H. Xingtai,1 X. Bai,1 D. Jizeng2 and H. Linghao3
1. Qinghai Academy of Animal and Veterinary Sciences, Xining 810003, Qinghai,
P.R. China
2. Department of Biology, Zhejiang University, Hangzhou 310027, Zhejiang, P.R. China
3. Qinghai Committee of Science and Technology, Xining 810000, Qinghai, P.R. China
The present experiment was conducted to quantify the net fluxes of both free (FAA) and peptide amino acids (PAA) across the mesenteric and stomach portions of the portal-drained viscera (PDV) of three yak cows (weighing 172.3 ± 18.6 kg) fitted with sampling catheters in the portal vein, mesenteric artery and mesenteric vein prior to its convergence with the gastrosplenic vein. Blood flow was determined by measuring the dilution of para-aminohippurate (PAH) infused constantly into a distal mesenteric vein. Amino acids in the deproteinised plasma were analysed before and after acid hydrolysis. The increased amino acids after acid hydrolysis were considered as PAA. Portal blood flow was 389 L/h or 2.32 L/h kg body weight (BW), of which 37% was contributed by the mesenteric vein. There was net appearance of a large quantity of PAA across PDV, which accounted for 92% of the total nonprotein amino acid flux. Net release of PAA and FAA in stomach-drained viscera (SDV) accounted for 78% and 42% of the net release in PDV, respectively. These results suggest that, in yak, the peptide possibly to be the primary form of amino acid absorption, and that the stomach area probably to be the major site of peptide absorption.
Keywords: Amino acids, flux, mesenteric, peptide, stomach, yak
Recent studies suggest that the peptide may be the primary form of amino acid absorption in the ruminants (Koelln et al. 1993; Webb et al. 1993; Seal and Parker 1996), and that the site of absorption of these peptides does not appear to be the mesenteric viscera but, instead, appears to be the stomach viscera (Webb et al. 1993). Further in vitro studies suggest that the forestomach has the ability to absorb dipeptide (Matthews and Webb 1995), and that the omasal epithelium contains mRNA that codes for protein capable of di-, tri-, and tetrapeptide transport (Matthews et al. 1996; Pan et al. 1997). Considerable evidence has been accumulated regarding the absorption of dipeptides and tripeptides. However, the nutritional and metabolic significance of peptide absorption is not fully understood, and the extent to which intact peptides may be absorbed into the blood stream is controversial (Webb et al. 1993; Backwell et al. 1997; Han 1999), especially in ruminants. The objective of the present study was to quantify the in vivo fluxes of both FAA and PAA across the mesenteric and stomach portions of the PDV in yak.
Three non-pregnant yak cows in late stage of lactation (1 kg of daily milk production) and at an average body weight of 172.3 ± 18.6 kg, were surgically fitted with in-dwelling catheters in the portal vein, the anterior mesenteric vein. The diet used consisted of field pea straw (500 g/kg), maize (335 g/kg), fish meal (80 g/kg), soy bean meal (70 g/kg), bone meal (10 g/kg) and salt (5 g/kg), and contained 133.5 g crude protein (CP) and 9.62 MJ ME/kg dry matter (DM). Each animal was offered 2.0 kg DM/day, which was calculated to meet maintenance energy (ME) requirement according to Han and Xie (1991), and this allotment was offered to the animals at 08:00 am and 20:00 pm in two equal meals.
On the sampling day, a primed (20 mL), and then a continuous mesenteric venous infusion of PAH (6%, wt/vol., pH 7.4) was initiated via the distal mesenteric vein catheter to determine portal and mesenteric venous blood flows. One hour after the infusion, simultaneous arterial, portal and mesenteric venous blood samples were collected slowly into syringes containing heparin. For PAA and FAA analyses, six mL of blood from each vessel were taken six times at 2-h intervals; and for PAH analysis, an additional 1.5 mL of blood was collected at 1-h intervals.
In the laboratory, packed cell volume was determined. Huntington (1982) described that the concentration of PAH in the whole blood was measured colorimetrically. In later analysis, pooled plasma samples were obtained from the six individual samples within yak and within sampling site, and deproteinised by the addition of an equal volume of sulfosalicylic acid (20%, wt/vol.). Precipitated protein was removed by centrifugation at 15,000 × g for 15 minutes at 4°C. The supernatant was divided into two portions, one of which was hydrolysed in 6 N HCl at 110°C for 24 h. Both samples were then subjected to analysis on an amino acid analyser (HITACHI 835–50). The difference before and after hydrolysis was considered as PAA.
Portal and mesenteric blood flows were calculated as described previously (Han et al. 1997). Net fluxes of FAA and PAA across PDV and mesenteric-drained viscera (MDV) were calculated as described by Seal and Parker (1996). Stomach flux was calculated as the difference between portal and mesenteric fluxes. Mean net fluxes across portal, mesenteric and gastrosplenic veins were compared using Student's T-tests; a probability of P ≤ 0.05 was used to test for significance.
