University of Groningen
Mechanisms involved in malabsorption of dietary lipids
Kalivianakis, Mini
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RIJKSUNIVERSITEIT GRONINGEN
MECHANISMS INVOLVED IN
MALABSORPTION OF
DIETARY LIPIDS
PROEFSCHRIFT
ter verkrijging van het doctoraat in de
Medische Wetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. D.F.J. Bosscher,
in het openbaar te verdedigen op
woensdag 23 september 1998
om 14.45 uur
door
MINI KALIVIANAKIS
geboren op 25 februari 1970
te Utrecht
Promotor: Prof. dr. R.J. Vonk
Referent: Dr. H.J. Verkade
ISBN: 90 367 0941 5
Promotiecommissie: Prof. dr. P.J.J. Sauer
Prof. dr. G.L. Scherphof
Prof. dr. L.T. Weaver
The studies presented in this thesis were performed within the program of the GUIDE
research school and were made possible by grants from the BIOMED Research Program
(BMH1-CT93-1239, SIGN), Numico BV, Zoetermeer, and a starting grant from University
Hospital Groningen.
The printing of this thesis was financially supported by ARC Laboratories, Amsterdam,
Campro Scientific BV, Veenendaal, Glaxo Wellcome BV, Zeist, Hope Farms BV, Woerden.
Printer: Ponsen & Looijen BV, Wageningen, The Netherlands.
Contents
Chapter 1
General introduction
1.1 Dietary lipids
1.2 Intestinal absorption and digestion of dietary lipids
1.3 Fate of lipids in the colon
1.4 Lipid malabsorption
1.5 Methods to measure lipid malabsorption
1.6 Scope of this thesis
7
8
10
14
15
17
21
Chapter 2
Detection of intestinal fat malabsorption due to impaired
lipolysis by the 13C-mixed triglyceride breath test in rats
Submitted
31
Chapter 3
The 13C-mixed triglyceride breath test in healthy adults:
determinants of the 13CO2 response
Eur J Clin Invest 1997;27:434-442
45
Chapter 4
The 13C-palmitic acid test with plasma sampling detects fat
malabsorption in bile-diverted rats
Submitted
61
Chapter 5
The 13C-palmitic acid test for detection of fat malabsorption
in healthy adults on calcium supplementation
77
Chapter 6
Fat malabsorption in cystic fibrosis patients on enzyme
replacement therapy is due to impaired intestinal uptake
of long-chain fatty acids
Am J Clin Nutr, in press 1998
89
Chapter 7
Increased fecal bile salt excretion is independent of the
presence of dietary fat malabsorption in two mouse models
for cystic fibrosis
105
Chapter 8
General discussion
119
Samenvatting
129
Nawoord
135
CHAPTER 1
General introduction
Chapter 1
CHAPTER 1
General introduction
1.1 Dietary lipids
On average, adult Western diets contain approximately 100 g of lipids per day, of which 92%
to 96% are long-chain triacylglycerols [1,2]. Triacylglycerols (also referred to as triglycerides)
are fatty acid triesters of glycerol (Figure 1.1). Triacylglycerols differ according to the identity
and position of their three fatty acid residues. Most triacylglycerols in nature contain longchain free fatty acids [1], although for example, triacylglycerols in human milk are mixtures
containing both medium- and long-chain fatty acids [3]. Fatty acids in biological systems
usually contain an even number of carbon atoms, typically between 14 and 24. The alkyl chain
may either be saturated or it may contain one or more double bonds. The predominant fatty
acid residues in nature are those of the C16 and C18 species palmitic, oleic, linoleic, and
stearic acids (Figure 1.2) [4]. Fatty acids can be divided into three main classes according to
their chain length: 1. short-chain fatty acids, less than 6 carbon atoms; 2. medium-chain fatty
acids, from 6 to 12 carbon atoms; 3. long-chain fatty acids, 14 or more carbon atoms [5]. The
properties of fatty acids are markedly dependent on their chain length and degree of
saturation. Unsaturated fatty acids are more fluid than saturated fatty acids of the same length.
By virtue of their smaller molecular size, medium-chain fatty acids are relatively soluble in
water [6].
8
General introduction
H
H
1
O
1
H C OH
H C O C
O
(A)
2
(B)
H C OH
2
H C O C
O
3
3
H C OH
H C O C
H
H
Figure 1.1 The structural formulas of (a) glycerol and (b) a triacylglycerol.
An adequate intake of dietary lipid is essential for life and well-being. Lipids serve
several important functions in the human body. Firstly, they represent the major source of
energy (9 kcal g-1), double that of sugars and protein [4]. In the average Western diet, lipids
provide approximately 40% of the caloric energy [1], which can be stored in the human body
for more than several days, in contrast to carbohydrates and proteins. The lipid content of
normal humans (21% for men, 26% for women) enables them to survive energy starvation for
2 to 3 months. Secondly, lipids are the major constituent of cell membranes in the form of
phospholipids, sphingolipids and cholesterol. Furthermore, lipids are the only source of
essential fatty acids, the precursors of eicosanoids such as prostaglandins, thromboxanes, and
leucotrienes [7,8]. Finally, lipids are necessary for the solubilization and uptake of the fatsoluble vitamins A, D, E, and K.
O
1
C OH
O
O
1
1
C OH
C OH
9
O
1
C OH
9
9
12
12
15
18
CH3
Stearic acid
18
CH3
Oleic acid
18
CH3
Linoleic acid
18
CH3
α−Linolenic acid
Figure 1.2 The structural formulas of some C18 fatty acids; stearic acid, oleic acid, linoleic acid, α-linolenic acid.
Lipids are particularly needed during periods of growth and development. In light of
the important physiological roles of lipids, an efficient high-capacity absorption mechanism is
9
Chapter 1
required. Impaired lipid absorption has been associated with physical complications, such as
diarrhea, retarded growth, and essential fatty acid deficiency. The aim of this thesis is to obtain
mechanistic information on the various pathophysiological processes involved in fat
malabsorption, with the purpose to increase diagnostic and eventually therapeutic possibilities
in patients with fat malabsorption. The mechanisms by which lipids are taken up will be
discussed in paragraph 1.2, and subsequently lipid malabsorption with special emphasis on the
disease cystic fibrosis will be discussed in paragraph 1.3. The distinct methods to diagnose and
quantify lipid malabsorption will be discussed in paragraph 1.4.
1.2 Intestinal absorption and digestion of dietary lipids
In order for intestinal lipid absorption to take place, lipids must undergo a number of physicochemical changes to enable transport from the intestinal lumen to the plasma compartment.
This is achieved by both mechanical and chemical means [5]. The overall process of intestinal
lipid absorption and digestion can be classified as a chain of events, including:
1. Emulsification; the dispersion of bulk fat globules into finely divided emulsion particle.
2. Lipolysis; the enzymatic hydrolysis of fatty acid esters at the emulsion-water interface.
3. Micellar solubilization; the desorption and dispersion of insoluble lipid products into an
absorbable form.
4. Membrane translocation; the transport of a lipid from the intestinal lumen across the
membrane of the intestinal mucosa cell.
5. Intracellular events; this intracellular phase of lipid absorption involves re-esterification of
fatty acids and monoacylglycerols into triacylglycerols, the packaging of the lipids into
chylomicrons and secretion of these chylomicrons at the basolateral side of the enterocyte
[1,2,5,9-13]. The overall process of lipid absorption is shown in Figure 1.3.
1.
Emulsification
2.
Lipolysis
3.
Solubilization
4.
Translocation
5.
Intracellular
events
Figure 1.3 Schematic of intestinal lipid absorption.
10
+
+
Lymphe
General introduction
Emulsification
Processing of lipids starts in the mouth with emulsification. The purpose of emulsification is to
increase the surface area of the lipid droplets, thereby increasing the area on which the
digestive enzymes can act effectively. Chewing breaks down large pieces of fat into smaller
sizes. Following ingestion, food enters the stomach, the major site for emulsification of dietary
lipids. Muscle contraction of the stomach - particularly peristalsis against a closed pylorus and
the squirting of lipid through a partially opened pyloric canal - produces the shear forces
sufficient for emulsification [1]. These peristaltic movements further grind the smaller pieces
of lipid into a fine emulsion [14], which together with other emulsified foodstuffs is referred to
as chyme. In addition to emulsifying food, the grinding action of the antrum mixes food with
various digestive enzymes derived from the mouth and the stomach. Similarly, intestinal
peristalsis continuously mixes luminal contents with digestive enzymes to ensure complete
digestion [5].
