V. Wizemann,
D. Ludwig,
function of the border in uremia1 R. Kuhi,
ABSTRACT
Amino
L-leucine
appears
absorption. chronic
Nut,.
31:
acid
I. Burgmann
absorption
to be inhibited
Brush renal
and
border
insufficiency.
1642-1646,
activity The
was
with
mild
of intestinal same
holds
studied uremic
in chronic
uremic
intoxication,
maltase
and
whereas disaccharidases
for -y-glutamyl-transpeptidase
rats.
Intestinal severe
transport
uremia
is higher activity.
of
enhances in rats Am.
with
J. Clin.
1978.
Although morphological disorders of the gastro-intestinal tract in uremia are well documented (1-3), conificting data have been reported concerning intestinal sugar and amino acid transport as well as mucosal disaccharidase and dipeptidase activities in renal failure (2, 4-8). The contradicting results may be explained by different grades of uremia and by the experimental design. Specific digestive enzyme activity had not been measured in the mucosal brush border where most of the enzymes are located, but in total mucosal homogenate. Following the concept of Craine (9) the intestinal brush border membrane is a “digeslive-absorptive surface”, where the enzymes located at the luminal membrane of the mucosa are involved in the transepitheial transport of sugar and amino acids. Using a brush border preparation, disaccharidase and dipeptidase activity of intestinal mucosa was determined in normal and in 90% nephrectomized rats. Referring to the hypothesis of Orlowski and Meister (10) that membrane bound -y-glutamyl transpeptidase plays a role in amino acid transport the enzyme was measured too. In order to estimate the overall effect of uremia on intestinal amino acid absorption, a preparation of isolated perfused ileal segments was used.
The brush border membrane was isolated according to the method of Hopfer et al. (1 l).2 In the membrane fraction, activities of the disaccharidases (lactase, cell lobiase, saccharase, maltase) were determined by the method of Dahlqvist (12), the dipeptidases (L-leucyl-Lleucine, L-methionyl-L-methionine, L-methionyl-L-leucine, L-leucyl-glycine, glycine-L-leucine, L-proline-L-leucine) according to Caspary et al. (14). The transport studies had been performed with isolated perfused ileal segments of normal and uremic rats. ‘4C-L-leucine was used.
Results In the brush border preparation, disaccharidases were enriched 13- to 32-fold cornpared to the activity in the total homogenate. The enrichment of dipeptidases in the membrane fraction was comparatively smaller (Fig. 1). The measurements of disaccharidases showed that chronic renal failure only had an effect on brush border maltase activity, which was enhanced (P < 0.01) (Fig. 2). Five of the six determined dipeptidases showed a higher activity in the intestinal brush border of the chronic uremic rats (P < 0.05, Fig. 3). The same held for -y-GT activity (Fig. 4). When the membrane fraction was incubated with so called uremic toxins, 2-hydroxyphenolic acid and 4-aminobenzoic acid
Methods weight 338 ± 22 g) were used as experimental animals. Uremia was induced by 90% nephrectomy. About 6 to 8 week&”after the operation, serum urea level was 222 ± 27 mg/ 100 ml (control group 48 ± 8 mg/l00 ml). The animals had free access to Altromin-diet and water.
1 From the Department of Internal Medicine, Mcdizinische University, 63 Giessen, Federal Republic of Germany. 2 The authors thank Dr. H. Murer (Max-Planck-Institut f#{252}r Biophysik, Frankfurt) for his aid in the isolation method.
1642
31: SEPTEMBER
One
hundred
sixteen
The
male
American
rats
of
Journal
a Siv
strain
ofClinical
(body
Nutrition
1978,
pp.
1642-1646.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
Digestive-absorptive intestinal brush
AMINO
ACID
ABSORPTION
IN
CHRONIC
UREMIA
1643
enrichment -.
