15N-leucine and 15N-isoleucine isotope dilution techniques versus the 15N-isotope dilution technique...

"N-Leucine and 15NN-Isoleucine Isotope Dilution Techniques Versus the 15N-Isotope Dilution Technique for Determining the Recovery of Endogenous Protein and Amino Acids

in Digesta Collected from the Distal Ileum in Pigs'

C.F.M. de Lange*p2, W. C. Sauer*.3, W. B. Souffrantt, and K. A. Lien*

*Department of Animal Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada and

+Academy of Agricultural Science, Oskar Kellner Institute, Rostock, Federal Republic of Germany

ABSTRACT: Twelve gilts, with an average initial BW of 38 kg, were fitted with simple T-cannulas at the distal ileum and two catheters in the external jugular veins. The animals were fed twice daily (0800 and 20001 700 g of one of four diets containing either soybean meal, canola meal, wheat, or barley as the sole protein source. 15N-leucine was infused continuously for 9 d at a rate of 40 mg/kg of B W daily via one of the jugular catheters. Blood samples were taken during feeding. Ileal digesta samples were collected continuously for 24 h on d 7 and 9 of the infusion period. 15N-enrichment excess in leucine and isoleucine in the trichloroacetic acid (TCAl-soluble fraction of blood and in digesta were measured using single-ion monitoring gas chromatography-mass spectrometry. As-

Key Words: Pigs, Endogenous Protein, Leucine,

suming that the 15N-enrichment excess in leucine and isoleucine in the TCA-soluble fraction of the blood is similar to that in endogenous protein secreted into the digestive tract and that the amino acid composition of endogenous protein is constant, the contribution of endogenous to total protein in ileal digesta was calculated using the 15N-isotope dilution technique for leucine and isoleucine, respectively. These contributions were much smaller for the 15N-leucine than for the 15N-isoleucine isotope dilution technique: 7.1 to 11.0 vs 21.8 to 24.9 g of protein/kg of DM intake. The values obtained with the 15N-isoleucine isotope dilution technique were close to those obtained with the 15N-isotope dilution technique as described in a previous publication.

Isoleucine, Ileal Digesta, Nitrogen Metabolism

Introduction

The 15N-isotope dilution technique seems to be one of the most valid methods for determining the recovery of endogenous protein at the distal ileum in pigs fed protein-containing diets (Souffrant et al., 1981, 1986; de Lange et al., 1990). By estimating

'Financial support was provided by the Farming for the Future Program of the Alberta Agric. Res. Council and the Natural Sciences and Eng. Res. Council of Canada.

'Present address: Ralston Purina Canada Inc., Woodstock, ON.

3To whom correspondence should be addressed. Received February 11, 1991. Accepted January 9, 1992.

J. Anim. Sci. 1992. 70:1848-1856

the 15N-enrichment excess in endogenous protein previously labeled via a continuous intravenous infusion of 15N-leucine, and in digesta, the contribution of endogenous to total protein in digesta can be determined. The enrichment excess in N in the trichloroacetic acid (TCAI-soluble fraction of blood is considered similar to that in endogenous protein secreted into the digestive tract.

The 15N-enrichment excess is not similar for all amino acids and other N-containing molecules, such as urea, present in the TCA-soluble fraction of the blood when 15N-leucine is infused continuously (Matthews et al., 1979). This may represent a source of error when using the 15N-isotope dilution technique, especially if a considerable portion of the total 15N-enrichment excess is present in

1848

ISOTOPE DILUTION AND

nonprotein N. The 15N-enrichment in individual amino acids, which would provide a more direct measure of the enrichment excess in endogenous protein, can be determined by gas chromatography-mass spectrometry (GCMSI. Assuming that endogenous protein composition is constant, the recovery of endogenous protein can then be determined from the recoveries of endogenous amino acids.

The objectives of the present studies were to determine, by use of alternative 15N-leucine and 15N-isoleucine isotope dilution techniques, the contribution of endogenous leucine, isoleucine, and protein to total leucine, isoleucine, and protein, respectively, in ileal digesta of pigs fed different protein-containing diets and to compare these results to those obtained with the 15N-isotope dilution technique (de Lange et al., 1990).

Materials and Methods

Detailed descriptions of the experimental procedure and composition of diets were presented previously (de Lange et al., 1990). Twelve gilts, fitted with a simple T-cannula a t the distal ileum and two catheters in the external jugular veins, were fed one of four experimental diets, which contained either soybean meal, canola meal, wheat, or barley as the sole protein source. I5N-1eucine was infused continuously for 9 d at a rate of approximately 40 mg/kg of BW daily via one of the jugular catheters. Blood samples were taken twice daily during feeding. Digesta samples were collected continuously for 24 h on d 7 and 9 of the infusion period.

Samples of blood and digesta were prepared for analyses as described previously (de Lange et al., 1990). Immediately after collection, blood samples were centrifuged, and the plasma was separated into TCA-soluble and TCA-precipitable (plasma protein) fractions. The TCA-soluble fraction (6 mL), containing free plasma amino acids and other plasma molecules such as urea, was diluted to 25 mL with 10% TCA. Digesta samples were frozen immediately after collection, freeze-dried, and ground (Model 4 Laboratory Wiley Mill, Arthur H. Thomas Co., Philadelphia, PA) through a 1-mm mesh screen before further analyses.

