Detection of intestinal fat malabsorption due to impaired lipolysis by

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CHAPTER 2

Detection of intestinal fat malabsorption due to
impaired lipolysis by the 13C-mixed triglyceride
breath test in rats

M. Kalivianakis, J. Elstrodt, R. Havinga, F. Kuipers,
F. Stellaard, R.J. Vonk, H.J. Verkade
Chapter 2

CHAPTER 2

Detection of intestinal fat malabsorption due to
impaired lipolysis by the 13C-mixed triglyceride
breath test in rats

Abstract

Background & Aim: The 13C-mixed triglyceride (13C-MTG) breath test has become popular
for the detection of impaired intestinal lipolysis as a cause for fat malabsorption. However, the
diagnostic value has been questioned because the relation between the extent of fat
malabsorption and the corresponding result of the 13C-MTG breath test has not been
established. We characterized the 13C-MTG breath test in rats with variable degrees of fat
malabsorption, achieved by feeding the lipase inhibitor orlistat. Methods: Rats were fed high
fat chow (35 en% fat) to which orlistat was added in amounts of 0, 50, 200, and 800 mg kg-1
chow for 5 days. Breath 13CO2 recovery was determined for 6 h after oral administration of
13
C-MTG (13 mg kg-1 BW). Total dietary fat absorption was measured by means of a 3-day
fecal fat balance. Results: Upon orlistat administration, total fat absorption decreased in a
dose-dependent way from 80.2 ± 2.2% to 32.8 ± 3.7% (mean ± SEM; 0 mg and 800 mg
orlistat kg-1 chow, respectively; P<0.001). Correspondingly, breath 13CO2 recovery from 13C-
MTG at 6 h decreased from 84.5 ± 7.8% to 42.0 ± 1.5% of the dose (P<0.001). The 6-h
recovery of breath 13CO2 appeared highly correlated with total fat absorption for the different
dosages of orlistat (r=0.88, P<0.001). However, in rats with fat absorption higher than 70%,
the coefficient of variation of cumulative breath 13CO2 excretion was large (15%) compared
with that of fat absorption (5%). Conclusion: The 13C-MTG breath test correlates significantly
with the extent of fat malabsorption in a rat model of impaired intestinal lipolysis. However,
the considerable interindividual variation of the 13C-MTG breath test does not support its
application for diagnostic purposes in individual patients.

32
The 13C-MTG breath test in rats fed orlistat

Introduction

Reduced secretion of pancreatic lipase into the intestine is a common feature of pancreatic
insufficiency. This condition may lead to fat malabsorption due to incomplete intestinal
hydrolysis of dietary triacylglycerols [1,2]. Intestinal fat malabsorption in patients can be
quantified by means of a fat balance, but this method does not discriminate between the
potential causes, such as impaired intestinal lipolysis, disturbed intestinal solubilization of long-
chain fatty acids, or decreased chylomicron formation. Measurement of maximal pancreatic
lipase output by means of an invasive, marker-corrected perfusion technique is considered to
be the gold standard for pancreatic insufficiency tests [3,4]. A non-invasive test has been
described to characterize pancreatic insufficiency in a functional way. In this test, a 13C-labeled
mixed triglyceride (13C-MTG; 1,3-distearoyl, 2[carboxyl-13C]octanoyl glycerol) is orally
ingested and the amount of 13C in expired air is determined [5]. 13C-MTG contains a 13C-
labeled medium-chain fatty acid (octanoic acid) at its sn-2 position, and long-chain fatty acids
(stearic acid) at the sn-1 and sn-3 positions of the glycerol backbone. The two stearoyl
acylchains have to be hydrolyzed by the pancreatic enzyme lipase before 13C-octanoate can be
absorbed, either in the form of a free fatty acid or as a mono-acylglycerol [6]. After its
absorption, octanoate is rapidly oxidized [6,7]. Thus, the principle of the 13C-MTG test is
based on lipolysis-dependent 13CO2 excretion via the breath.
Since the original description of the 13C-MTG breath test, the test has become
popular in clinical practice [5,8-11]. However, widely variable results have been obtained in
children [9], healthy adults [12], and in cystic fibrosis patients with or without pancreatic
enzyme replacement therapy [10,11]. The reason for this variability has not been elucidated: in
fact, quantitative relationship between the extent of fat malabsorption due to impaired lipolysis
and the corresponding result of the 13C-MTG breath test has never been demonstrated in
humans or in defined animal models.
A reliable way to decrease the lipolysis activity dose-dependently is with the use of
orlistat, an inhibitor of pancreatic lipase [13,14]. Orlistat, the chemically synthesized derivative
of the natural product lipstatin, is a selective and potent inhibitor of lipases, among which,
pancreatic lipase [15-23]. Orlistat inactivates pancreatic lipase by reacting covalently with
serine (Ser-152) in the active site of the catalytic domain [24,25].
In the present study we aimed to determine the relationship between the extent of fat
malabsorption and the results of the 13C-MTG breath test in a defined controlled animal
model. We applied the dietary supplementation of orlistat as a reproducible inducer of various
degrees of fat malabsorption in rats, in analogy to previous studies in mice and humans [26-
28]. To ensure that orlistat-induced fat malabsorption was exclusively due to impaired
lipolysis, we performed control experiments in which the absorption of the fatty acid
[1-13C]palmitic acid was determined, a substrate independent of lipolysis.

