AMERICAN
JOURNAL
OF
PHYSIOLOGY
Vol. 229, No. 4, October
1975.
Nitrogen
Printed in U.S.A.
utilization
J. F. WOOTTON Ithaca,
AND
of Physiohgy,
l)efm$nmt New
York
within
protein protein
large intestine
R. A. ARGENZIO
Biochemistry,
and Pharmacology,
New
York
State
Veterinary
College,
Cornell
University,
I4853
WOOTTON, J, F., AND R. A. ARGENZIO. Mtrogen utili can be determined directly. Therefore, net appearance of a constituent in a compartment due to processes other than flow along the tract can be estimated for each time interval and compartment from the following relationship (3): (P a - Pd)At = Qt2 - Qtl - PiAt + P.At where P, and Pd represent the (indeterminant) total rates of appearance and of disappearance and At = t2 - fl. The estimated errors expressed are calculated from the combined errors for the terms in the previous equation as follows : 2 6 (p
s2 a -
p&t
=
-
n(Qt2)
where
8 = standard
deviation.
a2 +
-
n(QtJ
+
a2 n(PiAL)
a2 +
1063
INTESTINE
-
n(~,A
t)
f
Sl,
SECTION
$1,
C OF
RVC
LVC
LDC
RDC
SC,
TRACT
FIG. 1, Concentrations of urea nitrogen in gastrointestinal tract of ponies fed control (A) or experimental (B) diet. Symbols represent time after last feeding (A, 2 h; 0, 4 h; x, 8 h, and l , 12 h or prefeeding). Letters shown on abscissa denote sections of tract separated by ligatures : cranial stomach (S,), caudal stomach (S 2) ; proximal (SIJ, middle (SI,), and distal (SI 3) thirds of small intestine; cecum (C); right ventral (RVC) and left ventral (LVC) colon; left dorsal &DC) and right dorsal (RDC) colon; proximal (SC,) and distal (SC,) halves of small colon. Concentrations represent mean & SE for 3 animals at each time period.
RESULTS
Figure 1 illustrates the concentrations of urea N in the mixed contents of each of the 12 segments of the digestive tract at each time of measurement. Animals ingesting the urea-containing experimental diet exhibited greatly elevated concentrations in the stomach and proximal small intestine 2 h after feeding. Detectible elevation persisted in these segments at 4 h as well. However, significant enhancement was not observed within the large intestine at any of the times of measurement. Furthermore, ammonia N concentrations (Fig. 2) tended to be lower in all segments of the intestinal tracts of these animals compared with those which received the urea-free control diet. The differences were especially pronounced in the colon. Concentrations of free amino acids followed patterns similar to those of ammonia within the small intestine, falling progressively and reaching minimum values of approximately 3 mg-atoms N/kg H20 in the cecal contents. The mean concentrations in the colon remained at similar levels. However, accurate estimations by the method employed were prevented by the high ammonia concentrations present in this organ. Net disappearance of amino nitrogen from the cecum was observed with both diets throughout the 1Z-h interval between feedings. Figure 3 illustrates the content and net appearance rates of ammonia plus urea N in the four compartments of the large intestine. The analogous data for protein N are shown in Fig. 4. Marketi differences between the cecum and the colon are apparent with respect to the distribution
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1064
J. F. WOOTTUN
AND
R. A. ARGENZIO
were similar for the two diets, and little, if any net appearance of protein could be detected in this compartment; 3) in the colon both net appearance and net disappearance of protein appear to have been greater in the ponies which consumed the urea-containing diet, whereas the loss of nitrogen to the feces was only modestly increased. Daily net changes observed within the entire large intestine are compared with dietary intake in Table 1. The changes in total and protein nitrogen paralleled one another closely+ This relationship extended to each individual compartment and to each time interval examined. Thus metabolic interconversion between protein and pools of low molecular weight nitrogenous constituents present within the intestinal lumen cannot be invoked to explain net appearance and disappearance of protein. Net disappearance was equivalent on the two diets, although, as shown in Fig. 4, the contributions of the various compartments differed. The overall net appearance of protein, essentially all within the colon, was enhanced in animals receiving the protein-deficient diet and exceeded the dietary protein intake. 3
s,
SI,
$I2
SI,
SECTION FIG. 2. concentrations Of tract of ponies fed control (A) described in legend to Fig. 1.