AnaZyticaZ Procedure. The methods for analyses of DM, chromic oxide, CP (N x 6.251, and amino acids in feed and digesta were described previously (de Lange et al., 19901. The I5N-enrichrnent excess in the TCA-soluble fraction of blood was determined using an emission spectrometer (Isonitromat RFT 520 1, VEB Statron, Fuersten- walde, FRG) (de Lange et al., 19901.

ENDOGENOUS N IN PIGS 1849

The l5N-enrichment excess in leucine and isoleucine was determined according to procedures adapted from those described by Haymond et al. (1980) and Krishnamurti and Schaefer (1987). To avoid complications during derivitization, TCA was removed from plasma samples before analyses by extraction from 800 pL of the diluted TCA- soluble fraction of blood by vigorously mixing with an equal volume of diethyl ether. After separation, the ether layer was removed and discarded. This procedure was repeated three times. Negatively charged ions and neutral organic compounds were then removed from the samples using anion and cation exchange columns. The aqueous layer (pH 2 to 31 was applied to 2 mL of AGl-X8 anion- exchange resin (200 to 400 mesh, formate form; Bio- Rad Laboratories, Richmond, CAI placed in 3-mL syringes from which the stopper was removed. The effluent was applied directly to 2 mL of AG 50-X8 cation-exchange resin (200 to 400 mesh, hydrogen form; Bio-Rad), also placed in 3-mL syringes. The anion- and cation-exchange resins were washed previously with neutralized, deionized water. After the samples were applied to the anion-exchange column, the columns were washed five times with 1-mL portions of neutral, deionized water. The anion-exchange column was discarded and the cation-exchange column was washed twice with 1 mL of neutral, deionized water. The effluent from the cation-exchange column was discarded. The amino acids were eluted from the cation-exchange column with freshly prepared 4 N NH40H solution. First, 1 mL of the NH40H solution was applied to the column and the effluent was discarded. Thereafter, 4 mL of the NH40H solution was applied to the column and the effluent was collected in 5-mL reaction vials, frozen, and freeze-dried. Trimethylsilyl (TMS) derivatives were prepared with the addition of 50 pL of N,O-bis-trimethylsilyl-trifluoracetamide (BSTFA) (Sigma Chemical, St. Louis, MO) and 100 pL of acetonitrile (Sigma). The reaction vials were tightly capped and kept at room temperature for 48 h. The vials were then stored at 4°C. The samples were injected into the GCMS within 5 d after derivatization.

Each digesta sample (1 g) was hydrolyzed in 100 mL of 6 N HC1 for 24 h (Blackburn, 1968). After hydrolysis an aliquot of approximately 250 pL, containing approximately . 2 O pmol of leucine, was applied to the anion- and cation-exchange columns in the same manner as described for the blood plasma samples. Subsequent procedures for the preparation of digesta samples for GCMS were similar to those described for blood plasma samples.

For 15N-enrichment analyses by GCMS (Varian Vista 6000 gas chromatograph [Varian, Sunnyvale, CAI connected to a 7070E Vg analytical organic

1850 DE LANGE ET AL.

mass spectrometer [VG Analytical, Wythenshaw, Manchester, UKI), approximately 1 pL of derivi- tized sample was injected onto a DB5 gas chrom- atographic column (.32 mm i.d. x 30 m) (J&W Scientific, Folsom, CAI. Helium was used as the carrier gas at a flow rate of approximately 1 mL/s. The column temperature was isothermal at 140' C with injector port and interface temperatures at 290 and 300" C, respectively. Electron impact ionization, source temperature of 25OoC, and energy set at 40 eV were used for mass spectrometry. Ion fragments at mass-to-charge ratios (rn/z1 of 158.2 and 159.2, corresponding to the 14N and 15N ion fragments, respectively, of both leucine and isoleucine were measured using single ion monitoring. Because molecular weights of derivatives of leucine and isoleucine are similar, no additional adjustments were necessary for simultaneous measurement of the enrichment in leucine and isoleucine. Run time was 4 min with a dwell time of 60 ms. Samples were injected every 5 min.

The observed peak areas of the labeled species (l5N-1eucine and 15N-isoleucine) were corrected for natural abundance according to procedures described by Campbell (1974). Because isotopic enrichment does not have a significant effect on the ionization efficiency, the 15N-enrichment excess in leucine and isoleucine were derived directly from the peak area ratios (Campbell, 1974; Matthews et al., 19811. For leucine, the 15N-enrichment excess was calculated using the following formula:

PA m / z 159 (1 + .0036591 x PA m / z 158 + PA m/z 159

15N-leu excess 14N-leu + I5N-leu

- -

where PA m/z 159 = peak area at m / z 159, corrected for natural abundance (.14751 x PA m/z 158); PA rn/z 158 = peak area at m/z 158; '5N-leu excess = 15N-leucine in sample, corrected for natural abundance in N COO3659 x 14N); 14N-leu = 14N-leucine in sample; and 15N-leu =

15N-leucine in sample. The same formula was used to calculate the 15N-enrichment excess in isoleucine. The natural abundance of 15N was determined to be .3673%. Therefore, it was calculated that .3659% [.003673/(1 + .00367311 of the peak area at m/z 158 attributed to the peak area a t m/z 159 was due to natural abundance of I5N. The contributions of the natural abundance of isotopes of all atoms present in leucine and isoleucine derivatives to the peak area at m/z 159 were determined daily from peak area ratios in unlabeled standards. The determined peak area ratios in the standards were very similar to those calculated based on the natural abundance of isotopes of the

atoms present in derivatized leucine and isoleucine (.14751 of the peak area at m/z 158, Campbell, 1974). The formula, however, was adjusted daily for the peak area ratios in the standards.