30
1
20
‘5
0
FIG. mU/mg
1.
total
Enrichment
0
hemogenat.
brush
of disaccharidases
and
border
membrane
dipeptidases
in the
brush
border
of rat
intestine.
pro? cpiiobiase
lactose
r’ailase
saccharini
2O0
no effect on enzyme were not influenced In the L-leucine suffering sufficiency. absorption
2300
2200
activity. Disaccharidases by either of the toxins.
transport studies the absorption of appeared to be depressed in rats from a moderate chronic renal inIn end stage uremia, L-leucine seemed to be enhanced (Fig. 5).
2100
Discussion 2000
1900
00
300
200
100
0 FIG. brush
( l0 was urea ml),
control
2. Disaccharidase
jremic
activity
in
the
intestinal
border.
M/liter), the activity of all dipeptidases inhibited by 24 to 36%. The addition of (300 mg/lOO ml), creatinine (20 mg/l00 and methylguanidine (l0 M/liter) had
In contrast to the fmding of diminished dipeptidase activities in the whole homogenate of mucosal biopsy material from uremic patients (2, 6, 7), animal experiences indicate no alteration of enzyme activity in renal failure (with the exception of leucine-proline dipeptidase, which was enhanced) (5). In the present study, membrane bound intestinal dipeptidases were significantly increased in the uremic animals. A possible role of brush border located -y-GT is indicated in Figure 4. According to the hypothesis of Orlowski and Meister (10) the enzyme transfers the -y-glutamyl group of glutathione to an acceptor, which can be an amino acid or a small peptide. Corresponding to the dipeptidase results, -y-GT activity is higher in the uremic brush border. Small molecular uremic toxins (14) had no influence on brush border enzyme activity with the exception of phenolic compounds and aromatic amines. The concentration of
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
i
25
WIZEMANN
1644
ET AL.
9500
prot
9000
8500
control uremic
0
8000
7500
7000#{149}
6500
6000
5500
l#{149}u-lt’u
met-met
FIG.
met-ieu 3. Brush
border
leu-g(y activity
of
gly-ieu dipeptidases.
p.roi-ieu
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
mU/mg
AMINO
mg prof
1’
-Glu-Cys
ACID
ABSORPTION
IN CHRONIC
-acceptor.Cys-
Gly
-I
300#{149}
0 0
200
control
uremic
100
FIG. of the
4. -y-GT concept
activity. of Orlowski
The equation and Meister
gives (10).
.ig Ieucmne
an outline
transport
active
of
L- leucmne
in
the
isolated
ileum
r)gprot amin
(
2.5
urea
;-
628 mg
7
I
2.0-
I I I
15
I
0/
7
-
..
urea52mg/.
L
10-
1 I
05-
-‘At
_
urea 2lOmg’/.
.
urea
154
./‘ 0
I 0
equilibration 0#{149}
1
25
FIG.
5.
Absorption
of
L-leucine
T
35
45 at different
55
65
grades
of uremic
75
85 intoxication
mm in rats.
mg’Y.
./.
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
r-Glu
1645
the two toxins was about 10-fold higher than in the serum of uremic patients. The rat intestinal absorption of leucine showed a biphasic pattern in chronic renal failure, opposite to the behavior of the exocrine pancreas (15). At a moderate degree of uremic intoxication, amino acid resorption appears to be inhibited, whereas in terminal renal failure, transport is enhanced. In experimental diabetes of rats a similar constellation of an increased digestive and transport function of the intestinal mucosa was found (16). There are no data available on intestinal amino acid absorption in uremic patients. Based on the fact that uremics can maintain a neutral or even positive nitrogen balance under a strictly protein-restricted diet one can assume that amino acid absorption is either undisturbed or enhanced. Our rat experiments support this hypothesis.
-Gly#{149}acceptor (glurathione)
400
UREMIA
WIZEMANN
1646
ET
AL. of
References
DAHLQVIST.
Morphology,
H. EDER. AutoraUntersuchungen Enterocolitis. Vir340: 357, 1970.
N.
