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American Journal of Clinical Nutrition, Vol. 70, No. 6, 1046-1058, December 1999
© 1999 American Society for Clinical Nutrition

Original Research Communications

Incorporation of urea and ammonia nitrogen into ileal and fecal microbial proteins and plasma free amino acids in normal men and ileostomates1,2,3

Cornelia C Metges, Klaus J Petzke, Antoine E El-Khoury, Lidewij Henneman, Ian Grant, Shahinaze Bedri, Meredith M Regan, Malcolm F Fuller and Vernon R Young

1 From the Massachusetts Institute of Technology, Laboratory of Human Nutrition and Clinical Research Center, Cambridge, MA; the German Institute of Human Nutrition, Bergholz-Rehbrücke, Germany; and the Rowett Research Institute, Aberdeen, Scotland.

2 Supported by NIH grants DK 42101, P-30-40561, and RR88, and a grant (Me 1420/1-1) from the Deutsche Forschungsgemeinschaft, Bonn, Germany.

3 Address reprint requests to CC Metges, German Institute of Human Nutrition, Arthur-Scheunert-Allee 114–116, 14558 Bergholz-Rehbrücke, Germany. E-mail: metges{at}www.dife.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The importance of urea nitrogen reutilization in the amino acid economy of the host remains to be clarified.

Objective: The objective was to explore the transfer of 15N from orally administered [15N2]urea or 15NH4Cl to plasma free and intestinal microbial amino acids.

Design: Six men received an L-amino acid diet (167 mg N•kg-1•d-1; 186 kJ•kg-1•d-1) for 11 d each on 2 different occasions. For the last 6 d they ingested [15N2]urea or, in random order,15NH4Cl (3.45 mg 15N•kg-1•d-1). On day 10, a 24-h tracer protocol (12 h fasted/12 h fed) was conducted with subjects receiving the 15N tracer hourly. In a similar experiment, 15NH4Cl (3.9 mg 15N•kg-1•d-1) was given to 7 ileostomates. 15N Enrichments of urinary urea and plasma free and fecal or ileal microbial protein amino acids were analyzed.

Results: 15N Retention was significantly higher with 15NH4Cl (47.7%; P < 0.01) than with [15N2]urea (29.6%). Plasma dispensable amino acids after the 15NH4Cl tracer were enriched up to 20 times (0.2–0.6 15N atom% excess) that achieved with [15N2]urea. The 15N-labeling pattern of plasma, ileal, and fecal microbial amino acids (0.05–0.45 15N atom% excess) was similar. Appearance of microbial threonine in plasma was similar for normal subjects (0.14) and ileostomates (0.17).

Conclusion: The fate of 15N from urea and NH4Cl differs in terms of endogenous amino acid metabolism, but is similar in relation to microbial protein metabolism. Microbial threonine of normal and ileostomy subjects appears in the blood plasma but the net contribution to the body threonine economy cannot be estimated reliably from the present data.

Key Words: Ammonia • urea • nitrogen metabolism • dispensable amino acids • indispensable amino acids • gut • microbial protein • stable isotopes • transamination • ileostomy • men


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
At an adequate protein intake in human subjects, {approx}60–70% of newly synthesized urea is excreted in the urine, whereas the rest is degraded by intestinal urease (1–5). About 50–70% of the hydrolyzed urea nitrogen is returned to the urea pool via ureagenesis (6, 7), whereas the remainder is transferred into the metabolic nitrogen pool. However, it remains uncertain as to whether this urea nitrogen makes a quantitatively important net contribution to body nitrogen homeostasis and whether it spares the requirements for indispensable amino acids, especially when the protein or indispensable amino acid intake is low (8–14).

From urinary [15N]urea excretion data, it has been proposed that urea nitrogen can be salvaged via urea breakdown in the colon, followed by incorporation of the nitrogen into microbial proteins with the subsequent release of the amino acids that are made available for host metabolism via absorption from the colon (3, 15). Although microbial activity is principally associated with the large intestine, it is not confined to that part of the gastrointestinal tract (16), and results of studies in nonruminant animals indicate that amino acid and nitrogen absorption in the hindgut are quantitatively not important (17–21). Although some colonic and cecal amino acid absorption may occur, no attempt has been made to quantify this process (22, 23). However, in nonruminant animals it seems that absorption of intact amino acids from microbial sources is more likely to take place in the small intestine (19, 24).

Although numerous studies have been performed, using in vitro and animal models, on the incorporation of nitrogen into single amino acids (4, 25, 26), only a few human studies have been undertaken to provide information on either the conversion of urea nitrogen (9, 27) or of its breakdown product ammonia (28, 29) into plasma amino acids. Furthermore, most of these earlier studies focused on short-term events in which an isotopic equilibration would probably not have been reached in the whole-body free amino acid pools. Also, no detailed information is available on the comparative in vivo incorporation of urea and ammonia nitrogen into microbial amino acids in the human large and small intestines, with the latter presumably serving as the site for absorption of microbial intestinal amino acids.