Lipolysis
The main objective of lipolysis is to convert triacylglycerols, which are virtually insoluble in
the aqueous phase of the gastrointestinal tract, into other forms of lipid with an increased
ability to interact with water. Enzymatic hydrolysis of dietary triacylglycerols in humans
beyond the breast feeding period is mainly catalyzed by preduodenal and pancreatic lipases,
and therefore in this thesis only these enzymes will be discussed. Preduodenal lipase is secreted
from different tissues depending on species [15-17] and has therefore been assigned different
names, e.g. gastric lipase [18], pharyngeal lipase [19], and lingual lipase [20]. In humans the
lipase is entirely a product of the chief cells of the gastric mucosa, and is therefore called
gastric lipase [2]. Regardless of species or tissue origin, the preduodenal lipases share
molecular and kinetic properties and it is assumed that they all have a common physiological
function, i.e. to initiate triacylglycerol digestion in the stomach. Gastric lipase preferentially
acts on the sn-3 position of the triacylglycerol molecule to release diacylglycerols and free
fatty acids [21,22]. The level of hydrolysis in the stomach by gastric lipase in humans under
physiological conditions accounts for approximately 10 to 30% of total lipid ingested [23].
The lipid emulsion enters the small intestine as fine lipid droplets less than 0.5 µm in
diameter [1]. Pancreatic colipase-dependent lipase, secreted by pancreatic acinar cells,
completes dietary lipid digestion in the proximal small intestine [24]. Pancreatic lipase acts
mainly on the sn-1 and sn-3 positions of the triacylglycerol molecule to release 2monoacylglycerol and free fatty acids [21,22]. Pancreatic lipase is one of the most studied and
best characterized lipases, and is considered to be responsible for quantitative digestion of all
triacylglycerols in the adult. In healthy human adults, the level of enzyme secreted into the
intestinal lumen was calculated to be in 1000-fold excess of what would be required for
hydrolysis of daily lipid intake [2]. Pancreatic lipase is clearly essential for efficient dietary lipid
digestion as evidenced by the steatorrhoea present in patients with for example cystic fibrosis
[25] or congenital pancreatic lipase deficiency [26,27].
The recent description of the primary and tertiary structures of pancreatic lipase has
provided insight into the molecular detail of pancreatic lipase-catalyzed lipolysis [28,29].
Pancreatic lipase is an enzyme with a marked substrate preference for triacylglycerols.
11
Chapter 1
Enzymatic hydrolysis of lipids can occur only on the surface of a lipid droplet, that is, at the
interface between the lipid droplet and the surrounding aqueous solution. When an oil-water
interface is encountered, pancreatic lipase activity increases markedly, a property termed
interfacial activation [29]. Although pancreatic lipase is secreted into the duodenum along with
bile salts, the enzyme is inhibited by physiological concentrations of bile salts and is dependent
on another pancreatic protein, colipase, for activity in the presence of bile salts [30].
The action of both gastric and pancreatic lipase is facilitated for medium-chain
triacylglycerols when compared with long-chain triacylglycerols due to their more expanded
surface films in water [1]. Consequently, medium-chain triacylglycerols are hydrolyzed both
faster and more completely than long-chain triacylglycerols [6]. In the case of mixed
triacylglycerols the medium-chain fatty acids are liberated preferentially [6].
Micellar solubilization
Bile is secreted by the liver and enters the intestine through the biliary tract. One of the
important properties of bile is its ability to increase the solubility of lipolytic products (i.e. 2monoacylglycerols and free fatty acids) in the aqueous intestinal lumen by the formation of
mixed micelles. Micelles are structures in which the polar group projects into the aqueous
phase while the nonpolar hydrocarbon chain forms the center. This macromolecular structure
has a high water solubility. Micellar solubilization increases the aqueous concentration of fatty
acids and monoacylglycerols 100 to 1000 times [9].
Much of our current understanding on the uptake of dietary lipids was derived from
the work of Hofmann and Borgström [31,32] and subsequent studies [33-35], who describe
the importance of micellar solubilization of lipids in the uptake of lipid digestion products by
enterocytes. To understand the importance of micellar solubilization, it is important to discuss
the unstirred water layer, a concept introduced by Westergaard and Dietschy [36] (Figure
1.4). According to this concept, the brush border membrane of the enterocytes is separated
from the bulk fluid phase in the intestinal lumen by an unstirred water layer, which is relatively
impermeable for the lipolytic products, especially the long-chain fatty acids. The rate of longchain fatty acid monomer diffusion in water is greater than that of aggregates of mixed
micelles [12]. The increased concentration of fatty acids by micellar solubilization overcomes
the slower diffusion rate, so that the net effect of micelle formation is an increase in the
transfer of lipolytic products across the unstirred water layer [36]. Thus, mixed micelles would
act as lipid shuttles to overcome the unstirred water layer [36].
The validity of this concept was later challenged by Carey and his associates, who
discovered the coexistence of unilamellar liposomes with bile salt-lipid mixed micelles in the
small intestine [37]. They proposed that when the bile salt concentration in the lumen exceeds
the critical micellar concentration, the lipids in the intestinal lumen will be incorporated into
mixed micelles [1]. When the amount of lipids in the aqueous phase increases further and the
amount of bile salts does not increase, this eventually results in the formation of liquid
crystalline vesicles (liposomes) [1]. However, so far the relative roles of the micelle and the
liquid crystalline vesicle in the uptake of fatty acids and monoacylglycerols have not been
resolved [9].
12
General introduction
The lipolytic products of medium-chain triacylglycerols are absorbed faster than those
of long-chain triacylglycerols. As lipolysis of medium-chain triacylglycerols is more complete
than that of long-chain triacylglycerols, the medium-chain triacylglycerols (unlike long-chain
triacylglycerols) are absorbed mainly as free fatty acids, and only rarely as mono- and
diacylglycerols [6]. Because of the increased water solubility of medium-chain fatty acids,
absorption of medium-chain fatty acids is not dependent on micellar solubilization [38-40].
Thus, for long-chain fatty acids passage across the unstirred water layer is rate limiting,
whereas passage of medium-chain fatty acids is only limited by the brush border membrane
[41].
Bulk solution in
intestinal lumen
Diffusion barrier
overlying microvilli
Cytosolic
compartment of
intestinal cell
1
2
Figure 1.4 Diagrammatic representation of the effect of bile salt micelles (or vesicles) in overcoming the diffusion barrier
resistance offered by the unstirred water layer. In the absence of bile acids, individual lipid molecules must diffuse across the
barriers overlying the microvillus border of the intestinal epithelial cells (arrow 1). Hence, uptake of these molecules is largely
diffusion limited. In the presence of bile acids (arrow 2), large amounts of these lipid molecules are delivered directly to the
aqueous-membrane interface so that the rate of uptake is greatly enhanced [9].
Translocation
The mechanism by which lipids are taken up by the enterocyte across its apical membrane
remains unresolved. Previously, it has been accepted that the uptake of free fatty acids and
monoacylglycerols by the enterocytes is a passive diffusion process [10,42]. Recently, the
possibility has been raised that some lipids may be taken up by enterocytes by carrier-mediated
processes [43-47]. It was shown that fatty acid binding proteins and/or fatty acid translocase,
associated with the brush border membrane, seem to play a role in the uptake of fatty acids by
enterocytes [43,47]. However, the exact role of the protein has not been resolved yet, and the
issue of whether fatty acids are taken up by passive diffusion or by a carrier-mediated process
needs further investigation.
13
Chapter 1
Intracellular events
In the intestinal cell the various absorbed lipids migrate from the site of absorption to the
endoplasmic reticulum. It has been suggested that the migration of the lipids is mediated via
fatty-acid-binding proteins (FABP) located in the intestine (intestinal FABP and liver FABP)
[48]. Re-esterification of free fatty acids and monoacylglycerols into triacylglycerols takes
place at the cytoplasmic surface of the endoplasmic reticulum [49] mainly via the
monoacylglycerol pathway [50,51]. This involves reacylation to diacylglycerols and
triacylglycerols by monoacylglycerol-acyltransferase and diacylglycerol-acyltransferase,
respectively [9]. The other route of triacylglycerol synthesis, the alpha-glycerophosphate
pathway, involves conversion of glycerol-3-phosphate via phosphatidic acid to diacylglycerols
and, subsequently, to triacylglycerols by various enzymes [9]. Under physiological
circumstances, the monoacylglycerol pathway predominates relative to the alphaglycerophosphate pathway [9].
Triacylglycerols are then transferred by a transfer protein to the inside of the
endoplasmic reticulum [52] and packaged into lipoprotein particles called chylomicrons.
Chylomicrons are made exclusively by the small intestine, and consist mainly of phospholipids,
dietary triacylglycerols and apolipoproteins apo A-I, apo A-IV, and apo B-48 [9]. Data from
both animals and humans indicate that the fatty acid composition of the triacylglycerol of
chylomicrons closely resembles the dietary lipid fed [53,54]. The chylomicrons are released
into the bloodstream via the lymph system for delivery of triacylglycerols to the tissues.
1.3 Fate of lipids in the colon
The digested nutrients that enter the colon encounter a large population of bacteria capable of
a wide range of metabolic activities. For example, the colonic flora play a major part in the
fermentation of carbohydrates to produce short-chain fatty acids. Although these short-chain
fatty acids may play a role in the prevention of colonic inflammation, further discussion is
beyond the scope of this thesis and the interested reader is referred to [55,56].