33
Chapter 2

Materials and Methods

Rats
Male Wistar rats (Harlan, Zeist, The Netherlands), weighing approximately 400 g, were
housed in an environmentally controlled facility with diurnal light cycling and free access to
tap water and chow. Experimental protocols were approved by the Ethical Committee for
Animal Experiments, Faculty of Medical Sciences, University of Groningen.

Materials
The mixed triglyceride (1,3-distearoyl, 2[1-13C]octanoyl glycerol) was purchased from Euriso-
Top (Saint Aubin Cedex, France) and was 99% 13C-enriched. In previous articles [5,10,12],
the breath test performed with the use of this compound has been denominated as the mixed-
triglyceride breath test or as the 13C-MTG breath test. For reasons of consistency, we adhere
to this nomenclature. [1-13C]palmitic acid was purchased from Isotec Inc. (Matheson, USA)
and was 99% 13C-enriched. Orlistat (previously known as tetrahydrolipstatin, THL, Ro 18-
0647) is a synthetic product and was kindly provided by Hoffmann-La Roche (Basel,
Switzerland).

Study protocol
13
C-MTG breath test. Rats were fed ground high-fat chow (35 en% fat; 4.538 kcal kg-1 food;
fatty acid composition measured by GC analysis: C8-C12, 4.4 mol%; C16:0, 28.5%; C18:0,
3.9%; C18:1n-9, 33.2%; C18:2n-6, 29.3%; C18:3n-3, 0.2%) (Hope Farms BV, Woerden, The
Netherlands) mixed with water (3:2, w/w) to form a homogenous paste. After 2 weeks on the
diet, rats were divided into a control group (no orlistat added to the diet) and 3 orlistat groups
(50, 200 or 800 mg orlistat per kg chow). There were 4 rats in each experimental group.
Orlistat was ground together with the high-fat chow and mixed with water. Administration of
orlistat started 2 days prior to the fat balance experiments. Food intake was recorded and feces
was collected for 3 days, in order to perform a fat balance. Feces was stored at -20°C prior to
analysis. After the fat balance, rats were fasted overnight. The following morning they were
placed in an airtight container (volume ~ 4.5 L) through which CO2-free air was passed at a
continuous flow of 750 mL min-1. The air leaving the metabolic cage was partly diverted (50
mL min-1) to a CO2 monitor (Capnograph IV, Gould Medical BV, Bilthoven, The
Netherlands) for measuring percentage of total CO2 in the breath, and to 10 mL test tubes
(Exetainers; Labco Limited, High Wycombe, United Kingdom) for collection of breath
samples. The rats were placed in the container at least 30 min before administration of the test
meal containing the label, to have the rats adapted to the cage and to obtain background
breath samples. The test meal consisted of 13C-MTG (13 mg kg-1 body weight) mixed with
high fat chow (6 g kg-1 body weight), orlistat and water. All rats ingested the test meal within
5 min. After ingestion of the test meal, 1-min breath samples were collected in duplicates at
30-min intervals for a period of 6 hours.
[1-13C]palmitic acid test. After 1 week on high fat chow, rats were equipped with
permanent catheters in jugular vein, and duodenum as described by Kuipers et al. [29]. This
experimental model allows to obtain multiple blood samples in unanesthetized rats without the