BERG
dipeptidases
and
disac-
charidases ofsmall intestinal mucosa in chronic renal failure. Acta Med. Scand. 195: 465, 1974. 3. MASON, E. E. Gastrointestinal lesions occurring in uremia. Ann. Internal. Med. 37: 96, 1952. 4. GRIMMEL, K., AND H. KASPER. Carbohydrate, protein and fat absorption in chronic uraemia. In: Dialysis, Transplantation, Nephrology, edited by B. H. B. Robinson. Pitman Medical, 1976, pp. 566. 5. GRIMMEL, K., S. BONGARTZ AND H. KASPER. The activity
of
intestinal
dases
in rats
with
disaccharidases
chronic
renal 21: 383,
Hepato-Gastroenterol.
and
insufficiency.
dipepti-
SCHMIDT.
activity
Anwendung
11.
12. 13.
14.
1974.
bowel disachyperazotemic states. Prague: VIIIth International Congress of Gastroenterology, July 7-15, Prague, 1968. 7. MCNAIR, A., J. OLSEN. Disaccharidase activity in chronic renal failure. Acta Med. Scand. 195: 93, 1974. 8. POINTNER, H., U. FLEGEL, C. BAYER AND P. charidase
15.
in
des
Trehalase-Toleranztests
zur Untersuchung der D#{252}nndarmresorption bei Patienten unter chronischer H#{228}modilayse. Wien. Klin. Wochochr. 86: 691, 1974. 9. CRANE, R. K. Structural and functional organization
epithelial
brush
cell
border.
In:
edited by K. B. Warren. demic Press, 1967, pp. 71. ORLOWSKI, M., AND A. MEISTER.
New
cycle:
for
a possible
Proc.
Acta
I. Small
6. MADZAROVA-NOHEJLOVA,
10.
A.
AND
an
transport,
16.
transport
Intracellular York:
The
system
Aca-
y-glutamyl amino
acids.
Natl.
Acad. Sci. USA 67, 1248, 1970. HOPFER, U., K. NELSON, J. PEROTrO AND K. J. ISSELBACHER. Glucose transport in isolated brush border membranes from rat small intestine. J. Biol. Chem. 248: 25, 1973. DAHLQVIST, A. A method for assay of intestinal disaccharidases. Anal. Biochem. 7: 18, 1964. CASPARY, W. F. Intestinale Peptidhydrolasenaktivit at in menschlichem D#{252}nndarmbiopsiematerialeine einfache Bestimmungsmethode. Klin. Wochechr. 52: 341, 1974. HICKS, J., D. YOUNG AND I. WOOTON. The effect of uremic blood constituents on certain cerebral enzymes. Clin. Chim. Acta 9: 228, 1964. WIZEMANN, V. Exocrine pancreatic function in chronic renal failure. In: Dialysis, Transplantation, Nephrology, edited by B. H. B. Robinson. Pitman Medical, 1976, pp. 585.
W. F., A. M.
CASPARY, FELDT. of
Increase
of digestive
intestinal
rats.
mucosa
Europ.
from
J. Clin.
Invest.
17.
GULYASSAY,
Uremia.
P., Stuttgart:
J.
18.
KOPPLE,
J. D.,
and keto Nephron
acid diets 80: 1, 1977.
RHEIN and
M. E. for
CREUTZfunctions
streptozotozin-diabetic
2: 278,
PETERS AND Thieme Verlag,
AND
W.
AND transport
1972. P. SCHOENFELD. 1972, p. 163.
SWENDSEID.
therapy
Amino
in
renal
acid
failure.
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
I. CASTRUP, H. J., U. LOHRS AND diographische und histochemische zur Entstehung der sogenannten chows Arch. Abt. A Pathol. Anat. 2. DENNEBERG, T., T. LINDBERG,
D. Ludwig,
function of the border in uremia1 R. Kuhi,
ABSTRACT
Amino
L-leucine
appears
absorption. chronic
Nut,.