Therefore, in the present study of healthy young adult men receiving an adequate nitrogen intake, we examined the comparative incorporation and distribution of 15N into selected dispensable and indispensable amino acids of plasma and fecal microbial proteins when isonitrogenous amounts of [15N]urea or [15N]ammonia were administered. To explore the potential importance of microbial amino acid synthesis and release in the small intestine and their uptake by the host, we also studied a group of otherwise healthy subjects with ileostomies receiving [15N]ammonia using a comparable experimental protocol.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Massachusetts Institute of Technology subjects
Six male subjects (age: 21.7 ± 1.6 y; weight: 81.7 ± 13.3 kg; and height: 1.82 ± 0.04 m) participated in this study, which was performed at the Clinical Research Center of the Massachusetts Institute of Technology (MIT). All subjects were in good health according to medical history, physical examination, vital signs, blood indexes, and urinalysis. They were nonsmokers and reported no or only minimal alcohol consumption. Subjects were allowed to engage in their usual daily activities, but not to participate in competitive sports or perform strenuous exercise. Body weight was monitored daily and did not change during the experimental period. The purpose of the study and the potential risks involved were explained to each of the subjects. Written informed consent was obtained and the subjects received financial compensation for their participation. The study was approved by the MIT Committee On the Use of Humans as Experimental Subjects and the MIT Clinical Research Center Advisory Committee.

Ileostomates
A second group of 7 otherwise healthy ileostomates were studied at the Human Nutrition Unit of the Rowett Research Institute: 3 men and 4 women with terminal ileum ileostomies after ulcerative colitis (n = 6) or cancer of the colon (n = 1). Their mean age, weight, and height were 59.7 ± 12.3 y, 71.6 ± 15.0 kg, and 1.68 ± 0.13 m, respectively. They did not take any medications and had no known diseases of the upper gastrointestinal tract. Each subject gave written informed consent to participate in this study, which was approved by the Joint Ethical Committee of Grampian Health Board and University of Aberdeen.

Diets
An adequate semisynthetic diet (160 mg N•kg-1•d-1) was provided. It was based on a crystalline L-amino acid (Ajinomoto USA, Inc, Teaneck, NJ) mixture patterned essentially after hen eggs, except for smaller but adequate concentrations of lysine and leucine (45 and 40 mg•kg-1•d-1, respectively) (Table 1Go). The average diet of the MIT subjects is shown in Table 2Go. Carbohydrates were consumed as a flavored drink plus protein-free cookies, whereas the amino acid mixture was eaten as a mash with added sugar and water. The diet of the ileostomates was the same but was adjusted for their different energy requirements. No other foods or beverages were allowed, except tap water, decaffeinated tea or coffee with or without artificial sweetener, and bouillon.


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  		TABLE 1. Composition of the crystalline L-amino acid mixture1
 

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  		TABLE 2. Composition of the L-amino acid–based diet1
 
Total nitrogen intake by the MIT subjects was 167 mg•kg-1•d-1, including urea and ammonia tracer intakes and a lysine supplement to equal the lysine tracer given on days 10 and 11 (see below). The ileostomates had a total nitrogen intake of 163 mg•kg-1•d-1. The diets were weight maintaining: the energy intakes of the MIT subjects and ileostomates were {approx}186 and 134 kJ•kg-1•d-1, respectively. Two of the 3 daily meals were consumed under the supervision of the dietary staff.

Experimental design and sampling
MIT subjects
The experiment consisted of 2 studies conducted 4–8 wk apart. In the intervening period, the subjects consumed their usual diet. Each study lasted 11 d. Starting on day 5 of the first study, subjects received daily either 1000 mg 15NH4Cl [99.8 atom% (AP); Isotec Inc, Miamisburg, OH] or 580 mg [15N2]urea (99.1 AP; Isotec Inc) and in the second study they were given the other tracer. The 15N tracers were divided into 3 portions and administered together with the meal in small gelatin capsules throughout days 5–10. On average, each subject ingested daily 277.5 mg 15N as either 15NH4Cl or [15N2]urea ({approx}3.45 mg 15N•kg-1•d-1, or 2.1% of total nitrogen intake). On day 10, the subjects consumed their last meal at 1630 and at 1800 (zero time) a 24-h tracer protocol with hourly oral doses of 15NH4Cl or [15N2]urea (1/24th of the daily dose) was started. The hourly doses were weighed individually and dissolved in {approx}25 mL tap water immediately before ingestion. In addition, on this day an intravenous infusion of [1-13C]lysine and oral [6,6-2H2]lysine was administered. The results from this part of the investigation will be reported elsewhere.

The feeding regimen on day 11, which started at 0600, consisted of 10 small hourly meals (1/10 of the total 24-h intake) to achieve a steady metabolic fed state. Because the subjects remained in bed during the 24-h tracer protocol, the supply of energy but not of nitrogen was decreased to 83%.