The contributions of endogenous leucine and isoleucine to total leucine and isoleucine in ileal digesta were calculated in the same manner as the 15N-isotope dilution technique for measuring endogenous N (Souffrant et al., 1981, 1986; de Lange et al., 19901. The 15N-enrichment excess in endogenous leucine (or isoleucine) in digesta was assumed to be similar to that in free plasma leucine (or isoleucine) in the blood samples taken during collection of ileal digesta. The ratio in 15N-enrichment excess in leucine (or isoleucine) in digesta and in free plasma amino acids indicates the contribution of endogenous leucine (or isoleucine) to total leucine (or isoleucine) in ileal digesta. The endogenous recovery of the other amino acid(s1 and total protein in ileal digesta could also be calculated assuming that the amino acid composition of endogenous protein is constant (de Lange et al., 1989a,bl. In these calculations, the leucine and isoleucine contents of endogenous protein were assumed to be similar to those observed in pigs fed a protein-free diet and intravenously administered with amino acids si- multaneously (de Lange et al., 1989b). Real ileal protein, leucine, and isoleucine digestibilities were then calculated from the apparent protein and amino acid digestibilities and the recovery of endogenous protein and amino acids in ileal digesta. The real protein and amino acid digestibilities in these diets were determined simulta- neously using the 15N-isotope dilution technique (de Lange et al., 1990). Apparent protein and amino acid digestibilities, corrected for endogenous protein and amino acids, are referred to as real digestibilities (Krawielitzki et al., 1977).

Statistical Procedure. The observed 15N-enrichment excess in each of the different N-containing fractions was analyzed using three-way analyses of variance with diet, collection day, and animal as sources of variation. The recovery of endogenous protein and amino acids and the calculated real ileal protein and amino acid digestibilities, as determined with each of the isotope dilution techniques, were analyzed using two-way analyses of variance with diet and collection day as sources of variation (Harvey, 1960). Equality of variances for the 15N-enrichment excess in the different N- containing fractions were tested with an F-test using the mean square error (Steel and Torrie, 19801. The recoveries of endogenous protein and amino acids and the calculated real protein and amino acid digestibilities as determined with the different isotope dilution techniques were analyzed in a similar manner. Treatment means were

ISOTOPE DILUTION AND ENDOGENOUS N IN PIGS 1851

compared using a t-test for means of samples with unequal variance (Steel and Torrie, 1980).

Results and Discussion

The 15N-enrichment excess in free plasma leucine was approximately 25 to 30 times higher than that in total N of the TCA-soluble fraction of blood (Table 1). The 15N-enrichment excess in free plasma isoleucine was only 15 to 25% of that in leucine. Matthews et al. (19791, in studies with dogs, showed that the 15N incorporation into plasma amino acids varied largely among individual amino acids when 15N-leucine was infused continuously. The 15N-enrichment excess in leucine was much higher than that in other amino acids after a 9-h infusion period. In addition, alanine, valine, isoleucine, serine, glutamate, and ornithine showed a significant incorporation of 15N. 15N-glycine was administered orally to male human adults for a 60-h period in another study by Matthews et al. (198 1). The observed 15N-enrichment excess was highest in glycine, followed by serine and urea. This study supports the studies by Matthews et al. (1979, 19811, which illustrate that the 15N-enrichment excess is highest in the amino acid that is used to administer the 15N-isotope, and that metabolic processes, especially transamination, result in differences in the 15N-enrichment excess in urea and individual plasma amino acids. Amino acids, such as lysine and threonine, that do not participate significantly in transamination are expected to show little or no incorporation of I5N (Matthews et al., 1979, 1981; Rodwell, 1988).

The 15N-enrichment excess in total N, leucine, and isoleucine (Table 1) is a function of the infusion rate of labeled leucine, the flux rate of leucine, and the metabolic pathways through which N from leucine is distributed to other amino acids and the total N pool. Because the total N flux is much larger than the leucine flux, the l5N-enrichment excess in total N is expected to be lower than that in leucine (Fuerst, 1983). Because of the close metabolic association of branched- chain amino acids in various tissues (Harper et al., 19841, the 15N-enrichment excess in isoleucine would be expected to be closer to that of leucine than to that of total N. The advantage of the long infusion period used in the present study, as opposed to those used by Matthews et al. (1979, 19811, is that equilibrium is expected to be achieved in the labeling of amino acids and urea in the blood plasma pool as well as in the labeling of endogenous proteins. This will permit a more accurate measurement of the enrichment excess in endogenous protein in ileal digesta because proteins with more rapid turnover rates (e.g.,

pancreatic and salivary secretions) and proteins with slower turnover rates (e.g., epithelial cells) are expected to have a similar percentage enrichment excess. The long infusion period will not, however, minimize the differences in labeling among individual amino acids and urea.