34
The 13C-MTG breath test in rats fed orlistat

interference of stress or restraint. Animals were allowed to recover from surgery for 6 days
and were subsequently divided into 2 groups: 1 control group receiving no orlistat and an
experimental group receiving 200 mg orlistat per kg chow. On day 7, 1.67 mL liquid fat kg-1
body weight was slowly administered as a bolus via the duodenal catheter. The fat bolus was
composed of olive oil (25% v/v; fatty acid composition: C16:0, 14%; C18:1n-9, 79%; C18:2n-
6, 8%) and medium-chain triglyceride oil (75% v/v; composed of extracted coconut oil and
synthetic triacylglycerols; fatty acid composition: C6:0, 2% max.; C8:0, 50-65% max.; C10:0,
30-45%; C12:0, 3% max.) and contained 33 mg kg-1 body weight [1-13C]palmitic acid and
0.47 mg kg-1 body weight orlistat for the experimental group. The fat bolus represented
approximately 15% of the daily fat intake. Blood samples (0.2 mL) were taken from the
jugular cannula at baseline, 1, 2, 3, 4, 5, 6 and 24 h after administration of the label and were
collected into tubes containing heparin. Plasma was separated by centrifugation (10 min, 5000
rpm, 4°C) and stored at -20°C until further analysis. Feces was collected in 24-h fractions
starting 1 day before administration of the label and ending 2 days afterwards. Feces samples
were stored at -20°C prior to analysis. Food intake was determined for 3 days.

Analytical techniques
Breath sample analysis. 13C-enrichment in aliquots of breath samples was determined by
means of continuous flow isotope ratio mass spectrometry (Finnigan Breath MAT, Finnigan
MAT GmbH, Bremen, Germany). The 13C-abundance of breath CO2 was expressed as the
difference per mil from the reference standard Pee Dee Belemnite limestone (δ13CPDB, ‰). The
proportion of 13C-label excreted in breath CO2 was expressed as the percentage of
administered 13C-label recovered per hour (% 13C dose h-1), and as the cumulative percentage
of administered 13C-label recovered over the 6-h study period (cum % 13C).
Plasma fats. Total plasma fats (triacylglycerols, phospholipids, etc.) were extracted,
hydrolyzed and methylated according to Lepage and Roy [30]. Resulting fatty acid methyl
esters were analyzed by gas chromatography to measure the total amount of palmitic acid and
by gas chromatography combustion isotope ratio mass spectrometry to measure the 13C-
enrichment of palmitic acid, as detailed below. The concentration of 13C-palmitic acid in
plasma was expressed as the percentage of the dose administered per liter plasma (% dose/L).
Rat chow and fecal fats. Rat chow and feces were freeze-dried and mechanically
homogenized, after which aliquots were extracted, hydrolyzed and methylated according to
the method of Lepage and Roy [30]. Resulting fatty acid methyl esters were analyzed by gas
chromatography to allow calculation of total fat intake, total fecal fat excretion, and total
palmitic acid concentration in food and feces. Total fecal fat excretion of rats was expressed as
g fat day-1 and percentage of total fat absorption was calculated from the daily fat intake and
the daily fecal fat excretion and expressed as a percentage of the daily fat intake.