C RVC LVC LDC RDC SC, SC, OF TRACT
nitrogen in gastrointestinal or experimental (B) diet. Symbols are
ammonia
of each of these classes of nitrogenous constituents. In neither case were significant changes in quantity observed in the cecum with respect to times of measurement or diet. Net disappearance essentially was continuous. Although estimated variances are large, the rates of disappearance of both protein and ammonia from the cecum appeared to be greater during each time interval with animals fed the control diet than with those which received the proteindeficient, experimental diet. In contrast, the ammonia and protein content of the colon both show significant time-dependent changes. The patterns of these changes differed with the different diets. Adaptation to the urea diet was accompanied by marked increase in volume of the large intestinal contents (l), which was most striking in the ventral and dorsal compartments of the large colon. This increase in volume was accompanied by a proportionate increase in protein nitrogen content (Fig. 4). The ammonia content was not proportionately greater, as reflected by the lower concentrations shown in Fig. 2. The time-dependent changes in protein and ammonia content correspond to periods of net appearance and net disappearance, which in several cases appear to be significant. A summation of average daily net changes in protein nitrogen distribution is represented in Fig, 5. Inflow and outflow via digesta passage along the tract as well as net appearance and disappearance are indicated for each of the four compartments of the large intestine. Comparison of the two diets shows the following: 1) smaller quantities of protein entered the ceca of animals fed the experimental diet, and net disappearance from this compartment also was reduced; 2) the amounts leaving the cecum by flow
I
0
2 4
6
8
6
8 10122
1012
2
4
6
8
10
6
8
10122
12
2
4
6
8
1012
4
6
8
10
2
4
6
8 10
4
6
8 1012
2
80-
0 2
4
4
122
TIME ( Hr post feeding) FIG. 3. Quantities and net rates of appearance or disappearance (+ SE) of ammonia plus urea N in large intestine of animals fed control (A) or experimental (B) diet. Positive values designate net appearance rates, whereas negative vaIues signify disappearance. Calculations of net appearance are based on data describing passage of PEG (I).
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1975 American Physiological Society. All rights reserved.
NITROGEN
UTILIZATION
WITHIN
EQUINE
LARGE
I
0 2 4
6
8
l012
2
4
t
6
8
I012
2
i
6
8
IO12
2
4
1065
INTESTINE
6
8
1012
greater in those animals consuming the high-protein, control diet and occurred in the cecum and ventral colon, primarily the former compartment (see Fig. 5). Net disappearance of total and protein nitrogen from the entire large intestine (Table 1) was equal on the two diets, the reason being that net appearance due to processes other than flow along the tract was greater in the animals consuming the protein-deficient, experimental diet. Net appearance was observed only in the colon (see Figs. 4 and 5). The values. for appearance and disappearance should not be construed to represent absolute amounts. Rather they provide (within their limits of error) minimal estimates which are not necessarily comparable between two diets. They represent the sums of values for those time intervals during which either net appearance or disappearance was observed. Each time interval is of several hours duration, and asynchrony of flow or of cyclic events among individual animals would minimize the (absolute values of) apparent net rates of appearance and disappearance. Furthermore, the selected time intervals between measurements were arbitrarily chosen and do not necessari1y coincide with cyclic events occurring in the large intestine. The apparent cycles, in turn, differ with diet. Finally, since net differences only are observable, steadystate turnover within a compartment: is not detected. Such CONTROL
DIET
EXPERIMENTAL
DIET
CECUM -
1
0
2 4
6
8
lo,12
I
2
4
6 TIME
8
10 (Hrps?
12
I
2
4
6
10
12
2
4
I
I
r
6
8
to
VENTRAL COLON
12
feeding)
4. Quantities and net rates of appearance of protein nitrogen in large intestine of ponies experimental (B) diet. FIG.
8
--
-
and disappearance fed control (A) and
D%%!
DISCUSSION
Despite the fact that animals closely grouped with respect to size consumed meals of constant size and composition at fixed intervals, large variability was observed in digesta volumes ( 1) and in the concentrations and quantities of nitrogen present at any time of measurement. Estimations of outflow, accumulation and, hence, net appearance involve the combination of pooled data collected from two separate groups of animals. Therefore, as indicated in the figures, the estimated errors are large. Still it is clear that some general conclusions are justified with regard to the temporal changes in nitrogen distribution. Comparison between calculated increments due to flow and actual increments accumulated in a compartment during a series of limited, sequential time intervals permits the detection of transient appearance or disappearance, for example, of protein nitrogen. These changes, which are not detectable through comparisons of either flow or accumulation alone, can be of fundamental significance to the overall nitrogen metabolism of the animal. The estimates of net retention of total and protein nitrogen shown in Table I are based solely on differences between flow into and out of the large intestine. Net retention was
--
SMALL COLON
0.29
0.40
5. Net appearance, disappearance, and passage of protein nitrogen expressed as gram-atoms of nitrogen per 24 h. Net appearance per 24 h is calculated from sum of areas above abscissae in Fig. 4, whereas net disappearance is derived in same fashion from areas below these axes. FIG.