Because the amino acid composition of endogenous protein is different from the amino acid profile in blood plasma (Davey et al., 1973; Keith et al., 1977; Wuensche et al., 1987; de Lange et al., 1989a,b) and the 15N-enrichment excess is not similar for all amino acids, the I5N-enrichment excess in total N in the TCA-soluble fraction of blood may provide an incorrect estimate of the enrichment excess in endogenous protein recovered in digesta collected at the distal ileum. Moreover, ammonia and urea present in blood plasma contribute much more to total N in the TCA-soluble fraction of the blood than to total endogenous N recovered at the distal ileum. The maximum contribution of N in urea and ammonia to endogenous N in ileal digesta is approximately 25% (Wuensche et al., 1987; de Lange et al., 1989a,b). Ammonia in blood plasma contributed approximately 25% to total N present in the amino acids and ammonia measured in blood plasma of young pigs (Chavez and Bayley, 1977). Urea N in plasma of 40-kg growing pigs contributed approximately 40% to total N present in amino acids and

Table 1. The 15N-enrichment excess (%] in total N, leucine, and isoleucine in the TCA-soluble fraction of blood and in digesta collected from the distal

ileum in pigs fed the ex erimental diets and continuously administered P - N leucine intravenously

Diet

15N-enrichment [%I excess in

Total N Leucine Isoleucine

- TCA-soluble fraction of blooda - Soybean meal . I gd 4. 15e .98f

Wheat .18d 5.1Qe .88f Barley .2 ld 5.96e .89f

Canola meal . 1 gd 4.04e 1 .04f

SEb ,003 ,467 ,065 cvc 1.7 10.4 6.8

Ileal digesta Soybean meal .13d .88e .59f Canola meal .08d .62e .40f Wheat . lad 1 .25e .70f Barley .17d 1 .06e .56f SEb ,003 .057 ,048 c v c 2.1 10.4 8.5

&Based on values in blood samples taken during the collection of digesta from the distal ileum on d 7 and 9 of the infusion period.

bStandard error of the four means within each column. CCoefficient of variation of the four means in one column. d,e,fValues in the same row followed by different super-

scripts differ (P c ,051.


Table 2. The recovery of endogenous protein, leucine, and isoleucine (g/kg of DMI) in digesta collected from the distal ileum in pigs

fed the experimental diets and continuously administered 15N-leucine intravenously as determined with

different isotope dilution techniques

Soybean Canola SE Barley Item me a1 meal Wheat

Protein 1 5 ~ a 25.5d 30.5d 27.4d 27.7d 2.37 15N-leub 9.0e 11.0e 7.7e 7.1e 1.58 5N-ilec 23.0d 24.gd 21.8d 22.2d 4.34

Leucine 1 5 ~ a 1 .gd 15N-leub .5e 15N-ilec 1.2d

1 5 ~ a .9d 15N-leub .3e

Isoleucine

15N-ilec .Bd

1.6d 1 .qd 1.4d .12

1.3d l . ld 1.2d .22 .Be .4e .4e .08

1.0d .9d .9d .08 .4e .3e .2e .05 .Bd .7d .7d .15

~ _____ _____ ~ _ _ _ ~

a 15N-isotope dilution technique.

d,eValues within a column, within method, followed by different superscripts differ ( P <

15N-leucine isotope dilution technique. 15N-isoleucine isotope dilution technique.

051

urea (Davey et al., 19731. The 15N-enrichment excess in urea and ammonia in the present study was probably somewhat higher than that in other N-containing compounds in the TCA-soluble fraction of blood. The 15N-enrichment excess was higher in urine than in the TCA-soluble fraction of the blood (de Lange et al., 19901. Urea and ammonia N accounted for approximately 85 to 95% of N excreted in the urine of growing pigs (Reeds et al., 1980).

The 15N-enrichment excess in leucine and isoleucine was more variable than that in total N in the TCA-soluble fraction of blood (Table I), probably as a result of the shorter turnover times of the free leucine and isoleucine pools than of the N pool. Fuerst (1983) indicated that the turnover time of the free leucine pool is about 10-fold shorter than that of all free amino acids combined in adult human subjects. The turnover times of the free leucine and isoleucine pools were estimated to be .8 and .7 h, respectively. On the other hand, the urea pool, the single largest contributor of N to the TCA-soluble fraction of blood, has a relatively long turnover time. Mosenthin (1987) estimated that the turnover time of the urea pool in pigs with a BW of approximately 80 kg varied between 7.75 and 8.50 h. Short-term changes in the turnover rate of the pools of free leucine, isoleucine, or total N in the TCA-soluble fraction of blood will, therefore, have a larger effect on the 15N-enrichment excess in leucine, and probably also in isoleucine, than in total N. To reduce the variation in the estimated 15N-enrichment excess in free plasma leucine and

isoleucine, blood sampling should be carried out more frequently.

