Articles in PresS. Am J Physiol Gastrointest Liver Physiol (December 30, 2004). doi:10.1152/ajpgi.00408.2004
15
15
1
VALIDATION OF LACTOSE[ N, N]-UREIDE AS A TOOL TO STUDY COLONIC NITROGEN
2
METABOLISM.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Karen P Geboes, Vicky De Preter, Anja Luypaerts, Bert Bammens, Pieter Evenepoel, Yvo Ghoos, Paul Rutgeerts, Kristin Verbeke Department of Gastroenterology University Hospital Gasthuisberg Catholic University of Leuven Herestraat 49 3000 Leuven Belgium Corresponding author: Karen Geboes Laboratory of Digestion and Absorption Department of Gastroenterology University Hospital Gasthuisberg Herestraat 49 3000 Leuven Belgium
[email protected] Tel: 003216344390 Fax: 003216344399
25 26
Key-words: carbohydrate fermentation, colonic nitrogen, stable isotopes
27 28 29 30
Abbreviations: LU: lactose ureide PEG: polyethylene glycol
31 32
This study has been carried out with financial support from the Commission of the European
33
Communities, specific RTD programme “Quality of Life and Management of Living Resources”,
34
QLK1-2001-00431 “Stable isotope applications to monitor starch digestion and fermentation for
35
the development of functional foods”. It does not necessarily reflect its views and in no way
36
anticipates the Commission’s future policy in this area.
37 38 39 40 41 42
1
Copyright © 2004 by the American Physiological Society.
1 2
ABSTRACT
3
In vitro experiments have shown that fermentation of carbohydrates prevents accumulation of
4
nitrogen in the colon. Variable results have been obtained upon modulation of dietary intakes
5
in vivo.
6
Lactose[ N, N]-ureide has been proposed as a tool to study colonic nitrogen metabolism.
7
However, upon oral administration of the marker, different urinary excretion patterns of the
8
label have been found. In this study, 50mg lactose[ N, N]-ureide was directly instilled in the
9
colon through an orocaecal tube to investigate the colonic handling of this molecule in a direct
15
15
15
15
N-
15
10
way.
11
In basal conditions, 42 (37 – 48)% of labeled nitrogen administered as lactose[ N, N]-ureide
12
was retrieved in urine after 72h. A substantial variability in total urinary excretion of the label
13
was found, but the urinary excretion pattern of the label was similar in all volunteers. When
14
inulin, a fermentable carbohydrate, was administered together with the labeled marker, a
15
significant decrease in urinary excretion of
16
The effect of a smaller dose of inulin (250mg) on colonic handling of lactose-[ N, N]-ureide
17
(250mg), was investigated in another group of volunteers and this time faecal excretion of the
18
marker was also evaluated. The results seem to indicate that fermentation of inulin causes an
19
increased faecal excretion of the marker, thereby reducing urinary excretion but not retention
20
in the human nitrogen pool.
21
This instillation study shows that lactose[ N, N]-ureide is a tool with good properties to
22
investigate the effect of different types of carbohydrates on nitrogen metabolism in the
23
proximal colon in vivo.
15
15
15
N after 72h was found, to 29 (23 – 34)%. 15
15
15
15
24
2
1
The accumulation of ammonia in the colon is of great importance for human health. First, part of the
2
ammonia formed in the colon is salvaged for human metabolism. On a normal protein diet, it has been
3
estimated that every day, about 30% of the nitrogen present in urea produced in the liver is salvaged
4
from the colon and 70% of the salvaged nitrogen is utilized for amino acid and protein synthesis (7).
5
Secondly, the amount of ammonia produced in the colon is important in some pathological conditions,
6
such as chronic portosystemic encephalopathy and end-stage renal failure (3, 26). Thirdly, some of
7
the effects of ammonia described in in vitro studies may point to a possible local toxic effect upon the
8
colonic mucosa. Ammonia has been shown to affect the intermediary metabolism and DNA synthesis
9
in colonic epithelial cells and to reduce their life span. It increases the turnover of epithelial cells and in
10
this way, increases the probability of genetic damage occurring in the presence of oncogenic agents
11
since dividing cell populations are more susceptible to chemical carcinogenesis (22).