Fat intake (g day -1 ) − Fecal fat excretion (g day -1 )
Total fat absorption = × 100%
Fat intake (g day -1 )

A similar calculation was performed to measure the absorption of [1-13C]palmitic acid. Values
were expressed as percentage of the dose administered (% dose).

35
Chapter 2

Gas liquid chromatography. Fatty acid methyl esters were separated and quantified
by gas liquid chromatography on a Hewlett Packard gas chromatograph Model 6890 equipped
with a CP-SIL 88 capillary column (50 m x 0.32 mm; Chrompack, Middelburg, The
Netherlands) and an FID detector. The gas chromatograph oven was programmed from an
initial temperature of 150°C to 240°C in 2 temperature steps (150°C held 5 min; 150-200°C,
ramp 3°C min-1, held 1 min; 200-240°C, ramp 20°C min-1, held 10 min). Quantification of the
fatty acid methyl esters was done by adding heptadecanoic acid (C17:0) as internal standard.
Gas chromatography combustion isotope ratio mass spectrometry. 13C-enrichment of
the palmitic acid methyl esters was determined on a gas chromatography combustion isotope
ratio mass spectrometer (Delta S/GC Finnigan MAT, Bremen, Germany). Separation of the
methyl esters was achieved on a CP-SIL 88 capillary column (Chrompack; 50 m x 0.32 mm).
The gas chromatograph oven was programmed from an initial temperature of 80°C to 225°C
in 3 temperature steps (80°C held 1 min; 80-150°C, ramp 30°C min-1; 150-190°C, ramp 5°C
min-1; 190-225°C, ramp 10°C min-1, held 5 min).

Calculations and statistics
The experimental data are reported as means ± SEM. Differences between sample means were
calculated with the use of Student t-test or ANOVA followed by post-hoc analysis (Student-
Newman-Keuls). For correlating two variables, regression lines were fitted by the method of
least squares and expressed as the Pearson correlation coefficient r. Differences between
means were considered statistically significant at the level of P<0.05. Analysis was performed
using SPSS for Windows software (SPSS, Chicago, IL, USA).

13 13
Table 2.1 Nutritional data and breath CO2 data obtained from control and orlistat-fed rats during C-MTG experiments (mean ±
SEM).

Orlistat Fat intake Fecal fat Fat uptake Fat absorption Cum breath 13C
mg kg-1 chow (g day-1) (g day-1) (g day-1) (% intake) (% dose)
0 2.7 ± 0.2a 0.5 ± 0.1a 2.1 ± 0.1a 80.2 ± 2.2a 84.5 ± 7.8a
50 2.1 ± 0.2b 0.3 ± 0.0a 1.8 ± 0.2a 85.2 ± 0.8a 82.0 ± 4.9a
200 2.9 ± 0.1a 1.2 ± 0.1b 1.7 ± 0.1a 59.2 ± 2.1b 58.5 ± 5.3b
800 3.0 ± 0.2a 2.0 ± 0.2c 1.0 ± 0.1b 32.8 ± 3.7c 42.0 ± 1.5c
Unlike letters indicate a significant difference (P<0.05).

Results
13
C-MTG test
Fecal fat balance. Nutritional data of the control and orlistat-fed rats are shown in Table 2.1.
Rats fed 50 mg orlistat kg-1 chow showed significantly lower food intake than the other
groups. Administration of 50 mg orlistat kg-1 chow did not lead to a change in fecal fat
excretion. Fecal fat excretion in rats fed 200 and 800 mg orlistat kg-1 chow, however, was
significantly increased when compared with rats fed 0 or 50 mg orlistat kg-1. In addition, fecal
fat excretion in rats fed 800 mg orlistat kg-1 chow was significantly higher compared with rats

36
The 13C-MTG breath test in rats fed orlistat

fed 200 mg orlistat kg-1 chow. Net fat uptake, defined as fat intake minus fecal fat excretion,
was significantly lower in rats fed 800 mg orlistat kg-1 chow than in the other groups.
Percentage of total fat absorption was significantly decreased in the groups fed 200 and 800
mg orlistat kg-1 chow when compared with rats fed 0 or 50 mg orlistat kg-1. In addition,
percentage of total fat absorption in rats fed 800 mg orlistat kg-1 chow was significantly lower
compared with rats fed 200 mg orlistat kg-1 chow.