TABLE 1. comparison of daily nitrugen intake with overall net appearance, disappearance, and retention in large intestine Nitrogen/X Total Control
Daily intake* Net appearance Net disappearante Net retention
diet
h, g-atoms
nitrogen Exptl
diet
Protein
nitrogen
diet
Exptl
Control
diet
3.8 1.1+0.3 2.9+0.5
5.0 1.8zt0.4 2*9+0.5
3.8 1 .wzo.2 2.5*0.3
1.4 I .7*0.4 2.5&0.4
1 &to.4
1 Ato.5
1.5M.3
0.8M.5
Net quantities are means & SE. * Dietary total (crude protein) nitrogen and protein (crude protein excluding added urea) nitrogen were estimated from the percentage composition and daily consumption of each diet.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1975 American Physiological Society. All rights reserved.
1066 turnover of protein could be of high biological significance, if it were to involve a net flux and incorporation of urea nitrogen into protein within the intestinal lumen coupled with proteolysis and absorption of amino acids. The potential for underestimation appears to be illustrated in particular by the data concerning the cecum. Net appearance of protein N was not detected in the ceca of animals which were fed adequate amounts of protein. However, in this study (Z), cyclic appearance and disap pearance of volatile fatty acids was observed suggesting the probability of cyclic changes in microbial cell mass and, therefore, of necessity, microbial protein content of this organ. More direct evidence has been presented by Slade et al. (13), who have documented the incorporation of nonprotein nitrogen into microbial protein in the equine cecum and the appearance of essential and nonessential amino acids derived from this protein in the venous blood leaving this organ. Coupling these two lines of observation would lead one to anticipate periods of net appearance of cecal protein- Failure to observe this is presumed to reflect masking of microbial activity by the continuous entry, digestion, and absorption of dietary and/or endogenous protein from the ileum. In those ponies which consumed the protein-deficient diet, disappearance rates were much lower (Fig. 4), and a suggestion of net appearance was observed between hours 0 and 2. Note (Fig. 5) that the amount of protein N calculated to enter the cecum by flow was reduced considerably in these animals. A shift was observed in progressively aboral segments from continuous protein disappearance in the cecum toward a situation in which the appearance and disappearance of protein are equivalent. Appearance, in fact, seems to predominate over disappearance in the dorsal colon of the animals receiving the urea-supplemented diet, with the balance being restored in the small colon. The distribution of protein nitrogen in the equine large intestine thus appears to reflect the sum of two processes. The first is simply a continuation of the digestion and absorption of dietary and endogenous protein, which was initiated in the stomach and small intestine. The second seems to be the cyclic appearance and disappearance of protein independent of digesta protein flow along the tract. While no direct evidence concerning the source of this protein is provided by these data, the accompanying appearance and disappearance of products which characterize fermentation, i.e., the volatile fatty acids, argue strongly in favor of a microbial origin. If this is indeed the case, the process assumes special importance in those animals receiving the experimental diet, since it represents the generation of protein de nova within the large intestine from a nonprotein source, presumably urea, which is the major dietary nitrogen supply. Extensive urea N recycling has been demonstrated to occur in man (17), horse (6, lo), and other simple-stomached (1 1), as well as ruminant (9) animals. The incorporation of urea N into amino acids and proteins under conditions of protein deprivation also has been demonstrated to occur in man (4) and in other monogastric species, including the horse (13, 14). The process requires the ureasecatalyzed hydrolysis of urea to ammonia and CO2 within
J. F. WOOTTON
AND
R. A. ARGE:NZXO
the gastrointestinal tract. The subsequent incorporation of ammonia N into nonessential amino acids may occur through the mediation of hepatic or intestinal mucosal enzymes. Alternatively, incorporation into both essential and nonessential amino acids, and thence into microbial protein, can occur within the lumen of the intestine and contribute to the economy of the host animal following subsequent proteolysis. Direct, qualitative evidence for the occurrence of the microbial process in the equine cecum and for the absorption -of amino acids from this organ has been provided by Slade et al. (13). The present findings suggest that the large colon is an important site for occurrence of these processes While absorption of amino acids from the equine large colon has not been investigated, it seems to be entirely plausible. The ultrastructure of the columnar cells of the surface epithelium of this organ in the horse (7), as in several other mammals (16) including man, appears to be closely similar to that of the analogous cells of the cecum and small intestine, including the presence of a brush border. Furthermore, substantial rates of transport of volatile fatty acids by the equine colonic and cecal mucosa have been measured in vitro (2). In humans and other monogastric animals, including the horse, the anabolic role of urea appears to be limited to