Twenty-four–hour urine collections, in hydrochloric acid, and 24-h fecal collections were started on day 3 and continued throughout the experimental period (0800 on days 3 and 11). The subjects were instructed to collect feces directly into clean plastic bags and to freeze the sample immediately at -20°C. Fasting blood samples were drawn from an antecubital arm vein into heparin-containing tubes by venipuncture on days 3 or 4 (baseline samples) and day 8. Throughout the 24-h tracer protocol, blood samples were taken via a small catheter at 0, 180, 240, 360, 540, 600, 660, 720, 780, 840, 1020, 1080, 1140, 1200, 1260, 1380, and 1440 min. The technique used for catheter insertion was described elsewhere (30). The fasting blood samples on day 11 were taken at 720 min, ie, the end of the fasting period.

Ileostomates
The subjects consumed the same experimental diet for 10 d (Table 2Go). On day 5, administration of 1000 mg 15NH4Cl as 3 gelatin capsules per day was started and continued for 5 d. They ingested daily 3.9 mg 15N•kg-1•d-1 (2.5% of total nitrogen intake). For this group, no [15N2]urea was administered nor was a 24-h tracer protocol performed on day 10. Starting on day 5, all ileostomy fluids were collected and frozen immediately. One fasting blood sample was obtained on the morning of day 11.

Urine and ileal and fecal microbial fractions
Total urinary nitrogen was determined by a micro-Kjeldahl method. Urinary urea nitrogen measurements were performed by using a modified version of the procedure of Marsh et al (31). Urinary urea 15N enrichment was measured by emission spectrometry (NOI 6E; Fischer Analysen Technik, Leipzig, Germany) after isolation with a rotational microdiffusion device, essentially as described previously (32). N2 gas was subsequently generated by reaction with hypobromite, and total urinary 15N enrichment was determined by the Dumas method in an emission spectrometer.

The microbial fraction of ileal digesta or feces was obtained by differential centrifugation (250 x g for 15 min at 4°C and 14500 x g for 30 min at 4°C) as described previously (33). The total nitrogen content and 15N enrichment of fecal or ileal microbial protein was determined by emission spectrometry. The freeze-dried samples were combusted in an elemental analyzer (Elementar Vario EL; Elementar Analysen GmbH, Hanau, Germany), coupled to the emission spectrometer (NOI 6E). Plasma was separated by centrifugation at 1000 x g for 10 min and stored frozen at -20°C until analyzed. The isolation of free amino acids was performed as described previously (34). The microbial pellet was precipitated and the protein was hydrolyzed and purified by filtration. Plasma free and protein amino acids were derivatized to form N-pivaloyl-i-propyl esters for 15N analysis (24, 34).

Amino acid 15N analysis and concentration
15N Enrichment in amino acid esters of alanine, glycine, glutamic acid, aspartic acid, proline, serine, ornithine, threonine, histidine, leucine, and valine was determined by gas chromatography–combustion isotope ratio mass spectrometry (GC-C-IRMS; Finnigan Delta S, Bremen, Germany), which allows a reliable determination of enrichments below the sensitivity range of conventional gas chromatography–mass spectrometry (0.0005 AP above natural abundance; 24). All amino acids were determined in one run. Briefly, after separation of amino acids by gas chromatography, the effluent is directed online into a combustion interface. Amino acids are combusted in an oxidation furnace at 980°C to CO2, N2, NOx, and water. Combustion gases are reduced in a reduction oven at 600°C; carbon dioxide and water are eliminated and N2 is directed into the ion source of an isotope ratio mass spectrometer. The observed 15N enrichment of plasma free glutamic acid represents the 15N abundance of the {alpha}-nitrogen of both glutamine and glutamate as discussed elsewhere (34). All of the glutamine of ileal and fecal microbial protein samples, which underwent acid hydrolysis, was converted to glutamic acid. The same is true for asparagine, which was converted to aspartic acid. Because ornithine is not a proteinogenic amino acid, 15N enrichments for this amino acid are given for plasma only. No aspartic acid values are given for plasma because the signal was too small. The derivative used here did not allow measurements of arginine.

15N Abundances are expressed as 15N atom% excess (APE) above baseline 15N abundance; the latter was significantly different for the various amino acids at natural abundance (34). Although a washout period of 4–8 wk was observed between the two 15N tracer experiments in some subjects, the earlier 15N had not been totally eliminated from the body. In the second study, 15N APE before administration of the tracer (baseline sample) was, on average, in the range of 0.004–0.012 APE above natural abundance (0.3663 AP). The enrichments of amino acids (15N APE) were corrected accordingly.

Because no (unenriched) baseline plasma or ileal chyme samples of the ileostomates were taken, the 15N enrichments in amino acids (APE) were calculated on the basis of the natural 15N abundance in unenriched total urinary nitrogen of each subject (x ± SD: 0.3658 ± 0.0003 AP). The amino acid content in ileal and fecal protein was determined after acid hydrolysis by ion exchange chromatography as described previously (35).