12
Ammonia is derived from proteolysis and ureolysis in the lumen of the large intestine. On average, 0.3
13
to 4.1g of nitrogen, the majority in the form of protein and peptides, enters the colon daily from the
14
small bowel (11). The polypeptide chains in dietary and endogenous protein are hydrolyzed by
15
proteases and peptidases to amino acids, which are further metabolized through various reactions,
16
involving deamination with the production of ammonia. Ammonia can also be derived from urea. There
17
are two possible ways in which urea may reach the microflora of the large bowel: through the
18
ileocaecal valve or by diffusion of blood urea in intestinal contents. The flux of urea from the ileum to
19
the colon is estimated at only 0.75g/day (5). The flux of urea by diffusion from the blood through the
20
colonic wall depends upon protein intake, metabolic state of the host and energy supply to the colonic
21
flora. Intraluminal hydrolysis of urea into ammonia is so fast that very little intact urea molecules may
22
be detected in the colonic lumen.
23
The accumulation of ammonia in the large bowel can be decreased by reducing the dietary protein
24
intake, but also by administration of fermentable carbohydrates. In vitro experiments have shown that
25
carbohydrate fermentation affects bacterial protein metabolism in different ways. Carbohydrate
26
fermentation results in a decrease of the pH through production of short chain fatty acids. As a
27
consequence, protease activity will decrease, since large intestinal proteases have a neutral to
28
alkaline pH optimum (11,19). In addition, amino acid metabolism is decreased when fermentable
29
carbohydrates are present in the medium (18, 19, 21, 23). This effect is suggested to be caused by
30
catabolite repression, which implies that in the presence of a certain substrate – in this case
3
1
fermentable carbohydrates - the transcription of the genes involved in the bacterial utilization of
2
another substrate (amino acids) is repressed, and their corresponding products are inactivated and
3
degraded. Both mechanisms lead to a decreased production of ammonia. On the other hand,
4
fermentation of carbohydrates provides an energy source to the microflora, thereby expanding
5
bacterial mass and increasing the incorporation of ammonia and amino acid nitrogen into the bacteria
6
(20). It is not clear whether colonic bacteria prefer free luminal amino acids or ammonia as nitrogen
7
source (6, 27).
8 9
Until now, all studies concerning colonic nitrogen metabolism involving human volunteers have been
10
performed by measurement of nitrogen excretion in urine and faeces. These studies led to
11
contradictory results (1, 2, 9, 24, 25, 29). These differences could be attributed to the fact that a
12
variety of factors are implied in the regulation of colonic nitrogen metabolism, including protein intake
13
in the diet and the metabolic state of the host. Within the large bowel, differences in fermentation
14
processes have been described in the different regions. Carbohydrate fermentation occurs mainly in
15
the proximal colon (12). Upon exhaustion of fermentable carbohydrates, extensive proteolysis may still
16
occur in the distal colon, suggesting that fermentation of carbohydrates causes merely a delay in
17
protein fermentation, especially on high protein intakes (9). Nutrient availability and especially the ratio
18
of available carbohydrates to nitrogen, depending both on the amount and type of protein and
19
fermentable carbohydrate taken in the diet, may be the most important factors controlling nitrogen
20
metabolism in vivo (10, 17, 29). Fermentation of carbohydrates influences both protein degradation
21
and nitrogen uptake by bacteria.
22 23
The use of stable isotopes is a safe and elegant way to investigate physiological processes in vivo.
24
In this study, lactose-ureide was used as a source of labeled nitrogen to study the fate of colonic urea-
25
nitrogen in the proximal colon, first in normal conditions and secondly upon administration of
26
fermentable carbohydrates.
27
Upon oral administration, lactose-ureide reaches the colon unmodified, because the molecular bond
28
between the carbohydrate moiety and urea in lactose ureide has been shown to resist enzymatic
29
degradation in the human gastrointestinal tract (13). When lactose[ N, N]-ureide reaches the colon, it
30
is degraded to [ N, N]-urea by selected bacteria. The labeled urea undergoes fast hydrolysis with
15
15
15
15
4
15
1
production of
2
consequence, the variations observed in
3
However, in this study, lactose-ureide was not administered orally but directly introduced into the colon
4
in order to avoid confounding differences in orocaecal transit time. The substrate was administered
5
through instillation in order not to disturb the normal environment and physiology of the colon. In the
6
past, studies concerning colonic nitrogen metabolism in vivo have been performed on patients with a
7
colostomy or by accessing the lumen through colonoscopy (14, 15). As opposed to the method used
8
in this study, neither of these approaches can be used to assess normal function. People with a
9
colostomy have suffered from colonic disease and bowel preparation before colonoscopy deranges
10
NH3 (28). The labeled NH3 is mixed with the ammonia present in the colon and as a 15
N-measurement reflect the fate of total colonic NH3.
the normal colonic environment.