situations in which dietary supplies of protein and amino acids are less than optimal for meeting metabolic requirements. Given a surfeit of protein, the net effect of enterohepatic urea metabolism becomes an ATP- (and NADH) consuming, futile cycle required for continuous detoxication of ammonia. Ingestion of a 3 % urea-supplemented diet has been shown by Slade et al. (14) to produce elevated plasma urea N concentrations (measured 3 h after feeding). The reduced concentrations (Fig. 2) of ammonia observed in the large intestine coupled with the rapid disappearance of urea (Fig. 1) from the proximal portions of the tract under dietary conditions similar to Slade’s are interesting in light of this fact. Taken together these findings suggest the following chain of events: I) ingested urea is absorbed as such, primarily from the stomach and/or small intestine, and enters the circulating pool producing transient uremia and increased urinary loss ; 2) urea enters the large intestine, where it is degraded to ammonium ion by urease-catalyzed hydrolysis; 3) growth of the expanded microbial population present under this dietary regimen is limited by the supply of nitrogen available within the intestinal lumen; therefore ammonia, produced at increased rates in response to a postprandial entry of urea into the large intestine, is incorporated rapidly into additional microbial protein; 4) as a result the concentrations of free ammonia are maintained at low levels in the large intestine relative to those present in the protein-fed animal; 5) microbial protein hydrolysis results in amino acid generation and absorption. The smaller quantities of protein present in the colon of animals receiving the control diet are presumed to reflect microbial growth limitation by factors other than nitrogen. Since the control diet was consumed in smaller quantity than the experimental diet and also contained a much
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NITROGEN
UTILIZATION
WITHIN
EQUINE
LARGE
1067
INTESTINE
colonic contents of animals consuming compared to those fed the experimental colon should have been more limited. Under these circumstances degradation of proteins and amino acids as microbial energy sources could increase in magnitude. The concomitant deamination would explain the disproportionately high ammonia concentrations observed in the
the control diet.
diet as
The authors gratefully acknowledge the technical support of Ms. Loretta Y. Tu and the advice and counsel of Professor C. E. Stevens. This investigation was supported by Grant AM09280 from the National Institutes of Health and New York State Research Project 46 l-5300. Received
for publication
11 November
1975,
REFERENCES
1. ARGENZIO, R. A., J. E. LOWE, D. W. PICKARD, AND C, E. STEVENS. Digesta Am,
passage
J. Physiol.
and water 226:
1035-1042,
exchange
in the equine
large
intestine.
1974.
2, ARGENZIO, R. A., M. SOUTHWORTH, AND C. E. STEVENS. Sites of organic acid production in the equine gastrointestinal tract. Am. J. Physiol. 226: 1043-1050, 1974. 3. ARGENZIO, R. A., AND C. E. STEVENS. Cyclic changes in ionic composition of digesta in the equine intestinal tract. Am. J, Physiol. 228: 1224-1230, 1975. 4. GIORDANO, G., C. DEPASCALE, C. BALESTRIERI, D. CITTADINI, AND A. CRESCENZI.The incorporation of urea-15N into serum of uremic patients on low nitrogen diets. J. Clin. Invest. 45 : 10 13, 1966. 5. HORWITZ, W. C@cial Methods of Analysis of the Association of OficiaL Agricultural Chemists (11 th ed.). Washington, D.C. : Association of Official Agricultural Chemists, 1970, p. 858. 6. HOUPT, T. R., AND K. A. HOUPT. Nitrogen conservation by ponies fed a low-protein ration. Am. J. Vet. Res. 32: 579-588, 1971. 7. KANAKOUDIS, G. G. Secretory granules in the columnar cells of the cecum and the great colon of the horse. Anat. Pistol. Embryol. 2 : 295-299, 1973. 8. National Research Council. Nutrient Requirements of Domestic Animals. Nutrient Requirements of Horses. Washington, D.C, : National Research Council, vol. 6, 1966.
9, PACKETT, L. V., AND T. D. D. GROVES. Urea recycling in the ovine. J. Animal Sci. 24 : 341-346, 1965. 10. PRIOR, R. L., E-I. F. HINTZ, J. E. LOWE, AND W. J. VISEK. Urea recycling and metabolism of ponies. J. Animal Sci. 38 : 565-571, 1974. Il. ROGOECZI, E., L. IRONS, A. KOJ, AND A. S. MCFARLANE. Isotopic studies of urea metabolism in rabbits. Biochem. J. 95: 521-532, 1965. 12. RUSSELL, J. A. Note on the calorimetric determination of amino nitrogen. J. Biol. Chem. 156: 467468, 1949. 13. SLADE, L. M., R. BISIIOP, J. G. MORRIS, AND D. G. ROBINSON. Digestion and absorption of 15N-labelled microbial protein in the large intestine of the horse. &it, vet. J. 127 : 12-13, 1969. 14. SLADE, L. M., D, G. ROBINSON, AND K. E, CASEY. Nitrogen metabolism in non-ruminant herbivores. I. The influence of non-protein nitrogen and protein quality on nitrogen retention of adult mares. J. Animal Sci. 30: 753-760, 1970. 15. THORLACIUS, S. W., A. DOBSON, AND A, F. SELLERS. Effect of carbon dioxide on urea diffusion through bovine ruminal epithelium. Am. J. Physiol. 220: 162-170, 1971. 16. TONER, P. G., K. E. CARR, AND G. M. WYBURN. TIze Digestive System-An Ultrastructural Atlas and Review. London : Butterworths, 1971, p. 107-120. 17. WALSER, M., AND L. J. BONDENLOS. Urea metabolism in man. J. Gin. Invest. 38 : 16 17-1626, 1959.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1975 American Physiological Society. All rights reserved.