Statistics
Values are reported as means ± SDs. Comparisons of means were performed with two-sided paired and unpaired Student's t tests with a significance level of {alpha} < 0.01 (36). For MIT subjects, paired t tests were used to compare 15NH4Cl and [15N]urea tracers. Because the ileostomates were studied in a different laboratory for a related but not identical purpose, unpaired t tests were used to compare MIT subjects and ileostomy subjects receiving the 15NH4Cl tracer. We did not statistically compare enrichments or concentrations among amino acids, for example glutamic acid compared with alanine.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MIT subjects
The incorporation of urea and ammonia 15N into selected plasma amino acids of healthy MIT subjects is summarized in Table 3Go and Figures 1 and 2GoGo. The enrichment pattern was comparable with both 15NH4Cl and [15N2]urea, although the magnitude of enrichment was quite different. The pattern became more evident after 6 d of tracer intake (day 11) (Table 3Go). Glutamic acid and alanine showed the highest enrichments. Glycine and serine were considerably less enriched than were glutamic acid and alanine, on both days 8 and 11, with the 15NH4Cl tracer. On day 11, the enrichment of ornithine was between those of proline and serine; proline showed the lowest enrichment of all the dispensable amino acids examined.


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  		TABLE 3. 15N Enrichment of dispensable and indispensable free amino acids in fasting plasma of MIT subjects and ileostomates after 3 and 6 d of daily 15NH4Cl or [15N2]urea intake1
 


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  		FIGURE 1. 15N Enrichment of dispensable (A) and indispensable (B) plasma free amino acids in Massachusetts Institute of Technology subjects during the 24-h tracer protocol after ingestion of 15NH4Cl for 6 d. The arrow indicates the time when the feeding of small meals began (at 0600) on experimental day 11. Note that the scales of the y axes in panels A and B are different, as are those in Figures 1 and 2GoGo. APE, atom% excess.

 


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  		FIGURE 2. 15N Enrichment of dispensable (A) and indispensable (B) plasma free amino acids in Massachusetts Institute of Technology subjects during the 24-h tracer protocol after ingestion of [15N2]urea for 6 d. The arrow indicates the time when the feeding of small meals began (at 0600) on experimental day 11. Note that the scales of the y axes in panels A and B are different, as are those in Figures 1 and 2GoGo. APE, atom% excess.

 
Among the nutritionally indispensable amino acids studied, there were striking differences in 15N enrichment. After 3 and 6 d of 15N intake (days 8 and 11), leucine and valine reached about the same enrichment levels as glycine, serine, and ornithine with both tracers. In contrast, threonine and histidine were only 25–50% as enriched as proline, which was least enriched among the dispensable amino acids (Table 3Go; Figures 1 and 2GoGo).

Although isonitrogenous amounts of the two 15N tracers were given, plasma amino acids were 5–12 times more enriched on day 8 after administration of 15NH4Cl than after [15N2]urea, although the difference was only significant for serine and valine (P < 0.01). This difference between the tracers was even more pronounced, 8–21 times more, after 6 d (day 11; Table 3Go) and was highly significant (P < 0.0005).

15N Enrichments in plasma amino acids during the 24-h tracer protocol showed a similar pattern (Figures 1 and 2GoGo). For clarity, only the mean values are included in these figures. Enrichments of threonine and histidine with the 15NH4Cl tracers were far lower than those of all other amino acids (Figure 1Go); this was not as clear with the urea tracer (Figure 2Go). After the administration of [15N2]urea, differences in 15N enrichments among plasma amino acids were generally not as pronounced (Figure 2Go).

A decrease in the enrichment of all amino acids, except threonine and histidine, is evident between 720 (start of feeding) and 1320 (end of feeding) min, reflecting the absorption of unenriched amino acids from the dietary amino acid mixture (Figures 1 and 2GoGo). For the last 4 h of the fasting period and for some hours into the fed state, there was a striking difference between the tracers with respect to the enrichment of alanine compared with glutamic acid and of serine compared with glycine. With the 15NH4Cl tracer, the decreasing rank order of enrichment of the amino acids was as follows: glutamic acid, alanine, glycine, and serine; with the [15N2]urea tracer it was as follows: alanine, glutamic acid, serine, and glycine (Figure 1A and Figure 2AGoGo).

In contrast with the large difference between the tracers in 15N enrichment of the plasma amino acids, those amino acids isolated from fecal microbial protein were only approximately twice as enriched after 15NH4Cl administration than after [15N2]urea administration (Figures 3 and 4GoGo). The ranking of enrichments among amino acids was essentially the same as that for the plasma amino acids. 15N Enrichment of total fecal bacterial nitrogen in healthy subjects tended to be higher with 15NH4Cl administration than with [15N2]urea administration, but it was not significantly different (Table 4Go). A plateau of enrichment was apparently reached after {approx}3 d of 15N tracer administration.



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  		FIGURE 3. Mean (±SD) 15N enrichment of dispensable (A) and indispensable (B) fecal microbial protein amino acids in Massachusetts Institute of Technology subjects during ingestion of 15NH4Cl for 6 d. Tracer administration began on day 5. Note that the scales of the y axes in Figures 3 and 4GoGo are different. APE, atom% excess.

 


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  		FIGURE 4. Mean (±SD) 15N enrichment of dispensable (A) and indispensable (B) fecal microbial protein amino acids in Massachusetts Institute of Technology subjects during ingestion of [15N2]urea for 6 d. Tracer administration began on day 5. Note that the scales of the y axes in Figures 3 and 4GoGo are different. APE, atom% excess.