The small amounts of long-chain fatty acids escaping absorption and entering the
large bowel have been regarded as of trivial biological significance. However, there is evidence
that colonic bacteria can metabolize dietary fats: colonic bacteria secrete lipase enzymes [57],
they have active transport mechanisms for medium- and long-chain fatty acids, and are capable
of oxidation, desaturation and hydroxylation of fatty acids [58]. In the past several studies
showed that the unabsorbed fraction of lipid may have important effects on bacterial
metabolism of the colon [59,60], and may even play a role in the etiology of colonic cancer
[61].
Obviously, the daily input of lipids into the colon increases considerably in the case of
various lipid malabsorption syndromes. However, the role of large amounts of lipids in the
colon has only been partially resolved and further research is necessary [58,62].
14
General introduction
1.4 Lipid malabsorption
Fat malabsorption is characterized by increased fecal excretion of mostly dietary lipids.
Increased fat content of the feces is also known as steatorrhoea, which may be a first symptom
of underlying diseases affecting fat absorption. It has been convenient to divide fat
malabsorption into those disorders with an impaired digestion of triacylglycerols from those
disorders with impaired intestinal uptake, which includes impaired mixed micelle formation
and translocation of fatty acids over the intestinal mucosa.
Impaired lipolysis
Under physiological conditions, pancreatic lipase is present in pancreatic juice in abundance.
Its high concentration in pancreatic secretions and its high catalytic efficiency ensure the
efficient digestion of dietary lipid. However, impaired lipolysis of dietary triacylglycerols,
caused by a lack of sufficient pancreatic lipases, is a well-recognized cause of steatorrhoea.
Pancreatic lipase deficiency can either be due to the (relative) absence of the enzymes involved
or due to inactivity of these enzymes [63]. Steatorrhoea in lipase deficiency is usually not
severe, unless lipase concentration in the upper intestinal tract is less than 10% of normal.
Such impaired lipolysis may be secondary to cystic fibrosis, chronic pancreatitis, pancreatic
resection, or pancreatic carcinoma [38,63].
The most effective treatment of lipase deficiency is to restore lipase activity. This is
accomplished either by eliminating causes of lipase inactivation, such as correcting gastric acid
hypersecretion, or to supply exogenous lipase [38,64].
Impaired uptake of long-chain fatty acids
Patients with a biliary fistula, biliary obstruction, chronic liver disease, or an interruption of the
bile salt enterohepatic circulation by ileal resection or disease have a decreased bile salt
secretion rate. With less bile salt present in the intestinal lumen, fewer mixed micelles form,
impairing solubilization of ingested lipids. However, total absence of bile in the intestine does
not completely inhibit fat absorption. Even up to 80% of dietary lipids were found to be
absorbed in a study in adults with biliary fistula [33]. An explanation could be the observation
of liquid crystalline vesicles by Carey et al. [1]. They suggested that when the amount of fat in
the aqueous intestinal phase is high compared with the amount of bile, liquid crystalline
vesicles are formed. These vesicles may play an important role in the uptake of fats by
enterocytes in disease states [33]. The finding of liquid crystalline vesicles may have important
pathophysiological implications. Because patients with low intraluminal bile salt
concentrations or with bile fistulae can have reasonably good lipid absorption, it was proposed
that the liquid crystalline vesicles may play an important role in the uptake of fatty acids and
monoacylglycerols by enterocytes in these disease states [1]. In addition, in the absence or at
low concentrations of bile salts, the absorption of fatty acids occurs to a relatively lower and
slower extent [65]. Brand and Morgan [66] showed that fat absorption occurs largely from the
proximal small intestine in control rats, whereas, in the absence of bile distal small intestine is
also involved. Presumably, the absorptive reserve of the distal small intestine is called upon in
15
Chapter 1
the case of bile diversion and much of the fat which failed to enter the proximal intestinal
mucosa is absorbed more distally [67].
Therapy is directed toward either restoring the enterohepatic circulation of bile salts
or by substituting medium-chain triacylglycerols in the diet [38]. Because of the increased
water solubility of medium-chain fatty acids, bile salts are not as necessary for efficient
absorption of medium-chain fatty acids.
Cystic fibrosis
A frequently encountered genetic disorder associated with fat malabsorption is cystic fibrosis
[68,69]. The pathophysiology of fat malabsorption in cystic fibrosis patients involves both
pancreatic insufficiency and deficient intestinal uptake of long chain fatty acids. Cystic fibrosis
is an autosomal recessive disorder in which defective transepithelial chloride transport results
in the production of mucus with increased viscosity in various organs. Among the organs
commonly affected, the lungs and the pancreas frequently are involved in serious symptoms at
young age [70]. The basic defect is the cystic fibrosis transmembrane regulator (CFTR), a
protein responsible for chloride ion transport. Both pancreatic insufficiency and high energy
expenditure due to increased respiratory work are thought to contribute to the frequently
observed poor nutritional status of these patients [68,71]. The positive correlation between a
good nutritional status and long-term survival or well-being of cystic fibrosis patients is well
documented [72]. This observation has led to increased attention for optimization of nutrient
intake and absorption in cystic fibrosis patients [68]. Recommendations for treatment of cystic
fibrosis patients include consumption of 120-150% of the recommended daily allowance of
energy for healthy individuals [73], with a normal to high lipid (40 energy %) intake to offset
increased energy requirements [74].
Despite recent improvements in the pharmacokinetics of the supplementary
pancreatic enzymes, many patients continue to experience a certain degree of steatorrhoea
[75-77], with lipid absorption reaching 80 to 90% of their dietary lipid intake. It has not been
elucidated if the remaining lipid malabsorption is due to an insufficient dosage of pancreatic
enzyme replacement therapy. This possibility is not unlikely because a decreased pancreatic
bicarbonate secretion may negatively affect enzyme activity by sustaining a low pH in the
duodenum [64]. At a low duodenal pH, the release of the enzymes from the (micro)capsules is
inhibited and the denaturation of the enzymes is stimulated [64,78]. However, it has been
demonstrated that increasing the pancreatic enzyme dosages does not completely correct lipid
malabsorption [79]. In addition, attempts to increase lipolysis by high-strength pancreatic
enzyme supplements has led to the reported association with fibrosing colonopathy [80-82].
An alternative explanation for the continuing fat malabsorption in CF patients on
pancreatic enzyme replacement therapy may involve inefficient intestinal uptake of fatty acids
[75,83]. Impaired uptake in CF patients can be due to an altered bile composition, decreased
bile salt secretion by the liver, bile salt precipitation, a decreased bile salt pool size, and/or bile
salt inactivation at low intestinal pH [77,83-86]. Furthermore, small bowel mucosal
dysfunction or alterations in the mucus layer may contribute to inefficient intestinal uptake of
long chain fatty acids in CF patients [68,87].
16
General introduction
Although it is known that the pathophysiology of fat malabsorption in cystic fibrosis
patients involves both pancreatic insufficiency and deficient intestinal uptake of long chain
fatty acids, the relative contribution of these two processes frequently remains unclear. Insight
into the contribution of either of these processes would benefit cystic fibrosis patients,
however, it is difficult to obtain mechanistic information in patients. In an attempt to further
elucidate the pathophysiology of cystic fibrosis, several mouse models of cystic fibrosis were
developed [88,89].
1.5 Methods to measure lipid malabsorption
The efficiency of intestinal lipid absorption in patients is routinely determined by means of a
lipid balance, requiring detailed analysis of daily lipid intake and the complete recovery of
feces for 72 h. However, in the case of lipid malabsorption, this method does not discriminate
between the potential causes, such as impaired intestinal lipolysis or disturbed micellar
solubilization of long-chain fatty acids. Since different therapies are selected for the different
causes, it is important to know the etiology behind the fat malabsorption. In the development
of novel diagnostic strategies, stable isotope techniques have been introduced. In this chapter
the several aspects regarding the fecal fat balance will be discussed first. Thereafter, attention
will be paid to stable isotope tests measuring impaired lipolysis and/or disturbed uptake of
long-chain fatty acids.
Fecal fat balance
The conventional method by which lipid absorption is evaluated is the 3-day fecal fat balance.
Estimation of a fat balance is carried out as follows: the patient is kept on a diet containing a
known amount of lipid and dietary intake is recorded for a period of 3 days. The feces
excreted during the same period is collected accurately, and lipid is determined quantitatively.
Since its first description in 1949, the titrimetric procedure of Van de Kamer [90] has been
used as a reference method for the measurement of lipid in the feces. The percentage of total
dietary lipid absorption is calculated from the amount of lipid ingested and the amount of lipid
excreted via the feces by the following equation:
Fat intake (g day -1 ) − Fecal fat output (g day -1 )
Percentage of total fat absorption =
× 100%
Fat intake (g day -1 )
The 3-day fecal fat balance in Western adult humans shows that intake of major
dietary lipids, principally triacylglycerols, constitutes approximately 100 g day-1. In addition,
substantial amounts of endogenous lipids are delivered to the intestinal lumen from bile [91],
desquamated cells [92], and dead bacteria [93,94]. Intestinal epithelial cells are being sloughed
off into the lumen continuously [92] and it can be estimated to amount to about 450 g of cells
per day of which 2 to 6 g are membrane lipids that are mostly digested and absorbed [1,91].