JOURNAL
OF
PHYSIOLOGY
Vol. 229, No. 4, October
1975.
Nitrogen
Printed in U.S.A.
utilization
J. F. WOOTTON Ithaca,
AND
of Physiohgy,
l)efm$nmt New
York
within
protein protein
large intestine
R. A. ARGENZIO
Biochemistry,
and Pharmacology,
New
York
State
Veterinary
College,
Cornell
University,
I4853
WOOTTON, J, F., AND R. A. ARGENZIO. Mtrogen utili can be determined directly. Therefore, net appearance of a constituent in a compartment due to processes other than flow along the tract can be estimated for each time interval and compartment from the following relationship (3): (P a - Pd)At = Qt2 - Qtl - PiAt + P.At where P, and Pd represent the (indeterminant) total rates of appearance and of disappearance and At = t2 - fl. The estimated errors expressed are calculated from the combined errors for the terms in the previous equation as follows : 2 6 (p
s2 a -
p&t
=
-
n(Qt2)
where
8 = standard
deviation.
a2 +
-
n(QtJ
+
a2 n(PiAL)
a2 +
1063
INTESTINE
-
n(~,A
t)
f
Sl,
SECTION
$1,
C OF
RVC
LVC
LDC
RDC
SC,
TRACT
FIG. 1, Concentrations of urea nitrogen in gastrointestinal tract of ponies fed control (A) or experimental (B) diet. Symbols represent time after last feeding (A, 2 h; 0, 4 h; x, 8 h, and l , 12 h or prefeeding). Letters shown on abscissa denote sections of tract separated by ligatures : cranial stomach (S,), caudal stomach (S 2) ; proximal (SIJ, middle (SI,), and distal (SI 3) thirds of small intestine; cecum (C); right ventral (RVC) and left ventral (LVC) colon; left dorsal &DC) and right dorsal (RDC) colon; proximal (SC,) and distal (SC,) halves of small colon. Concentrations represent mean & SE for 3 animals at each time period.
RESULTS
Figure 1 illustrates the concentrations of urea N in the mixed contents of each of the 12 segments of the digestive tract at each time of measurement. Animals ingesting the urea-containing experimental diet exhibited greatly elevated concentrations in the stomach and proximal small intestine 2 h after feeding. Detectible elevation persisted in these segments at 4 h as well. However, significant enhancement was not observed within the large intestine at any of the times of measurement. Furthermore, ammonia N concentrations (Fig. 2) tended to be lower in all segments of the intestinal tracts of these animals compared with those which received the urea-free control diet. The differences were especially pronounced in the colon. Concentrations of free amino acids followed patterns similar to those of ammonia within the small intestine, falling progressively and reaching minimum values of approximately 3 mg-atoms N/kg H20 in the cecal contents. The mean concentrations in the colon remained at similar levels. However, accurate estimations by the method employed were prevented by the high ammonia concentrations present in this organ. Net disappearance of amino nitrogen from the cecum was observed with both diets throughout the 1Z-h interval between feedings. Figure 3 illustrates the content and net appearance rates of ammonia plus urea N in the four compartments of the large intestine. The analogous data for protein N are shown in Fig. 4. Marketi differences between the cecum and the colon are apparent with respect to the distribution
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1975 American Physiological Society. All rights reserved.
1064
J. F. WOOTTUN
AND
R. A. ARGENZIO
were similar for the two diets, and little, if any net appearance of protein could be detected in this compartment; 3) in the colon both net appearance and net disappearance of protein appear to have been greater in the ponies which consumed the urea-containing diet, whereas the loss of nitrogen to the feces was only modestly increased. Daily net changes observed within the entire large intestine are compared with dietary intake in Table 1. The changes in total and protein nitrogen paralleled one another closely+ This relationship extended to each individual compartment and to each time interval examined. Thus metabolic interconversion between protein and pools of low molecular weight nitrogenous constituents present within the intestinal lumen cannot be invoked to explain net appearance and disappearance of protein. Net disappearance was equivalent on the two diets, although, as shown in Fig. 4, the contributions of the various compartments differed. The overall net appearance of protein, essentially all within the colon, was enhanced in animals receiving the protein-deficient diet and exceeded the dietary protein intake. 3
s,
SI,
$I2
SI,
SECTION FIG. 2. concentrations Of tract of ponies fed control (A) described in legend to Fig. 1.