 

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  		TABLE 4. 15N Enrichment in total fecal microbial nitrogen of MIT subjects and ileostomates during administration of 15NH4Cl or [15N2]urea
 
With the 15NH4Cl tracer, enrichments of dispensable amino acids, leucine, and valine in fecal microbial proteins and in the plasma free amino acid pool were approximately in the same range (0.1–0.6 15N APE) (Figures 1 and 3GoGo). However, 15N enrichments of threonine and histidine in plasma were only 14% and 49% of the corresponding values in fecal microbial protein (Table 5Go). After [15N2]urea ingestion, dispensable free plasma amino acid enrichments were 15–40% of those in the amino acids of fecal microbial protein, whereas indispensable amino acid enrichments in plasma were only 8–20% of the corresponding values in microbial protein (Table 5Go).


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  		TABLE 5. Ratios of free plasma 15N amino acid enrichment to microbial protein 15N amino acid enrichment after ingestion of [15N2]urea or 15NH4Cl over 6 d in MIT subjects and ileostomates1
 
15N Excretion over the whole experimental period (6 d of labeling) was calculated as the percentage of the total 15N dose given, which indicated significantly higher 15N retention of ammonia nitrogen than of urea nitrogen (Table 6Go). Furthermore, the percentage of tracer excreted as urea nitrogen was significantly higher with the [15N2]urea tracer (57%) than with the 15NH4Cl tracer (39%) (Table 6Go).


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  		TABLE 6. Fraction of total 15N ingested ([15N2]urea or 15NH4Cl on days 5–11) excreted as urinary total nitrogen, urea nitrogen, or nonurea nitrogen, and nitrogen retention over the 15N dosing period in MIT subjects1
 
Comparison between ileostomates and subjects with an intact gastrointestinal tract after 15NH4Cl intake
On day 11, fasting plasma glutamic acid, serine, and threonine enrichments were significantly lower in ileostomates than in the MIT subjects (Table 3Go). If adjusted for the relatively higher 15N intake of ileostomates, because of their lower body weights (3.9 compared with 3.45 mg 15N•kg- 1•d-1), this difference would be even larger. However, the intact subjects were given a small amount of 15NH4Cl or [15N2]urea hourly from days 10 to 11, which was not the case for the ileostomates and may, at least partly, account for the difference.

The enrichment of total microbial nitrogen in the ileal chyme of the ileostomates was slightly, though not significantly, higher than that of the fecal microbial nitrogen of intact subjects (Table 4Go). However, with 15NH4Cl, the enrichment of amino acids in the fecal microbial protein of intact subjects was in the same range as that of the amino acids in the ileal microbial protein of ileostomates (Figure 3 and Figure 5GoGo).



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  		FIGURE 5. Mean (±SD) 15N enrichment of dispensable (A) and indispensable (B) microbial protein amino acids of ileostomy fluid in ileostomates during ingestion of 15NH4Cl. Tracer administration began on day 5.

 
Ratios of 15N enrichment of plasma free amino acids to the enrichment of the corresponding microbial amino acids are shown in Table 5Go. For the dispensable amino acids, these ratios were lower by a factor of 2 for the ileostomates than for the MIT subjects given 15NH4Cl; however, for the indispensable amino acids the ratios appeared to be similar in the 2 groups. When the MIT subjects received [15N2]urea, the ratios were far lower than when they received 15NH4Cl, except for threonine and histidine.

The amino acid composition of fecal and ileal microbial protein is summarized in Table 7Go. Although no difference was seen in the amino acid pattern of MIT subjects ingesting either 15NH4Cl or [15N2]urea, as was expected, lower concentrations of glutamic acid, alanine, isoleucine, and methionine were found in ileal microbial protein than in fecal microbial protein. On the other hand, higher concentrations of threonine, histidine, proline, and serine were found in ileal microbial protein.


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  		TABLE 7. Amino acid composition of fecal and ileal microbial protein in MIT subjects and ileostomates after 5 d of 15NH4Cl or [15N2]urea intake1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although earlier studies reported incorporation of 15N from urea (9, 27) or ammonia (28, 29) 15N into amino acids in human subjects, there has not been a direct comparison of ammonia and urea 15N as nitrogen sources or of details of their metabolic fates in the same subject. The present study showed several significant and intriguing differences in the metabolic behavior of these two 15N-enriched compounds, which are discussed and highlighted below. Our tracer protocol apparently permitted achievement of an isotopic steady state because 15N enrichments in fecal microbial protein reached a quasiplateau by {approx}3 d of tracer administration. This makes for easier comparisons within and between the 2 tracer groups.

In the MIT subjects, who had intact intestinal tracts, oral administration of 15NH4Cl resulted in a distinctly different degree of 15N retention and 15N distribution than did an isonitrogenous dose of [15N2]urea (Table 6Go). To begin with, only 38.7% of the administered 15N ammonia appeared in urinary urea compared with 56.7% of the urea 15N. The greater body retention of ammonia nitrogen (47.7% compared with 29.6%) was reflected in higher 15N enrichments in both the dispensable and indispensable free amino acids in the circulating plasma; after 6 d of isonitrogenous 15N intake, 15N labeling of the dispensable amino acids (and of leucine and valine) in plasma was between 10 and 20 times higher after 15NH4Cl than after [15N2]urea (Table 3Go). In contrast, the 2 other indispensable amino acids that we measured, threonine and histidine, were more equally labeled with 15N from the 2 sources, although the difference was still 4-fold.