11 12
Inulin was chosen to investigate the effect of carbohydrate fermentation on the excretion of colonic
13
nitrogen (4, 16).
14 15
SUBJECTS AND METHODS
16
Experimental design
17
The study was carried out in fourteen healthy volunteers (6 men, 8 women). Each of them performed
18
the experiment twice, once without inulin and once with inulin, with an interval of at least one week.
19
The volunteers kept diet records the week before each experiment in order to allow a qualitative
20
comparison of dietary intakes. They were allowed to eat their usual diets, but were urged to keep a
21
constant macronutrient composition.
22
On the day before the test, each subject swallowed a narrow polyvinyl tube (bowel decompression
23
catheter, 5.3 mm; Rusch Manufacturing Ltd., Lurgan, Armagh, N. Ireland). The position of the tube
24
was followed by fluoroscopy. When the tip of the tube had reached the ligament of Treitz, the
25
volunteer was allowed to return home and eat, whereupon the tube progressed down the intestine. All
26
subjects ingested 1g of unlabelled lactose ureide that evening and then fasted overnight.
27
The next morning, brief radiographic screening was performed to ensure that the tip of the tube was in
28
the right colon. The labeled substrates were dissolved in 40ml NaCl 0.9% and injected in bolus. The
29
tube was flushed with another 40ml NaCl 0.9%. Thereafter, the tube was removed. The volunteers
5
1
were not allowed to eat or drink during the first 4 hours of the test. After 4 hours, they received a
2
sandwich with ham or cheese.
3 4
Nine of the volunteers performed a 3-day urine collection on each test occasion. On their first visit a
5
40ml solution containing 50mg lactose[ N, N]-ureide was administered. Two gram inulin was added
6
to the infusion in the second experiment.
7
The other five volunteers performed a 3-day urine and stool collection in the two experimental
8
conditions. They received a 40ml solution containing 50mg lactose[ N, N]-ureide and 185 kBq of H-
9
PEG on their first visit and the same solution with an additionally 250mg inulin on the second visit.
15
15
15
15
3
10 11 12
Substrates
13
Fifty milligram of lactose[ N, N]-ureide ([ N, N] LU) (synthesized according to the method of
14
Schoorl as modified by Hofmann (8) with [ N, N]-urea obtained from Euriso-top, Saint-Aubin, Cédex,
15
France) was administered directly into the colon. One gram of unlabelled lactose ureide (LU) was
16
ingested on the evening before the test to induce the proper enzyme activity in the colonic bacteria (8,
17
28).
18
Raftilin HP (Orafti, Tienen, Belgium), a linear beta (2,1)-linked fructose polymer, terminated by a
19
sucrose residue, purified from chicory root, was used as a prototype for a fermentable carbohydrate.
20
Raftilin HP contains > 99% inulin with a DP ranging between 5 and 60 and < 0.5% glucose, fructose
21
and sucrose.
22
Whenever correction for transit time was required, 185 kBq of H-polyethylene glycol ( H-PEG) (NEN
23
Life Science Products Inc, Boston MA, USA) was added to the infusion as an inert radiolabeled transit
24
marker.
15
15
15
15
15
15
3
3
25 26
Sample collection
27
Urine samples were collected in plastic containers to which one gram of neomycin was added in order
28
to prevent bacterial growth. All volunteers were asked to void before the infusion and this urine
29
collection was used for measurement of basal nitrogen content and
15
N-enrichment. After the infusion,
6
1
urine was collected over three consecutive days in different fractions: 0-3h, 3-6h, 6-9h, 9-24h, 24-48h,
2
48-72h. After measurement of the volume, samples were taken and stored at –20°C until analysis.
3
Five volunteers also performed a 72h stool collection. Date and time of voiding of stools were noted in
4
a diary. The stools were frozen immediately after voiding, weighed and then stored at –20°C. All stools
5
collected on the same day were combined and homogenized before further analysis. Samples of
6
known weight were removed and freeze-dried. The dried material was weighed again and aliquots
7
were taken for analysis of nitrogen and radioactivity.