The recoveries of endogenous leucine, isoleucine, and protein in digesta collected from the distal ileum, as determined with their respective isotope dilution techniques, are presented in Table 2. The recoveries of the other amino acid(s1 or protein within the columns were calculated indirectly assuming a constant amino acid composition of endogenous protein. Protein recovery values obtained with the 15N-leucine isotope dilution technique (7.1 to 11.0 g per kg of DM intakel were considerably lower (P < .05) than those with the 15N-isotope dilution technique (25.5 to 30.5 g per kg of DM intake) and the 15N-isoleucine isotope dilution technique (21.8 to 24.9 g per kg of DM intake). As a result, the observed real ileal amino acid and protein digestibilities in the diets differed largely between the different isotope dilution techniques (Table 3). The highest real ileal protein and amino acid digestibilities were obtained with the 15N-isotope dilution technique, followed by the 15N-isoleucine and the 15N-leucine isotope dilution techniques, respectively. The values for protein recovery obtained with the 15N-leucine isotope dilution technique were even lower than those in studies in which pigs were fed protein-free diets (Wuensche et al., 1987; de Lange et al., 1989a,b). This indicates that the amount of endogenous protein recovered at the distal ileum was underestimated using this technique because protein deficiency, in the form of either mild starvation (Poullain et al., 1989) or restricted

ISOTOPE DILUTION AND ENDOGENOUS N IN PIGS

Table 3. Real ileal protein, leucine, and isoleucine digestibilities [ %) in the experimental diets determined with different isotope dilution techniques

1853

Soybean Canola Item meal meal Wheat Barley SE

Protein l 5 ~ a 97.5d 84.1d 99.0d 94.Zd 1.05 15N-leub 88.7e 72.5e 85.3e 75Be 1.69 15N-ileC 96.Zd 80.7d 95.2d 89.3d 3.18

15Na 94.5d 84.1d 98Bd 94.3d 1.07 15N-leub 88.7e 76.7e 88.ge 81.8e 1.06 I5N-ilec 93.7d 82.3d 96.0d 9 1 .od 2.17