Even if a correction is made for the lower 15N retention after urea administration, 15N enrichments of the plasma free amino acids with 15NH4Cl were still {approx}10 times higher than those achieved with the labeled urea. Therefore, it is apparent that both the source and presumably the site of delivery of 15N ammonia (direct ingestion versus liberation via urea hydrolysis) influence its fate, with ingested ammonia being incorporated into glutamate, via glutamate dehydrogenase, and then rapidly exchanged between amino acids via transamination and also, probably, by direct incorporation of NH4 to form glycine (37). Note the similar enrichment of glycine and serine after 15NH4Cl intake than after [15N2]urea indicated in Table 3Go. In contrast, the ammonia that is liberated from urea apparently achieves only 5–10% of the amount of labeling in the precursor pools for {alpha}-ketoglutarate amination. These different sources of 15N label entering the body as [15N]ammonia can presumably explain the different ratios of serine to glycine enrichments after 15NH4Cl (0.96) and [15N2]urea (1.58) ingestion (Table 3Go), further indicating that the enrichment of the transaminating amino acid pool after 15NH4Cl intake must be several times higher than after [15N2]urea ingestion.

Another relevant factor may be the tissue sites of amino acid synthesis. Thus, glutamate is considered to be an important precursor for proline synthesis in mammals, but its 15N enrichment was only 25% of that of glutamate. Note, however, that the 15N enrichment we observed in plasma glutamic acid was a mixture of {alpha}-nitrogen of glutamate and glutamine. Because the plasma glutamine concentration was {approx}10 times as high as plasma glutamate, the observed enrichment was presumably mainly due to {alpha}-nitrogen of glutamine. However, because there is a rapid exchange of {alpha}-nitrogen between glutamate and glutamine by transamination (38), we assumed that the enrichments of both {alpha}-nitrogens are similar. Murphy et al (39) provided evidence that proline is synthesized from glutamate during intragastric infusion but not via an intravenous supply, suggesting that in the present experiment there may have been a relatively greater synthesis of proline from unlabeled dietary glutamate in the splanchnic tissues than from circulating 15N glutamate. Additionally, Patterson et al (28) found that 15N enrichment of plasma proline was far lower than that of plasma glutamate after short-term oral administration of 15NH4Cl.

The branched-chain amino acids were labeled to {approx}50% of the enrichment achieved with glutamate and alanine. This reflects the fact that a significant fraction of the circulating plasma free branched-chain amino acids is derived from the unlabeled leucine entering via the diet and from tissue protein turnover. We interpret these observations to suggest that {approx}50% of circulating leucine and valine is derived from transamination of their ketoanalogues {alpha}-ketoisocaproate and {alpha}-ketoisovalerate, respectively, and the remainder directly from protein turnover, the diet, or intestinal microbial sources. This interpretation is consistent with earlier findings (40) in a study using L-[1-13C,15N]leucine as tracer.

Plasma threonine and histidine were labeled with 15N and to a greater extent when 15N was administered as the ammonium salt. It is generally accepted that there is a low rate of histidine synthesis in the body and our findings extend those of Sheng et al (41), who showed incorporation of 15N into the imidazole ring of histidine after oral ingestion of 15NH4Cl in an adult male who had received parenteral histidine-free nutrition for 22 d. Whether the labeled histidine was entirely of intestinal microbial origin in that or in the present study cannot be determined. However, because threonine appears not to undergo any significant transamination (if any at all) and it is generally accepted that it cannot be synthesized by the body tissues of mammalian organisms, including humans (42), the labeled threonine detected in the plasma of healthy subjects presumably arises entirely from the synthesis of threonine by the intestinal microflora.

Data on the comparative amino acid compositions of animal and human ileal and fecal microbial proteins are scarce. Comparisons between ileal microbial protein from ileostomates and endogenous ileal protein from pigs fed protein-free diets (43) showed lower concentrations of dispensable and indispensable amino acids in the ileal endogenous protein. However, the proline concentration in endogenous ileal protein was 3 times the proline concentration in microbial ileal protein in this study. Particularly large differences were found for threonine, proline, alanine, and serine concentrations, which were all much higher in pig ileal mucine than in human ileal microbial protein (44). The differences observed between ileal and fecal microbial amino acid composition were presumably due to fermentation in the large intestine. No difference was seen between the indispensable-to-dispensable amino acid ratio in fecal and ileal microbial protein.