8 9
Analyses 15
10
Total N content and
11
(ANCA-SL, PDZ, Cheshire, UK) coupled with both a thermal conductor detector (TCD, PDZ, Cheshire,
12
UK) and a Stable Isotope Ratio Mass Spectrometer (IRMS, PDZ, Cheshire, UK). A known volume of
13
urine (15 µL) or faeces (about 7 mg) was oxidized in the presence of oxygen at 1000° C. The
14
combustion products thereafter passed through a second furnace containing copper at 600° C where
15
excess oxygen was absorbed and nitrogen oxides were reduced to elemental nitrogen. Total nitrogen
16
content was measured by means of a TCD, whereas the
17
an IRMS detector, coupled to the combustion unit of the elemental analyzer. The aim of this validation
18
study was the evaluation of the cumulative urinary and faecal excretion of the label and the urinary
19
excretion pattern of the label and not the identification of the molecules in which the labeled nitrogen
20
was retrieved.
N-enrichment of urine and faeces were measured using an elemental analyzer
15
N enrichment was determined by means of
21 22
Creatinine and urea content in urine were measured by standard laboratory techniques.
23 3
24
The H-PEG content in stool was measured with liquid-scintillation counting (Packard Tricarb Liquid
25
Scintillation Spectrometer, model 3375, Packard Instruments Inc, Downers Grove, IL, USA) after
26
oxidation to H-H2O (Packard Sample Oxidiser, model 306, Packard Instruments Inc, Downers Grove,
27
IL, USA).
3
28 29
For each urine collection, the results were expressed as total nitrogen (g), urea (g) and creatinine (g)
30
and percentage of administered dose
15
N recovered. A correction for creatinine content was
7
1
performed to correct for mistakes due to dilution or differences in sampling times, which may have
2
occurred in the first short collection periods (g urea/g creatinine and µg
3
Both for urinary and faecal measurements total nitrogen (g) excretion and cumulative percentage of
4
administered dose
5
expressed as percentage of administered dose recovered over 72h and were used to correct for
6
gastrointestinal transit by dividing the cumulative percentage of administered dose
7
72h by the cumulative percentage of administered dose of H recovered over 72h.
15
15
N/g creatinine).
N recovered over 24/48/72 hours were evaluated. Tritium contents in stool were
15
N recovered over
3
8 9
Statistical analysis
10
Because of the low number of measurements, all results were expressed as median plus interquartile
11
range and non-parametric statistical analysis was used. Alfa was set at 0.05. (Wilcoxon test and
12
Mann-Whitney test, Statistica 6.0, Statsoft Inc. 1984-2001)
13 14
RESULTS
15
Urinary total nitrogen excretion and kinetics of urinary N15 excretion in normal conditions
16
(n=14)
17
The urinary excretion of labeled nitrogen is presented as a function of time in figure 1.
18
The excretion rate of labeled nitrogen was maximal between 3-9 hours after instillation in all
19
volunteers. The
20
that the urinary excretion of the label was completed. The cumulative urinary excretion of
21
increased up to 28% (22 – 38%) of the administered dose at 24h and 45% (34 – 51%) at 72h.
22
Total nitrogen excretion in urine varied around 10g/24h. A diurnal variation in urinary nitrogen and
23
urea excretion was noted.
15
N-recovery in urine had returned to baseline by the end of the test period, indicating 15
N
24 25
Effect of instillation of 2g of Raftilin HP on urinary excretion of total nitrogen and
26
The results are summarized in table1.
15
N (n=9)
27 28
No difference in urinary excretion of total nitrogen was noted: urinary nitrogen excretion varied around
29
10g/24h in all the collections. No difference in total urinary creatinine or urea excretion was measured
30
either.
8
1
No differences in diurnal variation of urinary nitrogen and urea excretion were noted after correction
2
for creatinine content (fig.2).
3 4
A consistently lower excretion rate of labeled nitrogen upon administration of inulin was noted in the
5
first two urine collections, leading to a statistically significant reduction in cumulative urinary excretion
6
of labeled nitrogen at 24h: 27% (23 – 30%) of administered dose was retrieved in urine in normal
7
conditions versus 20% (18 – 21%) after addition of 2g of Raftilin HP to the infusion (fig. 3) (Wilcoxon
8
test, p=0.01). The statistically significantly lower recovery of the label was still noted 72h after the
9
instillation (Wilcoxon test, p=0.02).