C RVC LVC LDC RDC SC, SC, OF TRACT
nitrogen in gastrointestinal or experimental (B) diet. Symbols are
ammonia
of each of these classes of nitrogenous constituents. In neither case were significant changes in quantity observed in the cecum with respect to times of measurement or diet. Net disappearance essentially was continuous. Although estimated variances are large, the rates of disappearance of both protein and ammonia from the cecum appeared to be greater during each time interval with animals fed the control diet than with those which received the proteindeficient, experimental diet. In contrast, the ammonia and protein content of the colon both show significant time-dependent changes. The patterns of these changes differed with the different diets. Adaptation to the urea diet was accompanied by marked increase in volume of the large intestinal contents (l), which was most striking in the ventral and dorsal compartments of the large colon. This increase in volume was accompanied by a proportionate increase in protein nitrogen content (Fig. 4). The ammonia content was not proportionately greater, as reflected by the lower concentrations shown in Fig. 2. The time-dependent changes in protein and ammonia content correspond to periods of net appearance and net disappearance, which in several cases appear to be significant. A summation of average daily net changes in protein nitrogen distribution is represented in Fig, 5. Inflow and outflow via digesta passage along the tract as well as net appearance and disappearance are indicated for each of the four compartments of the large intestine. Comparison of the two diets shows the following: 1) smaller quantities of protein entered the ceca of animals fed the experimental diet, and net disappearance from this compartment also was reduced; 2) the amounts leaving the cecum by flow
I
0
2 4
6
8
6
8 10122
1012
2
4
6
8
10
6
8
10122
12
2
4
6
8
1012
4
6
8
10
2
4
6
8 10
4
6
8 1012
2
80-
0 2
4
4
122
TIME ( Hr post feeding) FIG. 3. Quantities and net rates of appearance or disappearance (+ SE) of ammonia plus urea N in large intestine of animals fed control (A) or experimental (B) diet. Positive values designate net appearance rates, whereas negative vaIues signify disappearance. Calculations of net appearance are based on data describing passage of PEG (I).
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on August 31, 2018. Copyright © 1975 American Physiological Society. All rights reserved.
NITROGEN
UTILIZATION
WITHIN
EQUINE
LARGE
I
0 2 4
6
8
l012
2
4
t
6
8
I012
2
i
6
8
IO12
2
4
1065
INTESTINE
6
8
1012
greater in those animals consuming the high-protein, control diet and occurred in the cecum and ventral colon, primarily the former compartment (see Fig. 5). Net disappearance of total and protein nitrogen from the entire large intestine (Table 1) was equal on the two diets, the reason being that net appearance due to processes other than flow along the tract was greater in the animals consuming the protein-deficient, experimental diet. Net appearance was observed only in the colon (see Figs. 4 and 5). The values. for appearance and disappearance should not be construed to represent absolute amounts. Rather they provide (within their limits of error) minimal estimates which are not necessarily comparable between two diets. They represent the sums of values for those time intervals during which either net appearance or disappearance was observed. Each time interval is of several hours duration, and asynchrony of flow or of cyclic events among individual animals would minimize the (absolute values of) apparent net rates of appearance and disappearance. Furthermore, the selected time intervals between measurements were arbitrarily chosen and do not necessari1y coincide with cyclic events occurring in the large intestine. The apparent cycles, in turn, differ with diet. Finally, since net differences only are observable, steadystate turnover within a compartment: is not detected. Such CONTROL
DIET
EXPERIMENTAL
DIET
CECUM -
1
0
2 4
6
8
lo,12
I
2
4
6 TIME
8
10 (Hrps?
12
I
2
4
6
10
12
2
4
I
I
r
6
8
to
VENTRAL COLON
12
feeding)
4. Quantities and net rates of appearance of protein nitrogen in large intestine of ponies experimental (B) diet. FIG.
8
--
-
and disappearance fed control (A) and
D%%!