Although measurement of fecal lipid excretion during a standard lipid intake is
generally considered to be the most accurate screening test for detecting lipid malabsorption,
17
Chapter 1
the test is not widely used because of its poor acceptability by patient, physician, and clinical
chemist. For the patient, the test involves the inconvenience of eating a defined diet and the
mechanical and esthetic problems of collecting, storing, and transporting stools. For the
physician, the test involves scientific uncertainty as to the completeness of the fecal collection
and also may involve storage and transport problems. For the clinical chemist, the test involves
the storage of bulky specimens and the unpleasant task of sample homogenization and
sampling [95]. Finally, in the case of lipid malabsorption, the lipid balance method does not
discriminate between the underlying mechanisms, such as impaired intestinal lipolysis or
disturbed intestinal solubilization of long chain fatty acids. In order to investigate the
underlying mechanisms, stable isotope techniques have been introduced, which will be
discussed in the next paragraph.
Stable isotopes
The renaissance of interest in stable isotopes in the last ten years is based upon the
development of new instrumentation, such as the availability of the quadruple mass
spectrometers interfaced with the gas chromatograph (GC/MS) and the development of
isotope ratio mass spectrometers (IRMS), which made possible the convenient use of selective
ion monitoring for the quantification of isotope enrichment. An increased awareness of the
health hazards of radioactivity, as well as greater availability of stable isotopes, also stimulated
the use of stable isotopes.
The most obvious advantage of stable isotopes is that they are nonradioactive and
present little or no risk to human subjects and they are even suitable for the study of infants,
children, and pregnant women [96]. Carbon 13 is a naturally occurring isotope present to the
extent of approximately 1.1% of the major isotopic species, carbon 12 [97]. Since carbon 13
naturally contributes 1.1% of the carbon pool, and since it has not been possible to
demonstrate more than trivial in vitro isotopic effects on chemical reactions with carbon 13labeled substrates [98,99], significant side effects in vivo are not expected from administration
of tracer doses of carbon 13.
Among the numerous applications of stable isotopes in physiology and medicine, the
investigation of lipid absorption and metabolism poses considerable challenges because of the
complexity of the subject, the multitude of influencing factors and the demanding analytical
requirements [100]. Various labeled fatty acids and labeled triacylglycerols are available and
can be given orally. When a 13C-labeled fat is ingested, the substrate may be digested,
absorbed and enters metabolic pathways leading to enrichment of bicarbonate, protein, lipid
and carbohydrate within the body. Unabsorbed amounts of the 13C-labeled fat are excreted via
the feces. After absorption, the 13C-labeled fat enters the oxidative pathways and is excreted as
13
CO2 via the breath.
When stable isotopes are used to measure fat digestion and absorption, between
ingestion of the labeled fat and appearance of 13C in plasma and excretion of 13CO2 in the
breath, many factors can influence the outcome of the test and expression of the results, such
as gastric emptying rate, absorption rate, hepatic clearance etc. [101]. Choice of substrate and
choice of sampling compartment are the first factors in determining the sensitivity and
18
General introduction
specificity of the test. The rate-limiting step of interest in the handling of substrates by the
body determines the selection of a substrate.
13
C-TRIOLEIN
Since triolein is a long-chain triacylglycerol, its efficient absorption depends upon the
overall process of fat absorption, thus, adequacy of lipolysis, bile salt solubilization and
intact mucosal surface. Hence, this substrate is a sensitive indicator of steatorrhoea, but will
not distinguish between the underlying mechanisms [102-104]. It has been proposed that
the triolein test is preferred when compared to the trioctanoin or the tripalmitin test for the
screening of total lipid malabsorption arising from a broad spectrum of gastrointestinal
disorders because of its higher sensitivity and specificity [95,105,106]. However, it has
been shown that the test has not the ability to predict the severity of malabsorption [107].
So far 13C-triolein has only been used with collection of breath and analysis of 13CO2, but its
radioactive form, 14C-triolein, has also been used with measurements of postprandial serum
[108,109].
13
C-HIOLEIN
Naturally occurring hiolein provides a new tracer for lipid absorption studies. Hiolein is a
long chain triacylglycerols mixture obtained from algae which is uniformly labeled with 13C
and enriched by 98%. The major fatty acid composition of hiolein is oleic acid (51%),
palmitic acid (17%) and linoleic acid (20%) [110]. Since hiolein consists mainly of
triacylglycerols, efficient absorption depends on the same processes as triolein, i.e. lipolysis,
bile salts solubilization and intact mucosa. Patients with significantly impaired lipolysis, bile
salt deficiency, or mucosal disorders, excrete the substance in their stool, and have
decreased amounts of 13CO2 in their breath [110-113].
13
C-TRIOCTANOIN
Trioctanoin is a medium-chain triacylglycerol. Although both medium and long-chain
triacylglycerols require lipolysis by gastric and pancreatic lipase, medium-chain
triacylglycerols, being water soluble, do not depend critically on the presence of bile salts
for their digestion and absorption. The 13C-trioctanoin test thus focuses on lipase activity
and a reduction in trioctanoin absorption reflects the level of lipolytic activity present in the
patients digestive tract. Digestion and absorption of trioctanoin have been assessed by
13
CO2 excretion via the breath [105,114-119]. The choice of a medium-chain triglyceride
has an additional advantage in that the lipolytic products are rapidly absorbed and oxidized
[120], thus shortening the overall study period [114]. The 13C-trioctanoin test distinguishes
pancreatic from non-pancreatic causes of steatorrhoea, and it has been applied for
measurements of lipid maldigestion in adults [119] and in children with cystic fibrosis
[115,116], and for measurements of lipid utilization in preterm and full-term neonates
[116,121]. A disadvantage of the 13C-trioctanoin breath test is that the rate of lipolysis is
facilitated for medium-chain triacylglycerols when compared with long-chain
triacylglycerols [1,6]. Hence, the test does not exactly reflect lipolytic rate of dietary fats,
because they mainly consist of long-chain triacylglycerols.
19
Chapter 1
13
C-MIXED TRIGLYCERIDE
A substrate that has the advantages of the 13C-trioctanoin breath test (short study period)
and avoids the disadvantages (facilitated lipolysis for medium-chain triacylglycerols) is the
so-called 13C-mixed triglyceride breath test [122,123]. The mixed triglyceride used is 1,3distearoyl, 2[carboxyl-13C]octanoyl glycerol. This molecule contains a 13C-labeled medium
chain fatty acid (octanoic acid) at the sn-2 position, and long-chain fatty acids (stearic acid)
at the sn-1 and sn-3 positions of the glycerol backbone of the triacylglycerol [123]. The two
stearoyl chains have to be hydrolyzed by lipolytic enzymes in the intestine before 13Coctanoate can be absorbed, thereby avoiding the disadvantage of the 13C-octanoin substrate
which contains only medium-chain fatty acids. Thus, the principle of the mixed triglyceride
breath test is based on lipolysis-dependent 13CO2 excretion via the breath. The applicability
of the mixed triglyceride breath test has been demonstrated in healthy adults [124,125],
pancreatic insufficiency patients [123,126], and preliminary data on the potential
applicability in children are available [127]. The mixed triglyceride breath test has a
sensitivity of 89% and a specificity of 81% compared with direct measures of lipase in
patients with pancreatic and non-pancreatic causes of steatorrhoea [123].
CHOLESTERYL-[1-13C]OCTANOATE
The utilization of cholesteryl-[1-13C]octanoate is another attractive substrate for measuring
pancreatic exocrine insufficiency. This substrate differs little from the cholesteryl esters
naturally present in food. It undergoes hydrolysis by pancreatic cholesteryl esterase, and the
labeled octanoate molecule is rapidly absorbed and oxidized [114,120]. Because cholesteryl
octanoate is not hydrolyzed by gastric lipase it may be used to assess pancreatic exocrine
function alone [128]. However, pancreatic carboxyl ester lipase activity requires the
presence of bile salts and therefore, this test will not only measure pancreatic function but
also solubilization by bile [128]. The test has been successfully used to diagnose exocrine
pancreatic insufficiency [129] and to monitor pancreatic enzyme replacement therapy in
patients with pancreatic insufficiency [128,130,131].
[1-13C]PALMITIC ACID
Efficient absorption of long-chain fatty acids, e.g. palmitic acid, is not dependent on
lipolysis since free fatty acids are already hydrolyzed substrates. Thus, application of this
test to patients with gastrointestinal complaints would identify individuals with inadequate
solubilization of long chain fatty acids or intestinal mucosa disease [105,132,133]. It is
difficult to discriminate between the two processes, which may be due to the fact that
impaired solubilization is rarely an isolated event [105]. The test has been performed in
healthy controls [134,135] and in patients with gastrointestinal diseases [105,136]. The
advantage of using palmitic acid as a substrate instead of other fatty acids is that palmitic
acid is a saturated fatty acid and both solubilization and translocation across the intestinal
mucosa of saturated fatty acids are more difficult when compared to unsaturated fatty
acids. In addition, palmitic acid is the most predominant fatty acid in the Western diet, and
therefore experiments mimic dietary fat absorption as much as possible.