Leucine

Isoleucine 1 5 ~ a 95.4d 82.gd 99.5d 94.9d 1.22 15N-leub 89.2e 74.2e a7.ge 80.1e 1.17 15N-ilec 94.5d 80.4d 96.Zd 9 1 .od 2.45

~~~ ~ ~

a 15N-isotope dilution technique.

d,eValues in a column of three values followed by different superscripts differ ( P c ,051.

15N-leucine isotope dilution technique. 15N-isoleucine isotope dilution technique

protein intake (Syme, 1982; Syme and Smith, 1982; Sherman et al., 1985) results in lower protein synthesis and secretion in the intestine. Several factors may be responsible for the differences observed in the recoveries of protein, leucine, and isoleucine in ileal digesta and, therefore, in the observed real digestibilities. These factors would include the 15N-enrichment excess in leucine, isoleucine, and total protein in the actual and assumed precursor pools and the relative contribution of these amino acids to endogenous protein as well as non-digested dietary protein at the distal ileum.

A possible diurnal variation in 15N-enrichment excess in free plasma leucine (or isoleucinel could not be determined because of the pattern of blood sampling used in these studies. Blood samples were only taken during feeding to ensure that the animals were always in the same physiological state; however, dietary amino acids in the samples would be at a minimum a t this time. Amino acids in the TCA-soluble fraction of blood are diluted to a lesser extent by recently absorbed dietary amino acids during feeding compared with 3 to 5 h after feeding, when large quantities of dietary amino acids enter the portal blood (Rerat, 19851. There- fore, the 1SN-enrichment excesses in leucine and isoleucine in blood samples taken during feeding most likely yield an overestimation of the average 15N-enrichment excesses in leucine and isoleucine in the TCA-soluble fraction of blood throughout the 24-h period. As a result, the contributions of endogenous leucine and isoleucine to total leucine and isoleucine, respectively, in digesta were most likely underestimated. Because of the shorter turnover times of the leucine and isoleucine pools,

compared with that of the total N pool, the effect of the pattern of blood sampling will be more obvious for leucine and isoleucine than for total N. More frequent blood sampling would likely provide a more accurate estimate of the 15N-enrichment excess for the entire digesta collection period.

The question of whether the I5N-enrichment excess in leucine (or isoleucine) in the TCA-soluble fraction of blood is a valid indicator of that of leucine (or isoleucinel in endogenous protein that is secreted into the digestive tract also deserves considerable attention. This question is especially critical for endogenous protein that originates from the mucosal epithelium. The majority of mucosal protein within the intestinal lumen is derived either from secretions along the villus (mucin and digestive enzymes) or from the loss of cells from the tips of the villi. Amino acid absorption into enterocytes is restricted to the villus, whereas mucosal cells are produced in the crypt (Menge et al., 1982; Smith and Syme, 1982; Syme and Smith, 1982). Protein synthesis occurs along the entire villus (Alpers, 1987). The simultaneous administration of labeled amino acids into the intestinal lumen and into a vein indicate that luminal amino acids are incorporated into protein largely in the enterocytes on the villus, whereas systemic amino acids are preferentially used by crypt cells (Alpers, 1972). Hirschfield and Kern (1969) demonstrated that luminal amino acids are more readily used for intestinal protein synthesis than are systemic amino acids. Moreover, Bronk and Parsons (19661 demonstrated that incubated rings of rat jejunum accumulate amino acids in substantial quantities and incorporate these into


mucosal protein. It seems, therefore, that a substantial proportion of the maintenance of the intestinal mucosa and the synthesis of secreted proteins occurs at the expense of luminal amino acids. Intestinal atrophy and hypoplasia during luminal protein deficiency are indications that the mucosal epithelium may actually require luminal amino acids for maintenance and growth (Alpers, 1987).

The relative contribution of leucine and isoleucine, derived from the systemic circulation (either directly or indirectly) and from the diet, to endogenous protein secreted into the intestinal lumen remains to be elucidated. However, it is clear that intensive utilization of absorbed amino acids for protein synthesis in gut tissue will result in a dilution of labeled amino acids originating from the blood plasma pool by unlabeled amino acids from the diet. This will result in less labeling of endogenous amino acids from the intestinal wall relative to the labeling of amino acids in the blood plasma pool (Souffrant et al., 1986). For this reason, the actual contribution of endogenous leucine and isoleucine to leucine and isoleucine in the digestive tract, based on the 15N-enrichment excess in leucine and isoleucine in the TCA-soluble fraction of blood and in digesta, would be underestimated. This underestimation is likely to be greater for leucine than for isoleucine because of the higher I5N-enrichment in leucine.

Transamination among amino acids in intestinal tissue, which would not be reflected in the 15N-enrichment excess in free plasma amino acids, could lead to a smaller difference in the 15N-enrichment excess in leucine and isoleucine that is used for protein synthesis in intestinal tissue than that in the free plasma amino acids. There is, however, no evidence to suggest that intestinal tissue is an active site of transamination among branched-chain amino acids (Harper et al., 19841, especially when sufficient amounts of amino acids are available. The microflora in the stomach and small intestine may also contribute to transamination among amino acids and, therefore, to changes in the amino acid profile of ileal digesta. Several studies show that 25 to 30% of the total N in digesta recovered from the distal ileum of pigs may be of bacterial origin (Dierick et al., 1983; Poppe et al., 1983; Drochner, 1984). Transamina- tion, either in the intestinal wall or by intestinal microbes, could lead to an underestimation of the recovery of endogenous protein at the distal ileum (Table 2) and thus of the real ileal protein digestibilities (Table 3).