31:
acid
I. Burgmann
absorption
to be inhibited
Brush renal
and
border
insufficiency.
1642-1646,
activity The
was
with
mild
of intestinal same
holds
studied uremic
in chronic
uremic
intoxication,
maltase
and
whereas disaccharidases
for -y-glutamyl-transpeptidase
rats.
Intestinal severe
transport
uremia
is higher activity.
of
enhances in rats Am.
with
J. Clin.
1978.
Although morphological disorders of the gastro-intestinal tract in uremia are well documented (1-3), conificting data have been reported concerning intestinal sugar and amino acid transport as well as mucosal disaccharidase and dipeptidase activities in renal failure (2, 4-8). The contradicting results may be explained by different grades of uremia and by the experimental design. Specific digestive enzyme activity had not been measured in the mucosal brush border where most of the enzymes are located, but in total mucosal homogenate. Following the concept of Craine (9) the intestinal brush border membrane is a “digeslive-absorptive surface”, where the enzymes located at the luminal membrane of the mucosa are involved in the transepitheial transport of sugar and amino acids. Using a brush border preparation, disaccharidase and dipeptidase activity of intestinal mucosa was determined in normal and in 90% nephrectomized rats. Referring to the hypothesis of Orlowski and Meister (10) that membrane bound -y-glutamyl transpeptidase plays a role in amino acid transport the enzyme was measured too. In order to estimate the overall effect of uremia on intestinal amino acid absorption, a preparation of isolated perfused ileal segments was used.
The brush border membrane was isolated according to the method of Hopfer et al. (1 l).2 In the membrane fraction, activities of the disaccharidases (lactase, cell lobiase, saccharase, maltase) were determined by the method of Dahlqvist (12), the dipeptidases (L-leucyl-Lleucine, L-methionyl-L-methionine, L-methionyl-L-leucine, L-leucyl-glycine, glycine-L-leucine, L-proline-L-leucine) according to Caspary et al. (14). The transport studies had been performed with isolated perfused ileal segments of normal and uremic rats. ‘4C-L-leucine was used.
Results In the brush border preparation, disaccharidases were enriched 13- to 32-fold cornpared to the activity in the total homogenate. The enrichment of dipeptidases in the membrane fraction was comparatively smaller (Fig. 1). The measurements of disaccharidases showed that chronic renal failure only had an effect on brush border maltase activity, which was enhanced (P < 0.01) (Fig. 2). Five of the six determined dipeptidases showed a higher activity in the intestinal brush border of the chronic uremic rats (P < 0.05, Fig. 3). The same held for -y-GT activity (Fig. 4). When the membrane fraction was incubated with so called uremic toxins, 2-hydroxyphenolic acid and 4-aminobenzoic acid
Methods weight 338 ± 22 g) were used as experimental animals. Uremia was induced by 90% nephrectomy. About 6 to 8 week&”after the operation, serum urea level was 222 ± 27 mg/ 100 ml (control group 48 ± 8 mg/l00 ml). The animals had free access to Altromin-diet and water.
1 From the Department of Internal Medicine, Mcdizinische University, 63 Giessen, Federal Republic of Germany. 2 The authors thank Dr. H. Murer (Max-Planck-Institut f#{252}r Biophysik, Frankfurt) for his aid in the isolation method.
1642
31: SEPTEMBER
One
hundred
sixteen
The
male
American
rats
of
Journal
a Siv
strain
ofClinical
(body
Nutrition
1978,
pp.
1642-1646.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
Digestive-absorptive intestinal brush
AMINO
ACID
ABSORPTION
IN
CHRONIC
UREMIA
1643
enrichment -.