10 11
After correction for creatinine content, a statistically significant decrease in excretion rate of the marker
12
was observed in the 3-6h collection (15µg (11 – 22µg)
13
creatinine; Wilcoxon test, p=0.03) (fig. 4).
15
N/g creatinine versus 11µg (6 – 14µg)
15
N/g
14 15
Effect of colonic instillation of 250mg of Raftilin HP on urinary parameters (n=5)
16
The results are summarized in table 2.
17 15
15
18
Five volunteers performed a 3-day urine after colonic instillation of 50 mg [ N, N] LU and once again
19
after simultaneous administration of [ N, N] LU and 250mg Raftilin HP .
20
The urinary total nitrogen excretion remained unchanged, varying around 10g/24h in all 24h
21
collections with and without 250mg Raftilin HP .
22
A clear reduction in cumulative urinary excretion of labeled nitrogen was observed after simultaneous
23
administration of inulin and was mainly due to a lower excretion rate of labeled nitrogen in the first two
24
urine collections. However, statistical significance was never obtained.
15
15
25 15
26
Effect of the amount of Raftilin HP on changes in cumulative urinary
N excretion
27
Both doses of Raftilin HP caused a similar absolute reduction in cumulative urinary excretion of the
28
marker: 12% (8 – 16%) reduction after administration of 250mg of inulin and 7% (4 – 19%) after
29
administration of 2g of inulin (Mann-Whitney test, p=NS). This indicates that an amount of 250mg of
30
inulin is sufficient to influence the excretion route of ammonia derived from 50mg lactose-ureide.
9
1 2
Effect of colonic instillation of 250mg of Raftilin HP on faecal parameters (n=5)
3
The results are summarized in table 2.
4
The colonic transit time was not different in the two test situations: 49% (35 – 60%) of the
5
administered dose of H-PEG was retrieved after 72h in basal circumstances and 60% (22 – 65%)
6
when inulin was added to the solution. No difference in total faecal nitrogen excretion after 3 days was
7
measured either.
8
On the other hand, there was a clear, but not statistically significant increase in faecal excretion of
9
labeled nitrogen after 72h: 13% (6 – 18%) of administered
3
15
N was excreted in normal conditions
10
versus 16% (15 – 25%) after addition of 250mg of Raftilin HP to the infusion.
11
No difference in faecal dry weight or in total nitrogen density was observed, but a consistent increase
12
in faecal density of labeled nitrogen was withheld (Wilcoxon test, p=NS).
13 14
Effect of 250mg of Raftilin HP on total excretion of the label (n=5)
15
No difference in total cumulative excretion of the marker was measured: 62% (48 – 75%) in the first
16
part of the study versus 64% (54 – 70%) in the second, indicating no difference in retention of the
17
marker (retention = 100 – (urinary + faecal excretion)).
18 19
Complete stool collections on both occasions were obtained in four out of five volunteers. An inverse
20
relationship between the changes in urinary and faecal parameters was found consistently.
21 22
DISCUSSION
23
In this study, lactose[ N, N]-ureide (LU) was directly instilled in the colon through an orocaecal tube
24
in order to investigate colonic handling of ammonia nitrogen. A low interindividual variability in urinary
25
excretion pattern of the label was found. Simultaneous administration of inulin, a fermentable
26
carbohydrate, caused a consistent decrease in urinary excretion of
27
Due to the onerous protocol, the combined study of faecal and urinary parameters was performed in a
28
limited number of volunteers. The observations in this study suggest that the decrease in urinary
29
excretion of the marker is accompanied by an increase in its faecal excretion.
15
15
15
N.
30
10
15
1
After administration of LU, part of the label is excreted in urine. The form of
2
[ N, N]-urea, which has escaped hydrolysis in the colon or [ N, N]-urea when [ N, N]-urea is
3
hydrolyzed in the colon to
4
liver (14, 15, 28). The techniques used in this study do not enable differentiation between [ N, N]-
5
urea and [ N, N]-urea.