Breath 13CO2 excretion measurements. As shown in Figure 2.1A, the 13C excretion
rate in breath after ingestion of 13C-MTG increased rapidly and reached a maximum value of
approximately 16% dose/h at 4 h, in rats fed 0 or 50 mg orlistat kg-1. No difference in breath
13
C expiration was observed between rats fed 0 or 50 mg orlistat kg-1. The 13C expiration rate
was markedly different in the groups fed 200 and 800 mg orlistat kg-1 chow (Figure 2.1A).
The 13C excretion rates rose more slowly and did not reach the high levels observed in the
other two groups. The 6-h cumulative 13CO2 excretion data are summarized in Figure 2.1B
and Table 2.1. The 6-h cumulative 13CO2 excretion, expressed as a percentage of the dose
administered, was significantly lower when rats were fed 200 and 800 mg orlistat kg-1 chow
compared with rats fed 0 and 50 mg orlistat kg-1 chow. In addition, the 6-h cumulative 13CO2
excretion of rats were fed 800 mg orlistat kg-1 chow was significantly reduced when compared
with rats were fed 200 mg orlistat kg-1 chow.

24 100
% Dose h-1 13CO2 Expiration

% Cumul 13CO2 Expiration

A B a
20 a
80
16
60 b
12
40
c
8

4 20

0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (h) Time (h)

-1 13
Figure 2.1 Time courses for (A) the excretion rates (% dose h ) and (B) the cumulative CO2 excretion (% cum) in breath (mean
13 -1
± SEM) over the 6-h study period following oral ingestion of C-MTG (13 mg kg body weight) to control rats and rats fed varying
-1
amounts of orlistat: 0 mg (•), 50 mg (Ž), 200 mg (~), and 800 mg (±) orlistat kg chow. Unlike letters indicate a significant
difference (P<0.05).

Relationship between total fat absorption and breath 13CO2 excretion. If the result of
the 13C-MTG breath test is exclusively determined by intestinal lipase activity, total fat
absorption would be expected to correlate with recovery of 13CO2 in the breath after 13C-MTG
ingestion in these experiments. Corresponding to the literature [31-33], the relationship
between fat excretion and cumulative breath 13CO2 excretion was considered to be
exponential. A significant correlation was indeed observed between the percentage of total fat
absorption and 6-h cumulative 13CO2 expiration (r=0.88, P<0.001; Figure 2.2). However, as
can be derived from individual data in Figure 2.2, the interindividual variation between

37
Chapter 2

recovery of 13CO2 excretion was large. Especially the individual 13C-results in rats with a
dietary fat absorption higher than 60% showed strong overlap. In these rats, the coefficient of
variation for percentage of total dietary fat absorption was only 5%, whereas coefficient of
variation for cumulative breath 13CO2 excretion was 15%.

100
r = 0.88, P<0.001

Expiration
80

60
2
13CO

40
% Cumul

0
0 20 40 60 80 100
% Total Fat Absorption

13 13
Figure 2.2 Relationship between the percentage of total fat absorption and breath CO2 excretion after oral administration of C-
-1
MTG (13 mg kg body weight) in rats fed varying amounts of orlistat (r=0.88, P<0.001); 0 mg (•), 50 mg (Ž), 200 mg (~), and 800
-1
mg (±) orlistat kg chow.

13
C-palmitic acid test
Data of the 13C-palmitic acid experiment are shown in Table 2.2. No significant difference in
mean fat intake was observed between control rats and rats fed 200 mg orlistat kg-1 chow
(P=0.36). Orlistat-fed rats excreted significantly more fat into the feces when compared with
control rats (P<0.01). The percentage of total fat absorption was significantly decreased in
orlistat-fed rats when compared with controls (46.7 ± 5.4% and 74.6 ± 1.3%, respectively,
P<0.01).