The portal and mesenteric blood flows averaged 389 L/h and 144 L/h or 2.32 L/h kg BW and 0.84 L/h kg BW, respectively. The mesenteric blood flow accounted for 37% of the portal blood flow. Large differences (for all amino acids, P< 0.001) between FAA and PAA concentrations in the same blood vessel were found, and the concentrations of PAA were 5.6, 5.1 and 5.2 times higher than that of FAA in the portal, mesenteric veins and artery, respectively.
Net release of FAA across the gastro-intestinal tract was minimal (Table 1) and the net portal flux was 40 g/day, of which 23 g came from the mesenteric viscera and 17 g from the stomach viscera, accounting for 58.5% and 41.5% of the total FAA appearing in the portal vein, respectively. Net release of PAA is shown in Table 1. The net flux of total PAA across the portal-drained viscera was 459 g/day, of which 358 g came from the stomach viscera and 101 g came from MDV, accounting for 78% and 22% of the total PAA, respectively. Compared with FAA, PAA appeared the major contributor to the total amino acid flux. Unlike FAA, almost all PAA in the portal vein came from the stomach viscera.
It is accepted that peptide absorption is an important physiological process in ruminants and may constitute the primary source of absorbed amino acids. In the present study, the net appearance of small peptides in the portal circulation accounted for 92% of total amino acid flux. This result was in good agreement with the results (87% to 90%) observed by Koelln et al. (1993); Webb et al. (1993) in steers and sheep, but was somewhat higher than those (63% to 70%) reported by Koelln et al. (1993) and Seal and Parker (1996) in steers. The origin of these peptides could not be determined by the techniques applied in the present study. Absorption from the lumen may be a logical explanation for the net appearance of these peptides in the portal circulation. Another explanation might be that these peptides are possibly the result of synthetic activity in the tissues drained by the portal vein. A combination of these explanations seems likely. The net flux of FAA across PDV in the present study was low as compared with PAA; this result was also similar to the observations of Koelln et al. (1993) and Webb et al. (1993). An explanation may be that the tissues between the lumen and the portal vein used a large part of the absorbed FAA as indicated by Seal and Parker (1996) and MacRae et al. (1997).
In the present study, the total appearance of FAA and PAA in the portal vein was 500 g/day, which was 1.9 times the intake of dietary protein. This result agrees with the observation by Koelln et al. (1993). Based on the evidence that chemical deproteinisation overestimates the PAA concentration in the plasma (Bernard and Rémond 1996; Backwell et al. 1997), it is suspected that the high flux of peptides may be due to the effect of the deproteinisation procedure (Neutze et al. 1996; Backwell et al. 1997). However, Seal and Parker (1996) reported that, even after the treatments of both chemical deproteinisation and physical filtration, the PAA flux still accounted for 63% of the net portal appearance of total amino acids. In their study, the net flux of free and peptide amino acids also was 1.6 times the protein intake. In young calves, the total appearance of FAA and PAA was as high as 438 g/day even when the animals were deprived of feed for 72 h (Koelln et al. 1993). These observations suggest that a large part of the small peptides might be the degradation products resulting from the turnover of tissue protein in the gastro-intestinal tract, spleen, pancreas or a combination of these organs.
Table 1. Blood flow (L/h) and net fluxes (g/d) of free (FAA) and peptide amino acids (PAA) across the portal (PV), mesenteric (MV) and stomach (SV) viscera of yak cows fed a concentrate-straw diet at maintenance level (mean ± SE).
Free amino acids |
Peptide amino acids |
Difference | |||||
PV |
MV |
SV |
PV |
MV |
SV |
||
Blood flow |
388.9 (3.9) |
143.9 (3.5) |
245.0 (3.9) |
388.9 (3.9) |
143.9 (3.5) |
245.0 (3.9) |
|
Plasma flow |
240.7 (3.3) |
89.4 (2.9) |
151.3 (3.1) |
240.7 (3.3) |
89.4 (2.9) |
151.3 (3.1) |
|
Aspartate |
1.9 (0.6) |
1.4 (0.6) |
0.6 (1.8) |
42.3 (1.5) |
8.5 (1.0) |
33.8 (1.5) |
a, b, c, d |
Threonine |