6
However, literature data indicate that when [ N, N]-urea is administered directly in the colon at
7
colonoscopy, about 5% of the label is recovered intact in urine (25). In our study, 28% of the labeled
8
nitrogen was recovered after instillation of 50mg lactose[ N, N]-ureide. Assuming 5% of the label
9
recovered in urine to be in the form of [ N, N]-urea, the remaining 23% may be considered to be
10
derived from [ N, N]-urea. This figure correlates with the 24% of administered label which was
11
recovered as [ N, N]-urea in urine upon oral administration of labeled lactose-ureide (28).
15
15
15
N in urine can be either
14
15
15
15
15
14
NH3 which is subsequently absorbed and converted to [ N, N]-urea in the 15
15
15
14
15
15
15
15
15
15
15
15
14
14
12 15
13
Most importantly, the results of our study show that the urinary excretion pattern of
14
all volunteers. In a previously performed study, three different urinary excretion patterns of
15
been described upon oral administration of lactose[ N, N]-ureide (12). The variability in the results
16
was assumed to refer to differences in bacterial activity in different regions of the colon. However, our
17
study showed a similar excretion pattern of the label in all volunteers upon instillation of lactose
18
[ N, N]-ureide in the right colon. This indicates that there is no major interindividual variation in
19
degradation of the LU or in hydrolysis of the formed urea once lactose-ureide has reached the colon.
20
Therefore, we suggest that the variation in urinary excretion pattern of
21
ingestion of labeled lactose ureide may refer to differences in orocaecal transit and hence, variability in
22
arrival of the molecule in the caecum. This implicates that it is possible to study colonic nitrogen
23
metabolism upon oral administration of lactose[ N, N]-ureide.
15
15
N was uniform in 15
N have
15
15
15
15
N observed upon oral
15
24 25
In order to evaluate the influence of carbohydrate fermentation on nitrogen metabolism in the colon,
26
Raftilin HP was added to the infusion in a second experiment. The most important observation was a
27
decrease in urinary excretion of labeled nitrogen as compared to baseline values after administration
28
of two different doses of Raftilin HP in all volunteers but one. Both doses of Raftilin HP caused a
29
similar reduction in cumulative urinary excretion of the marker and similar changes in the excretion
30
pattern, suggesting that a dose of 250mg Raftilin HP is sufficient to influence the excretion route of
11
1
ammonia derived from 50mg lactose-ureide. An amount of 250mg of inulin is extremely low compared
2
to the colonic dietary carbohydrate load and it may appear surprising that this dose of Raftilin HP can
3
provoke measurable effects on the excretion of labeled nitrogen. However, the reproducibility of the
4
results urges us to attribute this effect to the administration of inulin. Both substrates are administered
5
simultaneously and therefore are more likely to influence eachothers metabolism.
6 7
The reduction in cumulative urinary excretion of the label was caused by a clear reduction in urinary
8
excretion rate of the
9
caused by fermentation of carbohydrates would have resulted in a change of urinary excretion pattern
10
of the label and not necessarily in a reduction of total urinary excretion of the marker. This means that
11
this result is suggestive for a decrease in colonic absorption of ammonia due to an increased removal
12
of nitrogen.
15
N-isotope in the first hours of the test. Temporary changes in bacterial activity
13 14
The reduction in urinary excretion of the label may have been caused by changes in the bacterial or in
15
the human metabolism. In order to exclude an increased retention of the label in the human nitrogen
16
pool as a cause of the decrease in urinary excretion, the recovery of labeled nitrogen in faeces was
17
measured in 5 of the volunteers. The retention of the label in the human body was defined as 100% -
18
(%dose of
19
that the retention of salvaged nitrogen for human metabolism after administration of Raftilin HP (36%)
20
was not different from the retention in baseline conditions (38%) and that the decrease in urinary
21
excretion of the label was accompanied by an increase in faecal excretion. A consistent negative
22
relationship between faecal and urinary excretion of the
23
urinary towards faecal excretion upon carbohydrate fermentation.
24
The changes in urinary and faecal excretion of the marker were not caused by changes in colonic
25
transit, since no difference was seen in the recovery of H-PEG in faeces. The consistent increase in
26
faecal N-density strongly suggests an increased incorporation of
15
N excreted in urine + %dose of
15
N excreted in faeces). From these results, it was clear
15
N-isotope was found, suggesting a shift from
3
15
15
NH3 into the bacteria.