The pattern or rank order of labeling of the amino acids from protein of microbial origin was generally similar to that observed for the plasma free amino acids with each 15N source. This order of labeling was also found in human plasma amino acids after short-term oral administration of 15NH4Cl (28). However, several differences between plasma and microbial amino acids are notable and might provide some further clues about nitrogen exchange between body tissues and the intestinal lumen. Thus, the degree of 15N labeling in the microbial amino acids was, as for plasma free amino acids, higher with 15NH4Cl than with [15N2]urea. However, the differences were far smaller than for plasma amino acids: 15N enrichments were only about twice as high with ammonium chloride as with urea, and this difference became much smaller when allowances were made for differences in 15N retention (Table 6Go). This suggests that an oral dose of urea is a relatively more effective source of nitrogen for microbial amino acid synthesis than it is for the synthesis of tissue endogenous amino acids. This is supported by the fact that a higher proportion of administered urea nitrogen appeared in the urinary urea pool. Furthermore, we observed previously in pigs, after a 10-d intake of [15N2]urea, that lysine and other amino acids of duodenal and jejunal proteins were only 10% enriched compared with the enrichment after isonitrogenous intake of 15NH4Cl (24; CC Metges, unpublished observation, 1996). It appears that the nitrogen used for synthesis of bacterial amino acids can be derived in quantitatively significant amounts both from urea, via hydrolysis, and from ammonia, either directly or indirectly. Indirect routes include both urea nitrogen salvage and endogenous protein secretions.

The importance of 15N-labeled amino acids in endogenous secretions as a source of label for microbial metabolism is suggested because, after 15NH4Cl administration, the 15N enrichment of most of the bacterial amino acids was similar to the plasma values (Figures 1 and 3GoGo), whereas the ratio of plasma 15N to microbial amino acid 15N was much lower after administration of [15N2]urea. It is firmly established that there is considerable nitrogen cycling via the gut lumen and the gastrointestinal tissues, with nitrogen reappearing in the gut as so-called endogenous nitrogen. Endogenous nitrogen is composed of amino nitrogen from various digestive secretions (saliva, pancreatic juice, and bile), mucins, sloughed-off mucosal cells, urea, and ammonia. Studies in pigs have shown that total endogenous secretions can range between 11 and 16 g N/d (18, 45) and this rate appears to be similar in human adults (46).

The lower ratio of plasma to microbial 15N enrichment for threonine and histidine, compared with all of the other amino acids, presumably means that these labeled amino acids were derived exclusively from microbial activity within the intestine. Whether the present data can be used to estimate the quantitative significance of this microbial source of circulating threonine and histidine is uncertain and is discussed further below, with particular reference to the findings for the ileostomates.

The amount of 15N labeling achieved in the free dispensable amino acid pool in plasma from the ileostomates, who were only given the 15NH4Cl tracer, was generally lower than that in plasma from the MIT subjects who were given this tracer (Table 3Go). This may have resulted because the administration of the 15N tracer was terminated on day 10, with blood sampling on day 11, whereas the 15NH4Cl tracer was continued until day 11 in the MIT subjects. This does not completely explain the difference because the 15N enrichments of leucine and valine were not significantly different between the 2 groups. Furthermore, the ratios of plasma to microbial enrichments of the dispensable amino acids for the MIT subjects were almost twice those of the ileostomates (Table 5Go), suggesting some nitrogen absorption by the human large intestine.

15N Labeling of the microbial nitrogen and amino acids in ileal fluid was not significantly different from that of fecal microbial protein (Table 4Go), which perhaps indicates that microbes in the upper small intestine generally use similar nitrogen sources for amino acid synthesis as do those in the lower gastrointestinal tract. What was surprising to us was the degree of labeling in the plasma free threonine and histidine pools of the ileostomates. In addition, the ratio of plasma to ileal microbial amino acid 15N enrichment for threonine and histidine was not significantly different from the ratio of plasma to fecal microbial 15N enrichment in the MIT subjects (Table 5Go). Note that the ratio of plasma to fecal microbial amino acid and of plasma to ileal microbial amino acid enrichment for histidine, leucine, and valine were not significantly different between MIT subjects and ileostomates. Because we have no reason to expect that there would be major differences in the kinetics of whole-body threonine and histidine metabolism between the 2 groups of subjects, these labeling data suggest a similar rate of appearance of threonine and histidine of intestinal microbial origin in both the body tissues of MIT subjects, who had intact gastrointestinal tracts, and of the healthy ileostomates, who had only a functional small intestine. This suggests the possibility that the main site of absorption of microbial amino acids is the small intestine.

If the ratio of plasma to bacterial threonine 15N is indicative of the fraction of plasma threonine flux derived from bacterial metabolism, then the 15NH4Cl tracer studies indicate the fraction to have been {approx}14–17% in both groups of subjects. If a threonine flux of {approx}100 µmol (285 mg)•kg-1•h-1 (47) is assumed, then threonine synthesized by the gastrointestinal microflora was absorbed and entered the peripheral circulation at a rate of 40–48 mg•kg-1•d-1. On the other hand, if this rate is calculated in the same way when the [15N2]urea tracer is given, it is apparently much lower, or 22 mg•kg-1•d-1. This in turn suggests that the site of synthesis, release, or absorption of labeled threonine may not be the same when 15N is given as ammonium chloride as when it is given as urea. This difference cannot be readily ascribed to the difference in the retention of urea 15N label because both plasma and microbial amino acid enrichments would be subject to the same adjustment. Rather, it points to a difference in the nitrogen source (endogenous amino acids when 15NH4Cl was used as the tracer and urea nitrogen when [15N2]urea was used as the tracer), the site of tracer appearance and of the microbial population utilizing it, or both.