13
Table 2.2 Nutritional data of control and orlistat-fed rats during [1- C]-palmitic acid experiment (mean ± SEM).
13
Orlistat Fat intake Fecal fat Fat absorption C16:0 absorption
mg kg-1 chow (g day-1) (g day-1) (% intake) (% dose)
0 2.2 ± 0.2 0.6 ± 0.0 74.6 ± 0.3 83.7 ± 2.0
200 2.5 ± 0.2 1.3 ± 0.2* 46.7 ± 5.4** 87.0 ± 1.0
A symbol indicates a significant difference from the control group (0 mg orlistat kg-1 chow). * P<0.05; ** P<0.01.

The amount of 13C-palmitic acid excreted into the feces was calculated for the 48-h
period following administration of [1-13C]palmitic acid. No significant difference in absorption
of [1-13C]palmitic acid over the 48-h period studied was observed between control and
orlistat-fed rats (P=0.71, Table II), demonstrating that administration of orlistat does not
affect the absorption of [1-13C]palmitic acid. This is supported by the fact that 13C-palmitic
acid concentrations in plasma after intraduodenal administration of [1-13C]palmitic acid were
similar in control and orlistat-fed rats (Figure 2.3). The data of the [1-13C]palmitic acid
experiment indicate that fat malabsorption in orlistat treated rats is solely due to impaired
lipolysis.

38
The 13C-MTG breath test in rats fed orlistat

conc. (%Dose L -1)
60

13C16:0
20
Plasma

0
0 1 2 3 4 5 6 24
Time (h)

13 -1
Figure 2.3 Time courses of C-palmitic acid concentration in plasma of control rats (•) and rats administered 200 mg orlistat kg
13 -1
chow (~) after intraduodenal administration of [1- C]palmitic acid (33 mg kg body weight).

Discussion

We investigated the potency of the 13C-MTG breath test to quantify fat malabsorption due to
impaired lipolysis in rats fed different dosages of orlistat. After 13C-MTG ingestion, a
significant correlation was observed between 6-h recovery of 13CO2 in breath and percentage
of total fat absorption as shown in Figure 2.2. Two interesting observations arise from this
figure. Firstly, rats fed 200 and 800 mg orlistat kg-1 chow have fat malabsorption to an extent
that, if seen in patients, would coincide with steatorrhoea or bulky amounts of fat in the feces.
Especially in these rats, the relation between breath 13CO2 recovery and fat absorption is
strong. Apparently, under these conditions, the 13C-MTG breath test is a powerful analytical
technique for the detection of fat malabsorption. Clinical studies in humans indeed have shown
that the sensitivity and specificity of the 13C-MTG breath test to detect severe pancreatic
insufficiency are high [5]. Secondly, from a clinical point of view, rats with fat absorption
higher than 70% are a very interesting group. The extent of fat malabsorption in these animals
reflects, in a sense, the distinction that has to be made between healthy subjects and patients
whose fat malabsorption may easily be missed by examining the amounts of feces. In these
rats, at a rather narrow range of fat absorption, the 13CO2 response after ingestion of 13C-MTG
varies considerably (Figure 2.2, Table 2.1). These results indicate that, even in a homogeneous
group of rats with the same genetic background and diet, a considerable variation exists under
controlled circumstances.
Widely variable results with the 13C-MTG breath test have also been obtained in
healthy children [9], healthy adults [12], and in cystic fibrosis patients with pancreatic enzyme
replacement therapy [10,11]. So far, this variation has been blamed on large intra- and
interindividual variation caused by differences in, e.g., gastric emptying, hepatic clearance and
metabolism, endogenous CO2 production or pulmonary excretion [34-37]. The present data
indicate that the high variability of the 13CO2 response is a rather intrinsic property of the 13C-
MTG breath test, for which no optimal standardization seems possible at this moment.