DISCUSSION
Despite the fact that animals closely grouped with respect to size consumed meals of constant size and composition at fixed intervals, large variability was observed in digesta volumes ( 1) and in the concentrations and quantities of nitrogen present at any time of measurement. Estimations of outflow, accumulation and, hence, net appearance involve the combination of pooled data collected from two separate groups of animals. Therefore, as indicated in the figures, the estimated errors are large. Still it is clear that some general conclusions are justified with regard to the temporal changes in nitrogen distribution. Comparison between calculated increments due to flow and actual increments accumulated in a compartment during a series of limited, sequential time intervals permits the detection of transient appearance or disappearance, for example, of protein nitrogen. These changes, which are not detectable through comparisons of either flow or accumulation alone, can be of fundamental significance to the overall nitrogen metabolism of the animal. The estimates of net retention of total and protein nitrogen shown in Table I are based solely on differences between flow into and out of the large intestine. Net retention was
--
SMALL COLON
0.29
0.40
5. Net appearance, disappearance, and passage of protein nitrogen expressed as gram-atoms of nitrogen per 24 h. Net appearance per 24 h is calculated from sum of areas above abscissae in Fig. 4, whereas net disappearance is derived in same fashion from areas below these axes. FIG.
TABLE 1. comparison of daily nitrugen intake with overall net appearance, disappearance, and retention in large intestine Nitrogen/X Total Control
Daily intake* Net appearance Net disappearante Net retention
diet
h, g-atoms
nitrogen Exptl
diet
Protein
nitrogen
diet
Exptl
Control
diet
3.8 1.1+0.3 2.9+0.5
5.0 1.8zt0.4 2*9+0.5
3.8 1 .wzo.2 2.5*0.3
1.4 I .7*0.4 2.5&0.4
1 &to.4
1 Ato.5
1.5M.3
0.8M.5
Net quantities are means & SE. * Dietary total (crude protein) nitrogen and protein (crude protein excluding added urea) nitrogen were estimated from the percentage composition and daily consumption of each diet.
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1066 turnover of protein could be of high biological significance, if it were to involve a net flux and incorporation of urea nitrogen into protein within the intestinal lumen coupled with proteolysis and absorption of amino acids. The potential for underestimation appears to be illustrated in particular by the data concerning the cecum. Net appearance of protein N was not detected in the ceca of animals which were fed adequate amounts of protein. However, in this study (Z), cyclic appearance and disap pearance of volatile fatty acids was observed suggesting the probability of cyclic changes in microbial cell mass and, therefore, of necessity, microbial protein content of this organ. More direct evidence has been presented by Slade et al. (13), who have documented the incorporation of nonprotein nitrogen into microbial protein in the equine cecum and the appearance of essential and nonessential amino acids derived from this protein in the venous blood leaving this organ. Coupling these two lines of observation would lead one to anticipate periods of net appearance of cecal protein- Failure to observe this is presumed to reflect masking of microbial activity by the continuous entry, digestion, and absorption of dietary and/or endogenous protein from the ileum. In those ponies which consumed the protein-deficient diet, disappearance rates were much lower (Fig. 4), and a suggestion of net appearance was observed between hours 0 and 2. Note (Fig. 5) that the amount of protein N calculated to enter the cecum by flow was reduced considerably in these animals. A shift was observed in progressively aboral segments from continuous protein disappearance in the cecum toward a situation in which the appearance and disappearance of protein are equivalent. Appearance, in fact, seems to predominate over disappearance in the dorsal colon of the animals receiving the urea-supplemented diet, with the balance being restored in the small colon. The distribution of protein nitrogen in the equine large intestine thus appears to reflect the sum of two processes. The first is simply a continuation of the digestion and absorption of dietary and endogenous protein, which was initiated in the stomach and small intestine. The second seems to be the cyclic appearance and disappearance of protein independent of digesta protein flow along the tract. While no direct evidence concerning the source of this protein is provided by these data, the accompanying appearance and disappearance of products which characterize fermentation, i.e., the volatile fatty acids, argue strongly in favor of a microbial origin. If this is indeed the case, the process assumes special importance in those animals receiving the experimental diet, since it represents the generation of protein de nova within the large intestine from a nonprotein source, presumably urea, which is the major dietary nitrogen supply. Extensive urea N recycling has been demonstrated to occur in man (17), horse (6, lo), and other simple-stomached (1 1), as well as ruminant (9) animals. The incorporation of urea N into amino acids and proteins under conditions of protein deprivation also has been demonstrated to occur in man (4) and in other monogastric species, including the horse (13, 14). The process requires the ureasecatalyzed hydrolysis of urea to ammonia and CO2 within
J. F. WOOTTON
AND
R. A. ARGE:NZXO