The plausibility of the foregoing estimates of the appearance of microbially derived threonine in host tissue pools and in body threonine metabolism should be questioned. Thus, the current international recommended threonine requirement of adult humans (48) is 7 mg•kg-1•d-1. This value, derived from nitrogen balance studies in men and women (49, 50), is about half that derived from measurements of [13C]threonine oxidation (15 mg•kg-1•d-1; 51). Nevertheless, it seems unlikely that there would be a net contribution of microbially derived threonine equivalent to the apparent daily irreversible disposal of threonine (51), which, in turn, is from 3- to 7-fold higher than the international requirement (48). It also must be borne in mind that the above estimates refer to the gross uptake of amino acid synthesized by the microflora and do not necessarily represent a net increase of the same magnitude in the total amount of threonine available for metabolism. To the extent that the growth of the microbes that gives rise to labeled threonine is supported or balanced by the degradation of endogenous protein, the absorption of microbial amino acids can be seen as part of the normal mechanism by which endogenous nitrogen and indispensable amino acids are recycled rather than used as a net source of amino acids in addition to those supplied in the diet. Indeed, support for this view comes from previous [1-13C]leucine oxidation studies (30). In these studies there was excellent concordance between both predicted and measured protein oxidation when estimated from leucine oxidation and nitrogen excretion values, which would not have been true if there was significant additional net uptake of leucine from the gut microflora. There seems little reason to expect that if there were a significant net entry of microbially derived threonine into body tissues that this would not also apply to the other nutritionally indispensable amino acids, including leucine.

Furthermore, these estimates of the uptake of microbially derived threonine from the intestinal tract are based on the assumption that 15N enrichment of the absorbed labeled threonine is known, ie, that in the microbial protein of fecal or ileal origin. In fact, 15N enrichment of absorbed threonine could not be measured for technical reasons and the true value might be expected to be closer to that of the ileal digesta than to that of feces, but it could conceivably be higher (22). For some bacteria, peptides derived from microbial protein breakdown may not be incorporated but released in the surrounding medium (52). It is of interest that peptide-bound amino acids contributed {approx}50% to the portal plasma amino acid pool in rats (53). However, the extent to which these peptides may be derived from microbial protein is not known. If 15N-labeled substrates entering the gut lumen (as plasma-derived amino acids, endogenous proteins, or urea) were used preferentially by microbes in juxtaposition to the intestinal wall, with turnover and release of their constituent proteins and amino acids in that spatial domain, then the enrichment of the 15N threonine being absorbed from the gastrointestinal tract would not be accurately reflected by either the ileal or fecal microbial protein-bound lysine. There is evidence for the diversity of domains within the gut (54). In the present study, the finding of very low 15N enrichment in cecal ammonia after 15N urea was infused intravenously (55) likewise points to the possibility that nitrogen transactions between the body and the gastrointestinal microflora may not be fully revealed by analysis of the bulk protein phase of intestinal contents.

It was shown in ileostomates treated with antibiotics, who consumed a protein-free diet, that threonine losses in ileostomy fluid were the highest ({approx}4 mg•kg-1•d-1) of the indispensable amino acids (56). Under the current experimental conditions in which there was an adequate dietary nitrogen intake, microbial threonine losses were of the same magnitude if based on an ileal nitrogen loss of 1.51 g N/d (57) and a threonine concentration of 6.5 g/100 g protein (Table 7Go). Hence, there is also the possibility that, in part, entry of threonine of microbial origin helps to counteract the endogenous threonine losses rather than contribute to the total exogenous (dietary) input of threonine.

On the basis of the above arguments it seems doubtful that we can reliably estimate, quantitatively, the total net contribution made by threonine (or other indispensable amino acids) of bacterial origin to host metabolism and homeostasis according to the present paradigm. Although our data might suggest a nutritionally significant uptake into body tissues of nontransaminating amino acids of microbial origin, the quantification of this uptake and its net contribution to the amino acid economy requires a better understanding of nitrogen and carbon transactions between the intestinal mucosa and the microflora that frequent the intestinal lumen and are adherent to the walls.

In conclusion, the fate of 15N when given as urea or ammonia differs; endogenous amino acids were labeled more extensively with ammonia than with urea, but these 2 nitrogen sources appear to be of equivalent importance in the labeling of microbial protein. Furthermore, it is evident from the comparisons between the MIT subjects and the ileostomates that both the small and large intestine play a significant role in the interplay between endogenous nitrogen metabolism and that of intestinal microflora. Finally, microbially synthesized threonine was detectable in the circulating blood plasma of both the healthy MIT subjects and the healthy ileostomates, but we could not accurately quantify the net input of threonine via this route from the present data.


   ACKNOWLEDGMENTS
 
We thank the staff of the Clinical Research Center for their technical support and the staff of the Protein Metabolism Unit at the German Institute of Human Nutrition for their skillful technical assistance.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication October 29, 1998. Accepted for publication May 10, 1999.




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