless, we would have expected a strong animal effect on the isobutyrate recovery if the errors were predominantly caused by anatomical differences and thereby dependent on catheter placement. Numerical differences between steers (data not shown) means that some minor bias from mixing problems at the level of the portal vein cannot be ruled out. The fact that the VFA absorbed from the ruminal buffers will be transported via the same route as the infused isobutyrate enabled us to use isobutyrate as a calibrator of the bias arising from both analytical and anatomical origin.

Metabolism of [2-13C]Acetate
The blood acetate was enriched with carbon-13 (Table 6Go) by jugular infusion of [2-13C]acetate to measure the unidirectional uptake of arterial acetate by the PDV, and liver as well as the whole-body ILR of [2-13C]acetate. The portal recovery of arterially supplied acetate increased (P < 0.01) with VFA-buffer incubation compared with 0-buffer incubation. The PDV tissues therefore extracted a larger proportion of arterial acetate with a low arterial concentration, indicating that tissue uptake of acetate is not entirely dependent on mass action. Though a lower fraction of arterial acetate was taken up with VFA-buffer incubation, the increased concentration of arterial acetate resulted in a threefold increase (P < 0.01) in total PDV uptake of arterial acetate for the VFA-buffer incubation compared with 0-buffer incubation (Table 6Go). The recovery of arterial and portal [2-13C]acetate in the hepatic vein increased (P < 0.01) with VFA-buffer incubation compared with 0-buffer incubation; however, the total unidirectional uptake of acetate in the liver did not differ (P = 0.38) between treatments. In sheep, it has been observed that the portal recovery of arterial acetate is 65 to 70% under most conditions (Bergman and Wolff, 1971Go; Kristensen et al., 1996aGo,bGo). However, the differences between the VFA-buffer incubation and the 0-buffer incubation seen in the current study were not found in sheep under washed rumen conditions (Kristensen et al., 2000aGo). The explanation for this difference is not known. In agreement with the current study, it has been observed in sheep that the fractional uptake of afferent acetate in the liver is considerably lower than the fractional uptake of arterial acetate by the PDV (Bergman and Wolff, 1971Go) and that the fractional acetate uptake by the liver increased in fasted animals.


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  		Table 6. Atomic fraction excess (AFE) of carbon-13 in blood acetate, portal recovery of arterial [2-13C]acetate, hepatic vein recovery of arterial and portal [2-13C]acetate, portal-drained visceral (PDV) uptake of arterial acetate, hepatic uptake of arterial and portal acetate, and irreversible loss rate (ILR) of [2-13C]acetate infused into the jugular vein in steers incubated with bicarbonate buffers in the washed rumen
 
Previous experiments with sheep have indicated that endogenous acetate production, especially in fasted animals or animals with 0-buffer incubation, could be a large fraction of the total ILR of acetate (Bergman and Wolff, 1971Go; Kristensen et al., 2000aGo). Defining endogenous acetate production as the difference between total ILR and the net portal flux + PDV uptake of arterial acetate gives a negative estimate for endogenous acetate production in the current study. However, the positive net hepatic flux of acetate with both 0- and VFA-buffer in the current study and in most other net flux experiments (Reynolds, 1995Go) does indicate that cattle would be expected to have net endogenous acetate production. The negative estimate for endogenous acetate production could indicate that the ILR of acetate is slightly underestimated in the current study or that the sum of net portal flux + PDV uptake of arterial acetate is overestimated.

Portal Recovery of Absorbed VFA
Isobutyrate was infused into the ruminal vein, and it was assumed that the most reliable estimate for recovery of carbon in ruminally absorbed VFA was obtained by correction of the portal fluxes of VFA, lactate, and 3-hydroxybutyrate by isobutyrate recovery. The total portal recovery of VFA acetyl units was 0.99 ± 0.03 and the recovery of VFA propionyl units was 1.03 ± 0.02 when the portal flux of all VFA as well as 3-hydroxybutyrate and lactate was taken into account (Table 7Go).