20
General introduction
[U-13C]LINOLEIC ACID
[U-13C]linoleic acid is a long-chain fatty acid and, thus depends on the same absorptive
processes as [1-13C]palmitic acid does, i.e. adequate solubilization by bile components and
intact mucosa of the intestine. In addition, linoleic acid is an essential fatty acid and
therefore may be used for studies with respect to the essential fatty acid status.
1.6 Scope of this thesis
As discussed before, the process of lipid absorption can be viewed as a chain of events
occurring after lipid ingestion, including emulgation, lipolysis, solubilization, uptake in the
enterocyte, and chylomicron assembly. Under physiological conditions, the efficacy of lipid
absorption ranges from 96 to 98% [1,2]. Until now, most attention has been paid towards the
efficacy of the overall process of lipid absorption, yet, insight into the individual mechanisms
causing fat malabsorption has remained rather incomplete. A detailed insight into the
underlying mechanisms would enable not only improvements in diagnostic methodologies, but
also treatment in individual patients by modulating diet therapy, pancreatic enzyme
replacement therapy and supplementation of antacids and/or bile salts. Thereby, it is a
reasonable expectation that the prognosis of (pediatric) patients with impaired lipid absorption
can be improved, given the positive correlation between a good nutritional status and longterm survival or well-being [72-74].
The aim of the thesis is to obtain mechanistic information on the various processes
involved in fat malabsorption, with the purpose to increase diagnostic and eventually
therapeutic possibilities in patients with fat malabsorption. The approach to achieve this aim
involves studies in experimental animals, in human volunteers and in patients. The studies were
chosen to investigate in detail the two most frequently occurring pathophysiological processes
involved in human fat malabsorption, namely, impaired lipolysis and disordered bile formation,
as well as the most frequently encountered disease in children associated with fat
malabsorption, cystic fibrosis. Since our purpose was to increase diagnostic and eventually
therapeutic possibilities in patients, we applied stable isotopes in our experiments, allowing
physiological studies in humans in a non-harmful way. The applicability of stable isotope
labeled lipids for quantitative studies on lipid absorption has only been investigated to a very
limited extent.
A non-invasive test that has been described to characterize pancreatic insufficiency in
a functional way is the 13C-MTG breath test [123]. However, widely variable results have been
obtained in children, healthy adults, and in cystic fibrosis patients with and without pancreatic
enzyme replacement therapy [125,126]. The origin of this variability has not been elucidated.
In fact, a 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. Therefore, in this thesis the efficiency and repeatability
of the 13C-MTG breath test were investigated in rats treated with the lipase inhibitor orlistat
(chapter 2) and in healthy adults (chapter 3), respectively.
21
Chapter 1
Few attempts to develop a specific test for the detection of impaired intestinal uptake
of long chain fatty acids have been reported [105]. Intestinal uptake involves solubilization of
lipolytic products by the formation of mixed micelles composed of bile components and
lipolytic products, followed by the translocation of the lipolytic products across the intestinal
epithelium [2,9,34,35]. Potential substrates for the detection of impaired intestinal uptake are
13
C-labeled long chain fatty acids. In this thesis, the potency of 13C-labeled palmitic acid to
detect impaired intestinal uptake was determined in rats with long-term diversion of the biliary
tract (chapter 4). In addition, the sensitivity of the 13C-labeled palmitic acid test was
investigated in healthy adults supplemented with calcium in order to achieve mild fat
malabsorption due to decreased amounts of bile in the intestine (chapter 5).
A relatively frequently encountered disorder in Caucasian populations associated with
fat malabsorption is cystic fibrosis. Although it is known that the pathophysiology of fat
malabsorption in cystic fibrosis patients involves both pancreatic insufficiency [68,69] and
deficient intestinal uptake of long chain fatty acids [75,83], the relative contribution of these
two processes frequently remains unclear. In order to obtain more insight into the impaired
processes of fat malabsorption in cystic fibrosis we performed a study in pediatric cystic
fibrosis patients treated with their usual pancreatic enzyme replacement therapy (chapter 6).
The substrates 13C-MTG and uniformly labeled 13C-linoleic acid were both applied to
determine whether the rate-limiting step behind their remaining fat malabsorption was either
impaired lipolysis or impaired intestinal uptake of long chain fatty acids, respectively. Based on
the results of this study, we further explored the mechanisms involved in deficient intestinal
uptake of long chain fatty acids in further detail in two recently generated cystic fibrosis mouse
models (chapter 7). 1. Mice with the ∆F508 mutation in the cftr gene, ∆F508/∆F508 mice.
The ∆F508 mutation is the most frequently observed mutation in cystic fibrosis patients. 2.
Mice with complete inactivation of the cftr gene, cftr -/- mice [137,138].
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30
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 13CMTG 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 longchain 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 13Clabeled 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 [2628]. 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 EurisoTop (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 mixedtriglyceride 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 180647) 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%; C18:2n6, 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 13Cenrichment 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 (StudentNewman-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).
Table 2.1
13
Nutritional data and breath CO2 data obtained from control and orlistat-fed rats during
13
C-MTG experiments (mean ±
SEM).
Orlistat
mg kg-1 chow
0
50
200
800
Fat intake
(g day-1)
2.7 ± 0.2a
2.1 ± 0.2b
2.9 ± 0.1a
3.0 ± 0.2a
Fecal fat
(g day-1)
0.5 ± 0.1a
0.3 ± 0.0a
1.2 ± 0.1b
2.0 ± 0.2c
Fat uptake
(g day-1)
2.1 ± 0.1a
1.8 ± 0.2a
1.7 ± 0.1a
1.0 ± 0.1b
Fat absorption
(% intake)
80.2 ± 2.2a
85.2 ± 0.8a
59.2 ± 2.1b
32.8 ± 3.7c
Cum breath 13C
(% dose)
84.5 ± 7.8a
82.0 ± 4.9a
58.5 ± 5.3b
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.
24
% Cumul 13CO2 Expiration
% Dose h-1 13CO2 Expiration
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.
A
20
16
12
8
4
0
0
1
2
3
4
5
6
100
B
80
a
a
60
b
c
40
20
0
0
1
2
Time (h)
3
4
5
6
Time (h)
-1
Figure 2.1 Time courses for (A) the excretion rates (% dose h ) and (B) the cumulative
13
13
CO2 excretion (% cum) in breath (mean
-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%.
% Cumul
13CO
2
Expiration
100
r = 0.88, P<0.001
80
60
40
20
0
0
20
40
60
80
100
% Total Fat Absorption
Figure 2.2 Relationship between the percentage of total fat absorption and breath
13
CO2 excretion after oral administration of
13
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).
Table 2.2
13
Nutritional data of control and orlistat-fed rats during [1- C]-palmitic acid experiment (mean ± SEM).
Orlistat
mg kg-1 chow
0
200
Fat intake
(g day-1)
2.2 ± 0.2
2.5 ± 0.2
Fecal fat
(g day-1)
0.6 ± 0.0
1.3 ± 0.2*
Fat absorption
(% intake)
74.6 ± 0.3
46.7 ± 5.4**
13
C16:0 absorption
(% dose)
83.7 ± 2.0
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
Plasma
13C16:0
conc. (%Dose L -1)
The 13C-MTG breath test in rats fed orlistat
80
60
40
20
0
0
1
2
3
4
Time (h)
5
6
24
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 13CMTG 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 doseresponse 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 [113
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 [26,4245]. 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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43
CHAPTER 3
The 13C-mixed triglyceride breath test in healthy
adults: determinants of the 13CO2 response
M. Kalivianakis, H.J. Verkade, F. Stellaard,
M. van der Werf, H. Elzinga, R.J. Vonk
Eur J Clin Invest (1997) 27, 434-442
Chapter 3
CHAPTER 3
The 13C-mixed triglyceride breath test in healthy
adults: determinants of the 13CO2 response
Abstract
Background & Aim: Defects in lipolysis due to pancreatic insufficiency can be diagnosed by
the mixed triglyceride 13CO2 breath test. However, the effects of various test conditions on the
13
CO2 response have only partially been elucidated. Methods: In healthy adults we performed
the 13CO2 mixed triglyceride breath test and we compared a) the inter- and intra-individual
variation in the 13CO2 response, b) the effect of two different test meals, c) the effect of an
additional meal during the test, and d) the effect of physical exercise during the test. Results:
Upon repeating the test in the same individual (test meal cream), repeatability coefficients
were large, either with respect to time to maximum 13C excretion rate (3.8 h), maximum 13C
excretion rate (4.9% 13C dose/h), or cumulative recovery of 13C over the 9-h study period
(22.7% 13C dose). The cumulative 13C expiration over 9 h obtained with the test meal
composed of cream was quantitatively similar to that obtained with bread and butter: 42.2 ±
8.4%, and 47.7 ± 6.3%, respectively. Fasting for 9 h during the test resulted in similar 13C
expiration rates and cumulative 13C expiration (43.4% ± 7.2%), when compared to
consumption of an additional meal at 3 h after the start of the test (38.3 ± 5.3%). The 13CO2
response increased in 5 out of 7 subjects, but decreased in the other 2, when moderate
exercise was performed (bicycle ergometer, 50W for 5 h). Conclusion: Repeatability of the
MTG test in healthy adults is low. The present results indicate that a solid and a liquid test
meal, containing similar amount of fats, give similar cumulative 13CO2 responses, and that
stringent prolonged fasting during the test is unnecessary. Standardization of physical activity
seems preferable, since unequivocal effects of moderate exercise on the 13CO2 response were
observed in the individuals studied.