39
Chapter 2

Therefore, we propose that the 13C-MTG breath test is useful for the detection of severe fat
malabsorption due to low lipase activity in groups of patients. However, the large variation in
the 13CO2 response at a mild degree of fat malabsorption limits the diagnostic possibilities of
the 13C-MTG breath test in humans [10,11].
No data concerning the 13C-MTG breath test in rats have been published so far. If we
compare the present results on the 13C-MTG breath test in control rats with data obtained in
healthy humans, the 6-h cumulative percentage of breath 13CO2 appears to be much higher in
rats: 85% compared with 30% in humans [5,10,12]. We speculate that the extended fasting
period of our rats directs the absorbed 13C-octanoic acid directly into the oxidation pathway.
However, it can not be excluded that part of the difference is based on species specificity.
In vitro studies with orlistat have shown that orlistat is insoluble in aqueous buffers,
very poorly soluble in micellar lipid phases, but exhibits good solubility in emulsified lipids
[13,38-40]. Therefore, in studies of fat absorption in mice, rats, and humans, inhibition by
orlistat was mainly determined by the concentration of the drug in the lipid phase [26,27,40].
In contrast, when the dose of orlistat was not pre-dissolved in the dietary fat, but simply
admixed to the diet or administered as suspension or in capsules in a meal-contingent manner,
the inhibitory effect on fat absorption was reduced to a variable extent [26,27]. Therefore, a
meaningful comparison between our results on inhibition of fat absorption by orlistat and
previously published studies is only possible if the experimental design and the mode of drug
administration are taken into account. Since in the present study, orlistat was admixed to the
diet, the major part of it was likely dissolved in the target dietary fat upon preparation and
mixing of the diet. Except for the 50 mg kg-1 experiment, the effect of orlistat on fat
absorption was dose-dependent up to the largest dose tested. Whether with dose escalation
the effect could be intensified or would level out is unknown. Previously, a similar dose-
response relationship has been described in mice to which orlistat was administered either
dissolved in the fat component of the meal or administered as suspension immediately after the
meal [26]. In these mice, excretion of fat in the feces increased exponentially when orlistat
dose was increased, until a plateau of 80% of the ingested amount [26]. The orlistat dose
required for half maximal elimination of fat (ID50) reported for mice was approximately 3.3 mg
orlistat per g of fat ingested [26]. In our study, the ID50 for rats was approximately 500 mg
orlistat per kg chow, corresponding to roughly 2 mg orlistat per g of dietary fat. Thus, despite
the different design of our study, the potency of orlistat expressed as dose per dietary fat
ingested was rather similar, indicating that the mode of action of orlistat in our study was very
efficient.
To investigate whether the orlistat-induced fat malabsorption was not partially due to
other intestinal effects of orlistat resulting in fat malabsorption, control experiments with [1-
13
C]palmitic acid were performed. The [1-13C]palmitic acid absorption test detects fat
malabsorption due to impaired intestinal uptake of long-chain fatty acids [41]. If fat
malabsorption in rats fed with orlistat were not solely due to the inhibition of intestinal
lipolysis, an impaired absorption of intraduodenal administered [1-13C]palmitic acid would be
expected. However, fecal 13C-palmitic acid excretion and plasma 13C-palmitic acid
concentrations were not affected at a dosage of 200 mg orlistat kg-1 chow, despite significantly
reduced absorption of dietary fats. These data indicate that the orlistat-fed rat model indeed is

40
The 13C-MTG breath test in rats fed orlistat

specific for impaired lipolysis as cause of fat malabsorption, as has been shown before [26,42-
45]. In addition, these data show that in this rat model lipolytic and non-lipolytic processes
regarding fat malabsorption can be dissected and measured separately with the use of different
stable isotope tests.
In summary, dietary orlistat administration to rats provides a model for fat
malabsorption, specifically due to impaired intestinal lipolysis. The 13C-MTG breath test in this
animal model correlates significantly with the extent of induced fat malabsorption. However,
variation in 13CO2 results between individual rats was large, especially in rats with dietary fat
absorption higher than 70%. The present data do not support the application of the 13C-MTG
breath test for diagnostic purposes in individual patients.

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