30
1
20
‘5
0
FIG. mU/mg
1.
total
Enrichment
0
hemogenat.
brush
of disaccharidases
and
border
membrane
dipeptidases
in the
brush
border
of rat
intestine.
pro? cpiiobiase
lactose
r’ailase
saccharini
2O0
no effect on enzyme were not influenced In the L-leucine suffering sufficiency. absorption
2300
2200
activity. Disaccharidases by either of the toxins.
transport studies the absorption of appeared to be depressed in rats from a moderate chronic renal inIn end stage uremia, L-leucine seemed to be enhanced (Fig. 5).
2100
Discussion 2000
1900
00
300
200
100
0 FIG. brush
( l0 was urea ml),
control
2. Disaccharidase
jremic
activity
in
the
intestinal
border.
M/liter), the activity of all dipeptidases inhibited by 24 to 36%. The addition of (300 mg/lOO ml), creatinine (20 mg/l00 and methylguanidine (l0 M/liter) had
In contrast to the fmding of diminished dipeptidase activities in the whole homogenate of mucosal biopsy material from uremic patients (2, 6, 7), animal experiences indicate no alteration of enzyme activity in renal failure (with the exception of leucine-proline dipeptidase, which was enhanced) (5). In the present study, membrane bound intestinal dipeptidases were significantly increased in the uremic animals. A possible role of brush border located -y-GT is indicated in Figure 4. According to the hypothesis of Orlowski and Meister (10) the enzyme transfers the -y-glutamyl group of glutathione to an acceptor, which can be an amino acid or a small peptide. Corresponding to the dipeptidase results, -y-GT activity is higher in the uremic brush border. Small molecular uremic toxins (14) had no influence on brush border enzyme activity with the exception of phenolic compounds and aromatic amines. The concentration of
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
i
25
WIZEMANN
1644
ET AL.
9500
prot
9000
8500
control uremic
0
8000
7500
7000#{149}
6500
6000
5500
l#{149}u-lt’u
met-met
FIG.
met-ieu 3. Brush
border
leu-g(y activity
of
gly-ieu dipeptidases.
p.roi-ieu
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
mU/mg
AMINO
mg prof
1’
-Glu-Cys
ACID
ABSORPTION
IN CHRONIC
-acceptor.Cys-
Gly
-I
300#{149}
0 0
200
control
uremic
100
FIG. of the
4. -y-GT concept
activity. of Orlowski
The equation and Meister
gives (10).
.ig Ieucmne
an outline
transport
active
of
L- leucmne
in
the
isolated
ileum
r)gprot amin
(
2.5
urea
;-
628 mg
7
I
2.0-
I I I
15
I
0/
7
-
..
urea52mg/.
L
10-
1 I
05-
-‘At
_
urea 2lOmg’/.
.
urea
154
./‘ 0
I 0
equilibration 0#{149}
1
25
FIG.
5.
Absorption
of
L-leucine
T
35
45 at different
55
65
grades
of uremic
75
85 intoxication
mm in rats.
mg’Y.
./.
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
r-Glu
1645
the two toxins was about 10-fold higher than in the serum of uremic patients. The rat intestinal absorption of leucine showed a biphasic pattern in chronic renal failure, opposite to the behavior of the exocrine pancreas (15). At a moderate degree of uremic intoxication, amino acid resorption appears to be inhibited, whereas in terminal renal failure, transport is enhanced. In experimental diabetes of rats a similar constellation of an increased digestive and transport function of the intestinal mucosa was found (16). There are no data available on intestinal amino acid absorption in uremic patients. Based on the fact that uremics can maintain a neutral or even positive nitrogen balance under a strictly protein-restricted diet one can assume that amino acid absorption is either undisturbed or enhanced. Our rat experiments support this hypothesis.
-Gly#{149}acceptor (glurathione)
400
UREMIA
WIZEMANN
1646
ET
AL. of
References
DAHLQVIST.
Morphology,
H. EDER. AutoraUntersuchungen Enterocolitis. Vir340: 357, 1970.
N.