4.0 (1.4) |
1.0 (0.5) |
3.3 (0.9) |
27.8 (2.0) |
4.5 (1.1) |
23.3 (1.3) |
a, c, d |
Serine |
2.1 (0.7) |
1.5 (0.6) |
0.6 (1.2) |
32.0 (1.8) |
4.3 (0.9) |
27.7 (1.3) |
a, c, d |
Glutamate |
4.4 (0.7) |
1.3 (1.0) |
3.1 (0.8) |
63.0 (2.6) |
0.3 (0.4) |
63.3 (1.5) |
a, c, d |
Glycine |
4.3 (1.0) |
2.5 (0.8) |
1.8 (1.8) |
38.6 (1.6) |
16.0 (1.4) |
22.6 (1.1) |
a, b, c |
Alanine |
5.8 (0.8) |
4.1 (1.1) |
1.7 (1.2) |
22.3 (1.5) |
6.0 (1.0) |
16.3 (0.9) |
a, c, d |
Valine |
2.8 (0.8) |
1.6 (1.0) |
1.2 (0.6) |
27.7 (1.6) |
4.0 (1.2) |
23.7 (1.2) |
a, c, d |
Methionine |
0.6 (0.4) |
0.4 (0.5) |
0.2 (0.9) |
3.2 (0.6) |
1.1 (0.5) |
2.1 (0.4) |
|
Isoleucine |
1.6 (0.6) |
0.9 (0.4) |
0.7 (0.8) |
21.5 (1.4) |
6.8 (1.2) |
14.6 (1.3) |
a, c, d |
Leucine |
1.4 (0.6) |
1.8 (1.2) |
-0.4 (2.2) |
39.4 (1.3) |
7.2 (0.9) |
32.2 (1.2) |
a, c, d |
Tyrosine |
1.4 (0.5) |
0.1 (0.4) |
1.4 (1.1) |
18.4 (1.6) |
4.0 (1.1) |
14.4 (0.69) |
a, c, d |
Phenylalanine |
3.2 (0.7) |
1.8 (1.1) |
1.5 (0.5) |
21.2 (1.5) |
5.0 (0.8) |
16.3 (1.0) |
a, c, d |
Lysine |
1.4 (0.6) |
1.6 (0.7) |
-0.2 (1.8) |
32.7 (1.8) |
6.6 (0.8) |
26.1 (1.5) |
a, c, d |
Histidine |
1.5 (0.6) |
0.7 (0.7) |
0.8 (1.2) |
11.4 (1.4) |
2.0 (0.8) |
9.3 (1.2) |
a, c, |
Arginine |
3.0 (0.7) |
2.2 (1.2) |
0.7 (0.9) |
22.6 (1.3) |
3.4 (1.4) |
19.3 (0.9) |
a, c, d |
Proline |
0.8 (0.4) |
1.0 (0.9) |
-0.1 (1.1) |
32.0 (1.8) |
3.4 (0.7) |
28.7 (1.6) |
a, c, d |
Essential amino acids (EAA) |
19.4 (1.5) |
11.6 (1.6) |
7.7 (1.7) |
207.5 (3.2) |
59.4 (2.2) |
148.1 (2.3) |
a, b, c, d |
Non-essential amino acids (NEAA) |
20.9 (1.2) |
11.7 (1.3) |
9.3 (0.9) |
252.1 (2.9) |
41.9 (1.5) |
210.2 (2.0) |
a, b, c, d |
Total amino acids (TAA) |
40.2 (1.4) |
23.3 (1.4) |
16.9 (1.3) |
459.5 (3.1) |
101.2 (2.7) |
358.3 (2.2) |
a, b, c, d |
| a, b and c indicate significant differences (P<0.05) between free and peptide amino acid fluxes in the portal, mesenteric and stomach visceras, respectively. d indicates significant difference (P<0.05) in flux of peptide amino acid between mesenteric and stomach viscera. | |||||||
The recent observation that the stomach region of the gastro-intestinal tract may be an important site of peptide absorption is quite significant. The work of Webb et al. (1993) indicated that 86% and 90% of PAA in the portal veins of sheep and calves, respectively, came from the stomach viscera. Seal and Parker (1996) measured the net flux of PAA using the fractionation method separating a low-molecular-weight (<10,000 Da) peptide fraction by filtration and then HPLC (high performance liquid chromatography) to separate four fractions containing peptides with molecular weights of <1500 Da. They found that some 40% of portal PAA were from MDV, and suggested that the remaining 60% were from the stomach viscera, and that the stomach tissue may be a major site of peptide absorption. In the present experiment, 78% of the absorbed PAA were from the stomach viscera. The ability of the forestomach to absorb small peptide has been demonstrated by in vitro (Matthews and Webb 1995) and in vivo (Bernard and Rémond 1999) studies.
Although only 8% of the total amino acids appearing in the portal vein were FAA, 42% of these FAA were from the stomach viscera. From this percentage it could be said that the stomach might be an additional site of amino acid absorption. Webb et al. (1993) also indicated that 22% and 11% of the total FAA appearing in the portal vein of calves and sheep, respectively, were from the stomach viscera. Matthews and Webb (1995) shown with in vitro procedures using both radiolabelled and nonradiolabelled methionine the potential for amino acid absorption across the mucosal tissues of rumen and omasum. In addition, Leibholz (1971) observed transfer of histidine, glycine, lysine and arginine across isolated ruminal epithelial tissues of sheep. These observations suggest that the forestomachs of ruminants have the ability to absorb amino acids.
This study was part work of project 39560062 supported by National Natural Science Foundation of China. The authors would like to thank A.Y. Xie and S.T. Chai for the assistance in surgery, Lajia and Y.J. Guo for the care and maintenance of animals, and X.W. Zhang for the assistance in analyses.
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