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  		Table 7. Portal recovery of VFA absorbed from the washed rumen of steers incubated with bicarbonate buffers in the washed rumen
 
The difference in the acetate recovery between values corrected for PDV uptake of arterial [2-13C]acetate and noncorrected values illustrates the difficulties posed by PDV metabolism relating nutrient absorption to net portal fluxes of arterially abundant metabolites. That the corrected acetate flux is greater than 100% of ruminal acetate absorption does not necessarily mean that the portal flux of acetate is overestimated because epithelial metabolism of butyrate, isovalerate, and valerate could lead to production of acetate by the epithelium. In sheep, it was observed that the corrected acetate recovery was greater than 100% in washed rumen experiments (Kristensen et al., 2000aGo). It has previously been observed that the specific activity of carbon-14-labeled acetate decreased across PDV, liver, and muscles (Pethick et al., 1981Go; Crabtree et al., 1987Go). If there is acetate–acetyl-CoA cycling in PDV tissues not related to acetate uptake from luminal acetate or breakdown of other VFA, it would lead to overestimation of PDV uptake of arterial acetate. However, proper correction for acetate background during sample preparation is important when comparing arterial and venous samples with different levels of acetate. In addition, acetate–acetyl-CoA cycling was found to be low in muscle (Crabtree et al., 1987Go); thus, it seems reasonable to assume that the correction made for PDV uptake of arterial acetate is valid for correction of the portal flux of acetate in order to calculate the total acetate release by the PDV.

The net portal flux of lactate was assumed to represent VFA propionyl units from ruminal VFA, although it is likely that part of the portal lactate is derived from glycolysis in the PDV (Perry et al., 1994Go). The net uptake of glucose in the PDV is not able to account for all lactate released by the PDV (Table 4Go) and the increased net portal flux of lactate with VFA-buffer incubation therefore agrees with the metabolism of a small percentage of absorbed propionate and a larger proportion of absorbed valerate into lactate by the ruminal epithelium. This relatively low extent of propionate metabolism into lactate by the ruminal epithelium is in good agreement with previous estimates of direct carbon transfer from propionate to lactate measured using carbon-14-labeled propionate (Weigand et al., 1972Go; Weekes and Webster, 1975Go).

Our data indicate that there is a large first-pass sequestration of the longer-chain VFA (i.e., butyrate and valerate), but a zero or low first-pass uptake of acetate and propionate. However, although the epithelium is taking up a large fraction of butyrate and valerate, these acids are not completely oxidized by the epithelium, but might be repacked into acetate, 3-hydroxybutyrate, and lactate followed by release to the portal blood. Owing to the high arterial concentration of 3-hydroxybutyrate, the estimate of the unidirectional PDV production of this compound is strongly influenced by PDV uptake of arterial 3-hydroxybutyrate. We have here assumed that the uptake is 15% of the arterial concentration as found in sheep (Kristensen et al., 2000cGo), although this still remains to be investigated in cattle.