Substantial quantities of urea and ammonia are secreted into the small intestine of the pig, some of which are incorporated into microbial protein

(Bock, 1986; Bolduan et al., 1986; Rerat and Buraczewska, 1986; Mosenthin, 1987). Because the 15N-enrichment excess in isoleucine in the TCA- soluble fraction of blood was lower than that in leucine and closer to that in total N (Table 11, the incorporation of urea or ammonia N into leucine and isoleucine by gut microbes would result in a lower 15N-enrichment excess in leucine than in isoleucine. If the contribution of amino acids synthesized by microbes (from urea or ammonia) to total amino acids in ileal digesta is significant, then the contribution of endogenous protein to total protein in ileal digesta will be underestimated to a greater extent when the 15N-leucine, rather than the 15N-isoleucine, isotope dilution technique is used. It is not known, however, to what extent the incorporation of urea or ammonia N into microbial protein occurs in the small intestine.

The relative contribution of leucine and isoleucine to endogenous protein is quite similar to the relative content of apparent and real digestible leucine and isoleucine in the diets (de Lange et al., 19901. Moreover, leucine and isoleucine share the same transport system from the small intestine, which probably does not allow for an independent regulation of the absorption of these amino acids (Karasov et al., 1987). This suggests that differences between the 15N-leucine and the 15N-isoleucine isotope dilution techniques are more attribut- able to factors discussed previously than to the relative contribution of leucine and isoleucine to endogenous protein and undigested dietary protein.

As can be inferred from the previous discussion, disputable results may be obtained with an 15N-isotope dilution technique in which the individual amino acids are used to determine the recovery of endogenous protein and amino acids in ileal digesta, assuming a constant composition of endogenous protein. The results of these studies illustrate the complexity of the 15N-isotope dilution technique. Carbon or hydrogen isotope dilution techniques are possible alternatives to this technique; however, these would not be expected to overcome some of the potential difficulties just discussed. These techniques would not account for the possible contribution of dietary N to endogenous N synthesized in the intestinal wall, nor would they account for the effect of dilutions in the intestinal lumen and in the blood on protein recovered at the distal ileum. They will, therefore, also lead to an underestimation of the recovery of endogenous N in digesta collected from the distal ileum. These techniques may, in fact, present additional difficulties because proportionally less carbon and hydrogen is associated with protein

ISOTOPE DILUTION AND

than is the case with nitrogen. It seems, therefore, that the '5N-isotope dilution technique would be the better method.

The best version of this method would be the one in which all endogenous N sources are uniformally labeled. To achieve this, a complete mixture of I5N-labeled amino acids should be administered. However, such a method would be very expensive, and, therefore, impractical. The alternative to the aforementioned method is an 15N-isotope dilution technique in which one amino acid, actively involved in transamination (such as leucine), is infused continuously. The N pool to use to determine endogenous protein is still questiona- ble, Measuring the recovery of individual endogenous amino acids in ileal digesta to determine the recovery of total endogenous protein is likely the best method. At the very least, measurement of the 15N-enrichment excess in total N minus that in urea in the precursor pool and in ileal digesta is necessary to obtain values for endogenous protein synthesis that are not confounded by nonprotein N sources that may contain a high level of labeling. However, unequal labeling of individual amino acids may still represent a problem when the latter method is used. Because endogenous protein that is secreted into the intestinal lumen is synthesized in several distinct organs, the N in the deproteinized fraction of blood is probably, as yet, the best defined precursor pool for endogenous protein synthesis. However, careful attention must be given to the method of blood sampling. Further studies are required to determine the role of dietary N in the synthesis of endogenous protein in the intestinal wall, the relative contribution of the different endogenous protein sources to endogenous protein recovered at the distal ileum, the effect of protein intake on the relative importance of the different sources, and the effect of microbial fermentation in the small intestine on the composition of protein recovered at the distal ileum.

Implications

The results of the present study seem to support the 15N-isotope dilution technique for determining the minimum recovery of endogenous protein in digesta collected from the distal ileum in pigs. If assumptions are made about the amino acid composition of endogenous protein, the 15N-isotope dilution technique can also be used to determine the minimum recovery of endogenous amino acids. The 15N-isotope dilution technique can thus be used to study factors that affect the recovery of endogenous protein and amino acids at the distal ileum of pigs fed protein-containing diets.

ENDOGENOUS N IN PIGS 1855

Literature Cited

Alpers, D. H. 1972. Protein synthesis in intestinal mucosa: The effect of route of administration of precursor amino acids. J. Clin. Invest. 51:167.

Alpers. D. H. 1987. Digestion and absorption of carbohydrates and proteins. In: L. R. Johnson (Ed.) Physiology of the Gastrointestinal Tract (2nd Ed.). pp 1469-1487. Raven Press, New York.

Blackburn, S. 1968. Amino Acid Determination Methods and Techniques. Marcel Dekker. New York.

Bock, H. D. 1986. Utilization of urea in pigs. Arch. Anim. Nutr.

Bolduan, G., R. Schneider, H. Jung, and B. Klenke. 1986. The ammonia content in the digesta of pigs. Arch. Anim. Nutr. 36:28 1.

Bronk, J. R., and D. S. Parsons. 1966. Amino acid accumulation and incorporation in rat intestine in vitro. J. Physiol. 184: 950.

Campbell, I. M. 1974. Review: Incorporation and dilution values: Their calculation in mass spectrally assayed stable isotope labelling experiments. Bioorg. Chem. 3:386.