46
The 13C-MTG breath test in healthy adults
Introduction
A common feature of pancreatic insufficiency is a reduced output of pancreatic lipase. This
condition may lead to lipid malabsorption due to reduced intestinal hydrolysis of
triacylglycerols [1,2]. Measurement of maximal pancreatic lipase output by means of an
invasive, marker-corrected perfusion technique is considered to be the gold standard of
pancreatic insufficiency tests [3,4]. A non-invasive test has recently been described in which a
13
C-labeled mixed triglyceride (MTG) was ingested together with a test meal, after which the
amount of 13C in expired air was determined [5]. The MTG used is 1,3-distearoyl, 2[carboxyl13
C]octanoyl glycerol. This molecule contains a 13C-labeled medium-chain fatty acid (octanoic
acid) at the sn-2 position, and long-chain fatty acids (stearic acid) at the sn-1 and sn-3
positions of the glycerol backbone of the triacylglycerol [5]. The two stearoyl chains have to
be hydrolyzed by lipolytic enzymes in the intestine (mainly of pancreatic origin) before
[13C]octanoate can be absorbed, either in the form of a free fatty acid or of a monoacylglycerol [6]. It has been known that, after absorption, octanoate is rapidly oxidized to a
considerable extent [6,7]. Thus, the principle of the MTG test is based on lipolysis-dependent
13
CO2 excretion via the breath. The applicability of the MTG test in pancreatic insufficiency
has been demonstrated in adults [5,8,9], and preliminary data on the potential applicability in
children are available [10].
A general problem of breath tests using labeled lipids for the diagnosis of pancreatic
insufficiency or fat malabsorption in general, is a relatively poor sensitivity and specificity,
probably due to the numerous steps involved in the metabolism of the tracer compound [11].
Differences in gastric emptying, solubilization by bile acids, mucosal absorption, hepatic
clearance and metabolism, endogenous CO2 production and pulmonary excretion may obscure
the relationship between the quantity of label expired and the aim of the study, for example
hydrolysis in the intestine [12-15]. Up to now, none of these breath tests has been clinically
validated in different disease states. Presently, the discriminating test parameters are only
poorly defined, which limits the application of these tests in clinical studies.
The aim of the present study was to further characterize the MTG breath test and to
identify some factors apart from pancreatic insufficiency that may influence the quantitative
recovery of 13CO2 in breath. We examined the variation of the 13CO2 response within and
between healthy human adults, in which no rate-limiting variation in pancreatic exocrine
function was expected. In addition, we evaluated various determinants of the 13CO2 response:
1. The test meal. A variety of test meals has been described for breath tests with a diversity of
substrates [5,14,16,17]. So far, no standardized test meal for clinical purpose of these
breath tests has been proposed. A disadvantage of a test meal such as bread and butter for
children could be the extended time it would take for consumption, and the risk of not
consuming it quantitatively. Furthermore, such a test meal is not applicable to small infants.
Therefore, we examined whether the 13CO2 response of a liquid test meal is similar to the
13
CO2 response of the mentioned solid test meal.
47
Chapter 3
2. The fasting condition during the test. Since it may be cumbersome to keep patients, in
particular infants, fasted for at least six hours, we determined to what extent consumption
of an extra meal during the test influenced the 13CO2 response.
3. Physical activity. It is known that physical activity considerably affects the production rate
of CO2 and nutrient oxidation [18-20], however it is not established to what extent it
influences the results of the 13C-MTG test.
Materials and methods
Subjects
The studies were conducted with four male and seven female volunteers with a mean age of 23
± 1 (SEM) years and a mean body mass index of 20.9 ± 0.3 kg/m2. The volunteers were
healthy according to medical histories and did not have symptoms of lipid malabsorption, such
as diarrhea or gastrointestinal complaints. Informed consent was obtained, and the study
protocol was approved by the Medical Ethics Committee of the University Hospital
Groningen.
13
C-labeled substrate
MTG (mixed triglyceride, 1,3-distearoyl, 2[carboxyl-13C]octanoyl glycerol) was purchased
from the Belgian Institute of Isotopes (IRE, Fleurus, Belgium) and from Euriso-Top, Saint
Aubin Cedex, France, and was 99% 13C-enriched. The chemical purity exceeded 98%. Both
the isotopic and chemical purity were checked by NMR.
Study protocol
The subjects were instructed to avoid consumption of naturally 13C-enriched foods (e.g. corn
or corn products, pineapple, cane sugar) for at least two days prior to the study. After an
overnight fast (approximately 10 hours), each subject consumed a test meal consisting of
either 75 ml cream or 2 slices of bread and 25 g butter, each mixed with 13C labeled MTG (4
mg/kg body weight). Breath samples were collected in duplicates before consumption of the
test meal to provide a value of baseline 13C-excretion in expired CO2, and were subsequently
collected at 30-min intervals for a period of 9 hours after the ingestion of the test meal. Unless
stated otherwise, all experiments were performed under standard conditions, which implied
that: 1. the test meal consisted of 75 ml cream, 2. no additional food or liquids were permitted
during the 9-h period except for water, tea and coffee without sugar and milk, and 3. the
subjects only performed light office tasks during the tests.
Analytical techniques
Breath was collected by expiration via a straw into a 10 ml tube (Exetainers; Labco Limited,
High Wycombe, United Kingdom), from which aliquots were taken to determine 13Cenrichment by means of continuous flow isotope ratio mass spectrometry (Finnigan Tracer
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
48
The 13C-MTG breath test in healthy adults
(δ13CPDB, ‰). The proportion of 13C-label excreted in breath CO2 was expressed as the
percentage of administered 13C-label recovered per hour, and as the percentage of
administered 13C-label recovered over the 9-h study period.
Mean values of whole body CO2 excretion were measured by indirect calorimetry
(Oxycon, model ox-4, Dräger, Breda, The Netherlands) at 3 separate periods of 5 minutes
during the 9-h study period.
Intra- and inter-individual variation
The intra-individual variation was examined in eleven subjects by repeating the study under
identical (standard) conditions within four weeks after the first test. The individual
repeatability of the test was examined with the use of repeatability coefficients according to
Bland and Altman [21]. Coefficients of repeatability are based on the mean of the differences
between repeated measurements on a series of subjects and the standard deviation of the
differences. The definition of a repeatability coefficient adopted by the British Standards
Institution is the expectation that 95% of differences is within two standard deviations of the
mean difference [22]. Repeatability coefficients were calculated with respect to three
parameters: time to maximum 13C excretion rate, tmax, maximum 13C excretion rate, and
cumulative recovery of 13C after 9 h [23].
Influence of two different test meals on 13CO2 expiration
In six subjects the influence of two different easily applicable test meals on the 13CO2 response
in breath after oral ingestion of MTG was examined: 1. 75 ml cream (1040 kJ, 26 g fat, 2 g
carbohydrate, 2 g protein), and 2. two slices of bread with 25 g butter (1550 kJ, 22 g fat, 32 g
carbohydrate, 6 g protein). In addition, the influence of either test meal in itself on 13Cenrichment of breath was examined in each subject, by repeating the test with a test meal to
which no 13C-MTG was added.
Prolonged fasting during the test versus consumption of an additional meal
In seven subjects the effect of prolonged fasting on the 13C expiration rate was examined.
Fasting represented the standard condition mentioned above, whereas in the non-fasting
condition an additional meal was consumed at 3 hours after the start of the experiment. The
additional meal consisted of 2 slices of bread and 30 g strawberry jam (960 kJ, 2 g fat, 49 g
carbohydrate, 6 g protein). The experiments were performed with the initial test meal (time 0
h) consisting of 75 ml cream. In five subjects a control study was performed, in which the 13Clabeled MTG was omitted from the test meal.
Influence of physical exercise on 13CO2 expiration
In seven subjects, the influence of physical exercise on the 13CO2 response was investigated.