27 28
The combined study of faecal and urinary parameters was performed in a limited number of volunteers
29
because of the onerous protocol, leading to restricted power. However, the marker can be
30
administered in an oral testmeal, because the molecule can only be broken down by bacterial
12
1
enzymes and no interindividual variability in urinary excretion pattern of the label was found upon
2
colonic instillation in baseline conditions. Oral administration of the marker will allow us to perform
3
studies considering the relation between urinary and faecal excretion of colonic ammonia and the
4
effect of different fermentable carbohydrates hereupon in larger cohorts.
5 15
15
6
The results of this study indicate that lactose[ N, N]-ureide seems a very valuable tool to investigate
7
the effect of different types of carbohydrates on urea-nitrogen metabolism in the proximal colon in
8
vivo.
9
We may conclude that colonic metabolism of lactose-ureide is rather uniform when a priming dose of
10
lactose-ureide has been given.
11
Although we only examined urinary and faecal excretion of the marker simultaneously in a small
12
group, we speculate that fermentation of carbohydrates shifts colonic ammonia towards bacterial
13
metabolism, resulting in a reduced absorption of colonic ammonia, given the consistent negative
14
relationship between urinary and faecal excretion of the label and the statistically significant decrease
15
in urinary excretion upon administration of inulin. Fermentation of carbohydrates does not influence
16
the amount of nitrogen used for human metabolism. Further studies based on oral administration of
17
labeled lactose-ureide are warranted.
18 19 20 21 22 23 24 25 26 27
13
3
2,5
%dose/h
2
1,5
1
0,5
0 0-3h
3-6h
6-9h
9-24h
24-48h
48-72h
urine collections
1 2
FIGURE 1: Urinary excretion pattern of labeled nitrogen expressed as percentage of administered
3
dose recovered per hour, upon colonic instillation of lactose[ N, N]-ureide in normal circumstances in
4
healthy volunteers. Values are median ± IQR, n=14.
15
normal circumstances
15
w ith inulin
26 24
g urea/g creatinine
22 20 18 16 14 12 10 0-3h
3-6h
6-9h
9-24h
24-48h
urine collections
5 6
FIGURE 2: Diurnal variation in urinary urea excretion (expressed as g ureum/g creatinine) in normal
7
circumstances and when 2g Raftilin HP
8
volunteers. Values are median ± IQR, n=9.
is administered through colonic instillation in healthy
14
normal conditions
with inulin
3
2,5
%dose/hour
2
1,5
1
0,5
0 0-3h
3-6h
6-9h
9-24h
24-48h
48-72h
urine collections
1 2
FIGURE 3: Urinary excretion pattern of labeled nitrogen (expressed as % of administered dose
3
recovered per hour) upon colonic instillation of lactose[ N, N]-ureide in healthy volunteers, in normal
4
circumstances and when 2g Raftilin HP is administered simultaneously. Values are median ± IQR,
5
n=9.
15
15
6
15
1 normal conditions
w ith inulin
0,025
mg 15N/g creatinine
0,02
0,015
0,01
0,005
0 0-3h
3-6h
6-9h
9-24h
24-48h
48-72h
urine collections
2 3
FIGURE 4: Urinary excretion pattern of labeled nitrogen upon colonic instillation of lactose[ N, N]-
4
ureide in healthy volunteers, in normal circumstances and when 2g Raftilin HP
5
simultaneously, after correction for creatinine content (expressed as mg
6
median ± IQR, n=9.
15
15
is administered
15
N/g creatinine). Values are
7 15
N excretion in urine after colonic instillation in healthy volunteers of
8
Table 1: Total nitrogen and
9
[ N, N] LU with and without 2g Raftilin HP
10
15
15
1
Baseline conditions
Addition of 2g inulin
Urinary nitrogen excretion (g/72h)
30 (25 – 31)
26 (18 –30)
Urinary urea excretion in the first 24h (g)
21 (15 – 29)
17 (14 – 22)
Total urinary urea excretion (g/72h)
54 (38 – 66)
45 (30 – 51)
Urinary creatinine excretion in the first 24h (g)
0.8 (0.7 – 1.3)
0.9 (0.8 – 1.1)
Cum%/24h of 15N recovered in urine (%)
27 (23 - 30)
20 (18 – 21)*
Cum%/72h of 15N recovered in urine (%)
42 (37 – 48)
29 (23 – 34)*
Values are median ± IQR, n=9, * value differs from baseline conditions, p