Chavez, E. R., and H. S . Bayley. 1977. Variations in the concentrations of blood plasma free amino acids in suckled and fasted neonatal pigs. J. Anim. Sci. 44:242.

Davey, R. J., J. W. Phelps, and C. H. Thomas. 1973. Plasma free amino acids of swine as influenced by diet protein level, animal age and time of sampling. J. Anim. Sci. 37:81.

de Lange, C.F.M., W. C. Sauer, R. Mosenthin, and W. B. Souffrant. 1989a. The effect of feeding different protein-free diets on the recovery and amino acid composition of endogenous protein collected from the distal ileum and feces in pigs. J. Anim. Sci. 67:746.

de Lange, C.F.M., W. C. Sauer, and W. Souffrant. 1989b. The effect of protein status of the pig on the recovery and amino acid composition of endogenous protein in digesta collected from the distal ileum. J. Anim. Sci. 67:755.

de Lange, C.F.M., W. B. Souffrant. and W. C. Sauer. 1990. Real ileal protein and amino acid digestibilities in feedstuffs for growing pigs as determined with the '5N-isotope dilution technique. J. Anim. Sci. 68:409.

Dierick, N. A,, J. A. Decuypere, J. Lannoye, I. Vervaeke, and H. K. Henderickx. 1983. Digestion ileale et fecale de la matiere seche, des proteines et des acides amines de quelques rations conventionelles pour les porcs a l'engrais. Rev. Agric. (Bruss.) 36:1713.

Drochner, W. 1984. The influence of changing amounts of crude fibre and pectic components on prececal and postileal digestive processes in the growing pig. J. Anim. Physiol. Anim. Nutr. 14:l.

Fuerst, P. 1983. Intracellular muscle free amino acids-their measurement and function. Proc. Nutr. SOC. 42:451.

Harper, A. E., R. H. Miller, and K. P. Block. 1984. Branched chain amino acid metabolism. Annu. Rev. Nutr. 4:409.

Harvey, W. R. 1960. Least squares analyses of data with unequal subclass numbers. USDA Publ. ARS 20-8.

Haymond, M. W., C. P. Howard, J. M. Miles, and J. E. Gerich. 1980. Determination of leucine flux in vivo by gas chromatography-mass spectrometry utilizing stable isotopes for trace and internal standard. J. Chromatogr. 183:403.

Hirschfield. J. S. , and F. Kern. 1969. Protein starvation and the small intestine. 111. Incorporation of orally and in- traperitoneally administered I-leucine 4,5-3H into intestinal mucosal protein of protein-deprived rats. J. Clin. Invest. 48: 1224.

Karasov, W. H., D. H. Solberg, and J. M. Diamond. 1987. Depen- dence of intestinal amino acid uptake on dietary protein or amino acid levels. Am. J. Physiol. 252:G614.

Keith, M. 0.. H. G. Botting, and R. W. Peace. 1977. Dietary

36:285.


effects on the concentrations of free amino acids in plasma and whole blood of pigs. Can. J. Anim. Sci. 57:295.

Krawielitzki, K., T. Volker, S. Smulkowska, H. D. Bock, and J. Wuensche. 1977. Weitere Untersuchungen zum Multikom- partment-Model1 des Protein-Stoffwechsels. Arch. Tierer- naehr. 27609.

Krishnamurti, C. R., and A. L. Schaefer. 1987. Measurement of plasma flux and protein synthesis in pregnant ewes using gas chromatography and mass spectrometry. Nutr. Rep. Int. 35:683.

Matthews, D. E., E. B. Ben-Galim, and D. M. Bier. 1979. Determi- nation of stable isotopic enrichment in individual plasma amino acids by chemical ionization-mass spectrometry. Anal. Chem. 51:80.

Matthews, D. E., J. M. Conway, V. R. Young, and D. M. Bier. 1981. Glycine nitrogen metabolism in man. Metabolism 30: 886.

Menge, H., R. Hopert, E. Alexopoulos, and E. 0. Riecken. 1982. Three-dimensional structure and cell kinetics at different sites of rat intestinal remnants during the early adaptive response to resection. Res. Exp. Med. 181:77.

Mosenthin, R. 1987. Untersuchungen zum Einfluss pflanzlicher Kohlenhydrate in Rationen wachsender Schweine auf die endogene Stickstoff- und Enzymsekretion in den Ver- dauungstrakt sowie auf &e praecaecale und postileale Um- setzungen N-haltiger Verbindungen. Habilitationsschrift. Universitaet Kiel, F.R.G.

Poppe, S., H. Meier, H. J. Benneke, and E. Stuewe. 1983. Zur Protein- und Aminosaeuren Verdaulichkeit in verschiede- nen Darmabschnitten beim Schwein. Arch. Tierernaehr. 33: 743.

Poullain, M. G., J. P. Cezard, C . Marche, L. Roger, F. Mendy, and J. P. Broyart. 1989. Dietary whey proteins and their peptides or amino acids: effects on the jejunal mucosa of starved rats. Am. J. Clin. Nutr. 49:71.

Reeds, P. J., A. Cadenhead. M. F. Fuller, G. E. Lobley, and J. D. McDonald. 1980. Protein turnover in growing pigs. Effects

of age and food intake. Br. J. Nutr. 43:445. Rerat, A. A. 1985. Intestinal absorption of end products of diges-

tion of carbohydrates and proteins in pigs. Arch. Tierer- naehr. 35:461.

Rerat, A. A,, and L. Buraczewska. 1986. Postprandial quantita- tive kinetics of urea and ammonia nitrogen exchanges between the digestive tract and the portal blood in con- scious plgs receiving a diet with or without urea. Arch. Anim. Nutr. 36:252.

Rodwell, V. M. 1988. Catabolism of amino acid nitrogen. In: Harper's Review of Biochemistry. Lange Medical Publica- tions, Los Altos, CA.

Sherman, P., J. Forstner, N. Roomi, I. Khatri, and G. Forstner. 1985. Mucin depletion in the intestine of malnourished rats. Am. J. Physiol. 248:G418.

Smith, M. W., and G. Syme. 1982. Functional differentiation of enterocytes in the follicle-associated epithelium of rat Pey- er's patch. J. Cell Sci. 55:147.

Souffrant, W. B., B. Darcy-Vrillon, T. Corring, J. P. Laplace, R. Koehler, G. Gebhardt, and A. Rerat. 1986. Recycling of endogenous nitrogen in the pig. Arch. Anim. Nutr. 36:269.

Souffrant, W. B., R. Koehler, R. Matkowitz, G. Gebhardt, and H. Schmandke. 1981. Ernaehrungsphysiologische Unter- suchungen an Schweinen zur Beurteilung von modifizier- ten Proteinen. Arch. Tierernaehr. 31572.

Steel, R.G.D., and J. H. Torrie. 1980. Principles and Procedures of Statistics: A Biometrical Approach (2nd Ed.). McGraw- Hill Book Co., New York.

Syme, G. 1982. The effect of protein-deficient isoenergetic diets on the growth of rat jejunal mucosa. Br. J. Nutr. 48:25.

Syme, G., and M. W. Smith. 1982. Intestinal adaptation to protein deficiency. Biol. Int. Rep. 6:573.

Wuensche, J., U. Hermann, M. Meinl, U. Hennig, F. Kreien- bring, and P. Zwierz. 1987. Einfluss exogener Faktoren auf die praezaekale Naehrstoff und Aminosaeurenresorption ermittelt a n Schweinen mit Ileo- Rektal-Anastomosen. Arch. Tierernaehr. 32431.

15N-leucine and 15N-isoleucine isotope dilution techniques versus the 15N-isotope dilution technique...

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