The results from the tests done under standard conditions were compared to those obtained
during moderate exercise on a bicycle ergometer. The physical exercise started at 10 minutes
before the consumption of the test meal (75 ml cream). The energy performance was 50 watt
for 5 hours, which represents an intensity of approximately 25 to 35% of the subjects’
maximal aerobic capacity (VO2max). Drinking of water was allowed ad libitum during the
49
Chapter 3
bicycle test for the whole 9-h period. The last 4 hours of the experiment (time 5-9 h) the
subjects were in rest. Five subjects underwent a control study during which the influence of
the test meal and exercise as mentioned above was examined under background conditions,
i.e. without the addition of the label.
Statistical methods
The experimental data are reported as means ± SEM. Statistical comparisons between the data
were performed with the use of the two-tailed non-parametric Wilcoxon signed-rank test for
pairs. Differences between means were considered statistically significant at the level of
P<0.05. For statistical analysis three different characteristics of the 13CO2 expiration were
analyzed according to Matthews et al. [23]: 1. the time to the maximum 13C excretion rate,
tmax, 2. the maximum 13C excretion rate (expressed as % 13C dose per hour), and 3. the
recovery of 13C over the 9-h study period (expressed as % cumulative 13C excreted).
Results
Background variation of 13CO2 expiration after an unlabeled test meal
The background variation of 13C in breath was examined in several subjects by performing the
test without administration of the label under 4 different experimental conditions: standard
condition, test meal consisting of bread and butter, additional meal at 3 h after the start of the
test, and moderate exercise during the test for 5 hours. There was no detectable change in the
average expiration of 13CO2 over the 9-h study period, in any of the experimental settings. The
inter-individual background 13C-variation in breath CO2 at the various time points was small
(average SEM 0.12‰), as was the intra-individual variation (average SEM 0.21‰, data not
shown).
Intra- and inter-individual variation
The baseline 13C-abundance in breath prior to consumption of the test meal was -26.2 ± 0.2‰
in test 1. The standard error of the analysis at this enrichment level was 0.03‰ (n=10). After
ingestion of the 13C-MTG containing test meal at time 0, different time-course patterns were
observed for the excretion of 13C-label in breath over the 9-h study period, with maximum
excretion rates varying between 3 and 8 h after administration of the 13C-labeled test meal
(Figure 3.1, closed squares). At the maximum excretion rate, the enrichment of 13C in breath
was -21.6 ± 0.5‰ and at the end of the 9-h study period, the enrichment of 13C in breath had
not yet returned to the level of baseline 13C-abundance (-24.6 ± 0.3‰). When expressed as a
proportion of administered 13C, the peak excretion rate of label in breath in the first test was
9.3 ± 1.3% 13C per hour of the administered dose, varying between 5.0 and 16.3%. Over the
9-h study period the excretion of 13C in breath was 40.3 ± 5.0% of that administered, ranging
between 16.7 and 66.4% (Table 3.1; the subject numbers in Table 3.1 are corresponding to the
subject numbers in the figures).
50
The 13C-MTG breath test in healthy adults
16
16
16
1
3
4
12
12
12
12
8
8
8
8
4
4
4
4
0
0
3
6
9
16
% 13C dose h-1
16
2
0
0
3
6
9
16
0
0
3
6
9
16
7
12
12
12
8
8
8
8
4
4
4
4
0
3
6
9
16
0
0
3
6
9
16
0
0
10
12
12
8
8
8
4
4
4
3
6
9
0
9
3
6
9
3
6
9
0
0
3
6
9
11
12
0
6
16
9
0
3
8
12
0
0
16
6
5
0
0
3
6
9
0
0
Time (h)
Figure 3.1 Time courses for the excretion of
13
C in breath over 9 h following oral ingestion of MTG (4 mg per kg body weight) at
time 0 in eleven healthy adults in test 1 ( ) and test 2 (Ž). The tests were done under standard conditions with the test meal 75 ml
cream.
On repeating the test under identical circumstances, the time-course patterns of most
individuals appeared rather similar to the first test with maximum excretion rates varying
between 3 and 9 h after administration of the 13C-labeled test meal (Figure 3.1, open squares).
However, in one subject (Figure 3.1, subject 1) a strikingly different time course of label
expiration was observed when compared to the first test: tmax of 3 h in the first study and a tmax
probably after 9 h. The mean results on 13CO2 expiration were not significantly different when
compared to the first test: the baseline 13C-abundance was -26.0 ± 0.1‰, the enrichment of
13
C in breath at peak excretion was -21.7 ± 0.5‰, and the enrichment of 13C in breath at the
end of the 9-h study period was, again, not yet at baseline 13C-abundance (-24.8 ± 0.6‰).
When expressed as a proportion of administered 13C, the peak excretion rate of label was 8.3 ±
1.0%, ranging from 4.5 to 14.5%, and excretion of label in breath over the 9-h study period
was 33.2 ± 3.6%, ranging from 18.8 to 48.3% (Table 3.1).
For the 3 parameters studied, the repeatability coefficients [21] of time to maximum
13
C excretion tmax, maximum 13C excretion rate, and cumulative recovery of 13C after 9 h were
3.8 h, 4.9% 13C dose/h, and 22.7% 13C cumulative excreted, respectively (Figure 3.2). Thus,
for example, since the repeatability coefficient of the cumulative percentage 13C excreted is
22.7%, the cumulative percentage 13C excreted in a second experiment performed under
identical circumstances may be 22.7% above or below the cumulative percentage 13C excreted
51
Chapter 3
Difference in tmax (test 1-2)
300
A
Mean + 2*SD
200
100
Mean
0
-100
-200
Mean - 2*SD
-300
0
100
200
300
400
500
Difference in recovery (test 1-2)
Average tmax (min)
Difference in max. excretion rate(test 1-2)
in the first experiment. This lack of repeatability is much less obvious when only Figure 3.1 is
considered.
8
B
Mean + 2*SD
6
4
2
Mean
0
-2
Mean - 2*SD
-4
-6
0
2
4
6
8
10
12
14
Average max. excretion rate (% 13C dose/h)
Mean + 2*SD
30
C
20
10
Mean
0
-10
Mean - 2*SD
-20
0
10
20
30
40
50
60
Average recovery (% 13C cum)
Figure 3.2 Repeatability of test results of test 1 and test 2 calculated according to Bland and Altman [21], in which (A) the time to
the maximum
recovery of
13
13
C excretion rate tmax, (B) the maximum
13
C excretion rate (expressed as %
C over the 9-h study period (expressed as % cumulative
13
13
C dose per hour), and (C) the
C excreted), were determined after oral ingestion of MTG
(4 mg per kg body weight).
Influence of two different test meals on 13CO2 expiration
Time courses for the excretion rate of 13C in breath for the subjects ingesting the two different
test meals (cream versus bread and butter) are shown in Figure 3.3. The maximum 13C
excretion rate occurred at similar time points (tmax 5.0 ± 0.6 h and 4.8 ± 0.5 h, for the cream
test meal and the bread-and-butter test meal, respectively), and values were not significantly
different from each other: 10.3 ± 2.2% and 9.7 ± 1.2% 13C dose/hour, respectively (P=0.84).
The 9-h cumulative 13C expiration amounted to 42.3 ± 8.4% (ranging from 16.7 to 66.4%) for
the cream test meal and to 47.7 ± 6.3% (ranging from 34.5 to 78.0%) for the bread-and-butter
test meal (P=0.69) (Table 3.1).
In order to compare our data with those of Vantrappen et al. [5], who performed the
MTG test for 6 hours, we also calculated the cumulative 13C excretion over a 6-h period. For
the cream test meal, this value was 28.4 ± 7.5% (n=6, ranging from 2.9 to 47.6%), and for the
bread-and-butter test meal 32.8 ± 5.1% (n=6, ranging from 22.0 to 55.5%). These recoveries
during 6 h after label administration were comparable to those described by Vantrappen et al.
52
The 13C-MTG breath test in healthy adults
[5], who obtained a recovery of 33.5 ± 1.4% (n=25, ranging from 23 to 52%). Yet, the interindividual variation between our data appeared considerably larger.
Table 3.1
Cumulative 9-h
13
CO2 excretion in breath after oral ingestion of MTG (4 mg per kg body weight) in healthy adults for
the 4 different experimental conditions mentioned in the text: a) standard experimental condition (test 1 and test 2), b) the test meal
consisted of 2 slices of bread and 25 g butter, c) three hours after the start of the test an additional meal was ingested consisting of
2 slices of bread and 30 g of strawberry jam, and d) during the first 5 h of the test subjects were bicycling at 50 watt. The subject
numbers are corresponding to the subject numbers mentioned in the figures.