BERG
dipeptidases
and
disac-
charidases ofsmall intestinal mucosa in chronic renal failure. Acta Med. Scand. 195: 465, 1974. 3. MASON, E. E. Gastrointestinal lesions occurring in uremia. Ann. Internal. Med. 37: 96, 1952. 4. GRIMMEL, K., AND H. KASPER. Carbohydrate, protein and fat absorption in chronic uraemia. In: Dialysis, Transplantation, Nephrology, edited by B. H. B. Robinson. Pitman Medical, 1976, pp. 566. 5. GRIMMEL, K., S. BONGARTZ AND H. KASPER. The activity
of
intestinal
dases
in rats
with
disaccharidases
chronic
renal 21: 383,
Hepato-Gastroenterol.
and
insufficiency.
dipepti-
SCHMIDT.
activity
Anwendung
11.
12. 13.
14.
1974.
bowel disachyperazotemic states. Prague: VIIIth International Congress of Gastroenterology, July 7-15, Prague, 1968. 7. MCNAIR, A., J. OLSEN. Disaccharidase activity in chronic renal failure. Acta Med. Scand. 195: 93, 1974. 8. POINTNER, H., U. FLEGEL, C. BAYER AND P. charidase
15.
in
des
Trehalase-Toleranztests
zur Untersuchung der D#{252}nndarmresorption bei Patienten unter chronischer H#{228}modilayse. Wien. Klin. Wochochr. 86: 691, 1974. 9. CRANE, R. K. Structural and functional organization
epithelial
brush
cell
border.
In:
edited by K. B. Warren. demic Press, 1967, pp. 71. ORLOWSKI, M., AND A. MEISTER.
New
cycle:
for
a possible
Proc.
Acta
I. Small
6. MADZAROVA-NOHEJLOVA,
10.
A.
AND
an
transport,
16.
transport
Intracellular York:
The
system
Aca-
y-glutamyl amino
acids.
Natl.
Acad. Sci. USA 67, 1248, 1970. HOPFER, U., K. NELSON, J. PEROTrO AND K. J. ISSELBACHER. Glucose transport in isolated brush border membranes from rat small intestine. J. Biol. Chem. 248: 25, 1973. DAHLQVIST, A. A method for assay of intestinal disaccharidases. Anal. Biochem. 7: 18, 1964. CASPARY, W. F. Intestinale Peptidhydrolasenaktivit at in menschlichem D#{252}nndarmbiopsiematerialeine einfache Bestimmungsmethode. Klin. Wochechr. 52: 341, 1974. HICKS, J., D. YOUNG AND I. WOOTON. The effect of uremic blood constituents on certain cerebral enzymes. Clin. Chim. Acta 9: 228, 1964. WIZEMANN, V. Exocrine pancreatic function in chronic renal failure. In: Dialysis, Transplantation, Nephrology, edited by B. H. B. Robinson. Pitman Medical, 1976, pp. 585.
W. F., A. M.
CASPARY, FELDT. of
Increase
of digestive
intestinal
rats.
mucosa
Europ.
from
J. Clin.
Invest.
17.
GULYASSAY,
Uremia.
P., Stuttgart:
J.
18.
KOPPLE,
J. D.,
and keto Nephron
acid diets 80: 1, 1977.
RHEIN and
M. E. for
CREUTZfunctions
streptozotozin-diabetic
2: 278,
PETERS AND Thieme Verlag,
AND
W.
AND transport
1972. P. SCHOENFELD. 1972, p. 163.
SWENDSEID.
therapy
Amino
in
renal
acid
failure.
Downloaded from https://academic.oup.com/ajcn/article-abstract/31/9/1642/4650724 by University of Rhode Island user on 08 December 2018
I. CASTRUP, H. J., U. LOHRS AND diographische und histochemische zur Entstehung der sogenannten chows Arch. Abt. A Pathol. Anat. 2. DENNEBERG, T., T. LINDBERG,