Implications for Rumen Epithelial Energy Metabolism
Bergman and Wolff (1971)Go suggested that the ruminal epithelium metabolized 30, 50, and 90% of ruminally absorbed acetate, propionate, and butyrate, respectively. So far, it has been difficult to account for all of the carbon supposedly metabolized by the epithelium, and it has largely been assumed oxidized; however, that would imply that ruminal epithelium has a metabolic activity that exceeds its expected energy requirement (Sutton, 1985Go; Kristensen and Danfaer, 2001Go). The data obtained in the current study, along with observations from a previous study with sheep (Kristensen et al., 2000aGo), point to the conclusion that the ruminal epithelium does not have superphysiological energy expenditure. The estimate for the epithelial uptake is, however, calculated as a relatively small difference between a large absorption from the rumen and an almost-as-large portal flux of VFA and VFA metabolites. It will therefore be difficult to obtain a precise estimate of the epithelial uptake unless this question is addressed more specifically (i.e., by applying catheterized animal models that more precisely attempt to isolate the rumen from the remaining parts of the PDV; Rémond et al., 1993Go). A major part of the VFA taken up by the epithelium is seemingly metabolized into 3-hydroxybutyrate and to a minor extent acetoacetate. However, even though it is assumed that epithelial ketogenesis is solely located in the mitochondria, and the FADH2 generated by acyl-CoA dehydrogenase therefore can drive ATP synthesis (2 ATP/FADH2), the total ATP production from ketogenesis might be as low as 0.75 ATP per butyrate metabolized. It is assumed that this lower ATP production would occur if butyrate is activated by acyl-CoA synthetase with consumption of two ATP (Aas, 1971Go) and if 75% of ketone bodies released is 3-hydroxybutyrate, whereby 75% of the NADH generated is used for reduction of acetoacetate and not ATP synthesis.

Net Splanchnic Flux of VFA Carbon
The increase in net splanchnic flux of acetyl units in acetate, butyrate, isovalerate, valerate, and 3-hydroxybutyrate could account for 0.64 ± 0.02 of ruminally absorbed VFA acetyl units. This value largely reflects that the uptake of arterial acetate by the PDV increased by more than 30% of the increase in the acetate absorption with the VFA buffer. It is also likely that the PDV uptake of arterial 3-hydroxybutyrate is reducing the net splanchnic acetyl flux.

The increase in net splanchnic flux of propionyl units with the VFA buffer accounted for only 0.34 ± 0.05 of absorbed VFA propionyl units. The hepatic propionate uptake accounted for 93% of net portal flux of propionate; however, even though the increased portal flux of propionate could account for the entire net hepatic flux of glucose, the net hepatic glucose flux did not change. The fact that the net splanchnic glucose flux remained unchanged without VFA absorption from the rumen means that the true change in net splanchnic flux of propionyl units has been masked by substitution between propionate and unidentified glucogenic substrate that is expected to be either amino acids or hepatic glycogen.

Conclusion
In conclusion, the ruminal epithelial first-pass uptake of absorbed acetate is not detectable. The epithelium might be producing acetate from other ketogenic VFA. The ruminal epithelium is metabolizing approximately 10% of the absorbed propionate, and the increased net portal flux of lactate points to lactate as the product of propionate metabolism. Butyrate and valerate are extensively metabolized during the first pass; however, they seem to be repackaged primarily into 3-hydroxybutyrate by the epithelium. Approximately half of the isovalerate was metabolized by the epithelium. The PDV uptake of arterial acetate was 35 to 45% of the unidirectional acetate uptake to the portal blood and had a large effect on the availability of acetyl units to peripheral tissues.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
Literature Cited
 
The current study shows that the ruminal epithelium of cattle does not have superphysiological energy metabolism and that the portal flux of volatile fatty acids, lactate, and 3-hydroxybutyrate can account for the volatile fatty acids being absorbed from the rumen. This points to the use of portal fluxes of volatile fatty acids and volatile fatty acid metabolites to quantify energy supply from volatile fatty acids in ruminants.


   Footnotes
 
1 The Danish Agric. and Vet. Res. Council (Grant No. 23-01-0156) and the Univ. of Kentucky Agric. Exp. Stn. supported the study. Publication No. 03-07-154 of the Univ. of Kentucky Agric. Exp. Stn. The skilful assistance of C. Adkins, J. Combs, K. Hanson, B. Kitts, J. Piel, and D. True is gratefully acknowledged. Back

2 Correspondence: Blichers Allé D20, DK-8830 Tjele (phone: +45-8999-1109; fax: +45-8999-1166; e-mail: nbk{at}agrsci.dk).

Received for publication December 10, 2003. Accepted for publication March 17, 2004.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 


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