the gastrointestinal tract. The subsequent incorporation of ammonia N into nonessential amino acids may occur through the mediation of hepatic or intestinal mucosal enzymes. Alternatively, incorporation into both essential and nonessential amino acids, and thence into microbial protein, can occur within the lumen of the intestine and contribute to the economy of the host animal following subsequent proteolysis. Direct, qualitative evidence for the occurrence of the microbial process in the equine cecum and for the absorption -of amino acids from this organ has been provided by Slade et al. (13). The present findings suggest that the large colon is an important site for occurrence of these processes While absorption of amino acids from the equine large colon has not been investigated, it seems to be entirely plausible. The ultrastructure of the columnar cells of the surface epithelium of this organ in the horse (7), as in several other mammals (16) including man, appears to be closely similar to that of the analogous cells of the cecum and small intestine, including the presence of a brush border. Furthermore, substantial rates of transport of volatile fatty acids by the equine colonic and cecal mucosa have been measured in vitro (2). In humans and other monogastric animals, including the horse, the anabolic role of urea appears to be limited to situations in which dietary supplies of protein and amino acids are less than optimal for meeting metabolic requirements. Given a surfeit of protein, the net effect of enterohepatic urea metabolism becomes an ATP- (and NADH) consuming, futile cycle required for continuous detoxication of ammonia. Ingestion of a 3 % urea-supplemented diet has been shown by Slade et al. (14) to produce elevated plasma urea N concentrations (measured 3 h after feeding). The reduced concentrations (Fig. 2) of ammonia observed in the large intestine coupled with the rapid disappearance of urea (Fig. 1) from the proximal portions of the tract under dietary conditions similar to Slade’s are interesting in light of this fact. Taken together these findings suggest the following chain of events: I) ingested urea is absorbed as such, primarily from the stomach and/or small intestine, and enters the circulating pool producing transient uremia and increased urinary loss ; 2) urea enters the large intestine, where it is degraded to ammonium ion by urease-catalyzed hydrolysis; 3) growth of the expanded microbial population present under this dietary regimen is limited by the supply of nitrogen available within the intestinal lumen; therefore ammonia, produced at increased rates in response to a postprandial entry of urea into the large intestine, is incorporated rapidly into additional microbial protein; 4) as a result the concentrations of free ammonia are maintained at low levels in the large intestine relative to those present in the protein-fed animal; 5) microbial protein hydrolysis results in amino acid generation and absorption. The smaller quantities of protein present in the colon of animals receiving the control diet are presumed to reflect microbial growth limitation by factors other than nitrogen. Since the control diet was consumed in smaller quantity than the experimental diet and also contained a much
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NITROGEN
UTILIZATION
WITHIN
EQUINE
LARGE
1067
INTESTINE
colonic contents of animals consuming compared to those fed the experimental colon should have been more limited. Under these circumstances degradation of proteins and amino acids as microbial energy sources could increase in magnitude. The concomitant deamination would explain the disproportionately high ammonia concentrations observed in the
the control diet.
diet as
The authors gratefully acknowledge the technical support of Ms. Loretta Y. Tu and the advice and counsel of Professor C. E. Stevens. This investigation was supported by Grant AM09280 from the National Institutes of Health and New York State Research Project 46 l-5300. Received
for publication
11 November
1975,
REFERENCES
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passage
J. Physiol.
and water 226:
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exchange
in the equine
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1974.
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9, PACKETT, L. V., AND T. D. D. GROVES. Urea recycling in the ovine. J. Animal Sci. 24 : 341-346, 1965. 10. PRIOR, R. L., E-I. F. HINTZ, J. E. LOWE, AND W. J. VISEK. Urea recycling and metabolism of ponies. J. Animal Sci. 38 : 565-571, 1974. Il. ROGOECZI, E., L. IRONS, A. KOJ, AND A. S. MCFARLANE. Isotopic studies of urea metabolism in rabbits. Biochem. J. 95: 521-532, 1965. 12. RUSSELL, J. A. Note on the calorimetric determination of amino nitrogen. J. Biol. Chem. 156: 467468, 1949. 13. SLADE, L. M., R. BISIIOP, J. G. MORRIS, AND D. G. ROBINSON. Digestion and absorption of 15N-labelled microbial protein in the large intestine of the horse. &it, vet. J. 127 : 12-13, 1969. 14. SLADE, L. M., D, G. ROBINSON, AND K. E, CASEY. Nitrogen metabolism in non-ruminant herbivores. I. The influence of non-protein nitrogen and protein quality on nitrogen retention of adult mares. J. Animal Sci. 30: 753-760, 1970. 15. THORLACIUS, S. W., A. DOBSON, AND A, F. SELLERS. Effect of carbon dioxide on urea diffusion through bovine ruminal epithelium. Am. J. Physiol. 220: 162-170, 1971. 16. TONER, P. G., K. E. CARR, AND G. M. WYBURN. TIze Digestive System-An Ultrastructural Atlas and Review. London : Butterworths, 1971, p. 107-120. 17. WALSER, M., AND L. J. BONDENLOS. Urea metabolism in man. J. Gin. Invest. 38 : 16 17-1626, 1959.
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