Subject
Sex
1
2
3
4
5
6
7
8
9
10
11
F
F
M
M
F
M
F
F
F
M
F
Standard condition
Test 1
Test 2
66.4
48.3
61.3
35.0
25.1
28.9
31.5
21.6
16.7
27.1
52.7
40.0
50.0
57.0
50.4
39.2
29.9
18.8
36.1
24.1
23.0
24.7
16
16
2
12
12
8
8
8
4
4
4
0
3
6
9
16
0
0
3
6
9
0
0
5
12
12
8
8
8
4
4
4
3
6
9
0
3
6
9
3
6
9
6
12
0
73.2
56.6
16
16
4
0
Bicycling
for 5 h
31.6
85.3
44.6
25.3
35.3
3
12
0
Additional
meal at 3 h
49.5
41.3
26.3
27.0
19.2
49.8
55.0
16
1
% 13C dose h -1
Test meal
bread/butter
34.5
78.0
43.5
40.3
44.5
45.1
0
3
6
9
0
0
Time (h)
Figure 3.3 The influence of two different test meals on the excretion rate of 13C in breath after oral ingestion of MTG (4 mg per kg
body weight) in 6 healthy adults. ( ) Test meal consisting of 75 ml cream; (Ž) test meal consisting of 2 slices of bread and 25 g
butter.
Prolonged fasting during the test versus consumption of an additional meal
Figure 3.4 shows the 13C expiration rate data, comparing the fasting and non-fasting
experimental condition. In the fasting condition, the maximum 13C excretion rates occurred at
tmax 4.7 ± 0.6 h, and, after consumption of an additional (low fat) meal at time point 3 h, at tmax
53
Chapter 3
4.4 ± 0.4 h, and did not differ between the two groups: 10.7 ± 1.9% and 9.4 ± 1.4% 13C
dose/hour, respectively (P=0.38). The 9-h cumulative 13C expiration amounted to 43.4 ± 7.2%
(ranging from 16.7 to 66.4%) for the fasting condition and to 38.3 ± 5.3% (ranging from 19.2
to 55.0%) for the non-fasting condition (not significantly different, P=0.38) (Table 3.1). The
consumption of an additional low-fat meal at 3 h after the start of the experiment neither
altered the form nor the height of the mean curve for all subjects.
16
16
16
% 13C dose h -1
1
16
3
2
4
12
12
12
12
8
8
8
8
4
4
4
4
0
0
3
6
9
16
0
0
3
6
9
16
0
6
12
12
8
8
8
4
4
4
3
6
9
0
6
9
3
6
9
0
0
3
6
9
7
12
0
3
16
5
0
0
0
3
6
9
0
0
Time (h)
Figure 3.4 The influence of the consumption of an additional meal 3 h after the start of the experiment on the excretion rate of
13
C
in breath after oral ingestion of MTG (4 mg per kg body weight) in 7 healthy adults. ( ) Prolonged fasting during the test; (Ž)
Consumption of an additional carbohydrate rich meal 3 hours after the start of the experiment.
Influence of physical exercise on 13CO2 expiration
The effects of physical exercise on the 13C excretion were not similar in the seven subjects
studied (Figure 3.5). In five of the seven subjects, physical exercise during the test induced an
increase in the maximal 13C excretion rate and cumulative 13C excretion when compared to the
test under standard conditions (i.e. resting during the entire experiment). However, one of the
seven subjects responded in the opposite direction (Figure 3.5, Table 3.1; subject 1), as no
increase of the peak excretion rate was observed after physical exercise, and an actual
decrease in the cumulative 13C excretion was noticed. The remaining subject (Figure 3.5, Table
3.1; subject 4) showed a small increase in the maximal 13C excretion rate, but still the
cumulative 13C excretion was decreased. The peak excretion of 13C-label in breath during the
rest session in the subjects occurred at tmax 5.2 ± 0.7 h after administration of the 13C-labeled
test meal and amounted to 9.8 ± 1.7% 13C dose per hour (n=7). The total excretion of 13C
over the 9-h study period was 43.1 ± 7.1%. Bicycling for 5 h at moderate intensity decreased
the time to maximum 13C excretion (tmax 3.9 ± 0.9 h), however, this effect was not significant
(P=0.22). Furthermore, the maximum 13C excretion rate in the seven subjects was significantly
increased (16.3 ± 2.6% versus 9.8 ± 1.7%, P=0.03), although total excretion of 13C over the
9-h study period was not significantly increased (50.3 ± 8.5% versus 43.1 ± 7.1%, P=0.47).
54
The 13C-MTG breath test in healthy adults
% 13C dose h -1
30
30
1
30
2
30
3
20
20
20
20
10
10
10
10
0
0
30
3
6
9
0
0
30
5
3
6
9
0
30
7
20
20
20
10
10
10
0
0
3
6
9
0
0
0
3
6
9
0
3
6
9
3
6
9
0
4
0
3
6
9
8
0
Time (h)
13
Figure 3.5 The influence of physical exercise on the cumulative excretion of C in breath after oral ingestion of MTG (4 mg per kg
body weight) in 7 healthy adults. ( ) Prolonged resting during the test; (Ž) Performance of physical exercise during the test on an
bicycle ergometer at 50W for 5 hours. During last 4 hours of the experiment subjects were in rest.
Discussion
The present investigations in healthy adults were designed to further characterize the
determinants of the 13C-MTG test in healthy volunteers. Intra- and inter-individual variations
were examined by repeating the study in the same individuals on a separate occasion. We also
determined to which extent the 13CO2 response was influenced by different test meals, by
prolonged fasting, and by physical exercise.
The variability of measurements in different subjects (i.e. the inter-individual
variation) is usually considerably larger than the variability between measurements on the same
subject (i.e. the intra-individual variation) [24]. Obviously, both kinds of variability are
important to assess the potential applicability of a diagnostic test. Only in one recent study the
repeatability of breath tests was investigated based on the ingestion of 13C-labeled lipid,
involving [1-13C]palmitic acid in healthy volunteers [17]. Also in this study a poor repeatability
was reported. No information is available on the repeatability of the MTG test under
physiological or pathological conditions. We examined the repeatability of the MTG test by
determining coefficients of repeatability [21]. The calculated repeatability coefficients from
data of 11 healthy adults for the 3 parameters studied are considerable (see Results section).
The values of the repeatability coefficients reflect the range of outcomes in which a repeated
test in the same individual will result with a 95% likelihood. Thus, for example, since the
repeatability coefficient of the cumulative percentage 13C excreted is 22.7%, the cumulative
percentage 13C excreted in a second experiment is predicted to be (with a 95% likelihood)
22.7% above or below the cumulative percentage 13C excreted in the first experiment. These
large repeatability coefficients are predominantly due to a considerable intra-individual
variation. The acceptability of a certain diagnostic test is based on the actual values obtained
and their repeatability coefficients in patients, compared to healthy controls. At present, only
55
Chapter 3
information on the mean values of 13CO2 expiration of pancreatic insufficiency patients is
available from the literature. It remains to be established whether the means and repeatability
coefficients of diseased individuals allow a clear discrimination from unaffected individuals
using the MTG-test.
Various test meals have been described for the use in breath tests with a diversity of
lipid substrates [5,14,16,17]. So far, no standardized test meal for clinical purpose of these
breath tests has been proposed. Theoretically, however, the test meal might influence the time
course of the 13CO2 response, for example by affecting the rate of gastric emptying [25-29]. In
our present study a liquid and a solid test meal with a comparable amount of fat were applied
to investigate the effect of different test meals on the 13CO2 response. The cumulative
percentage 13C of the dose excreted was comparable for the two test meals. This particular
feature is valuable for the potential application of the MTG test in children, since a liquid test
meal is more convenient to administer.
The originally described MTG breath test [5] lasted for 6 hours. Cumulative excretion
might be the most discriminative parameter when MTG is used as a clinical test. However,
also the cumulative excretion of 13CO2 depends on the end point (e.g. 6, 9 or more hours),
which has not yet been defined by validation studies. In order to evaluate possible diagnostic
advantages we extended the collection period of breath sampling up to 9 hours. In subjects,
who reached their maximum excretion rate before 6 h, an increase in oxidation by
approximately 50% was observed when the cumulative percentage 13C excreted after 9 hours
was compared to that after 6 hours. However, in 2 subjects (Figure 3.1, subjects 5 and 8), the
cumulative percentage 13C excreted after 9 hours was more than twice the amount excreted
after 6 hours. It is tempting to speculate that these particular subjects have a decreased gastric
emptying rate, compared to the others. If so, especially subjects with slow gastric emptying
will be diagnosed incorrectly when the time period of the breath test is not sufficiently long.
The originally described MTG-test [5] involved prolonged fasting for 6 hours. This
feature of the test would probably limit its applicability in (very young) pediatric patients. To
allow eating during the test would alleviate the applicability. However, the results of the 13CO2
response should then not be influenced by eating. Present data indicate that the consumption
of an additional low-fat meal 3 hours after the start of the experiment does neither change the
form of the 13C expiration curve nor the height of the curve. These observations suggest that
eating an additional meal during the test does not influence the results, which is in agreement
with the findings of Schwabe et al. [7]. They concluded that oral administration of glucose did
not seem to inhibit 14C-labeled octanoic acid oxidation [7].
In five out of seven individuals studied, the performance of physical exercise during
the test increased the cumulative recovery of 13C in breath, when compared to performance of
the test under resting conditions. Exercise enhances the oxidation of carbohydrates and lipids,
which is associated with a higher CO2 and 13CO2 response. Available data suggest that gastric
emptying during exercise is subject to a number of factors including calorie count, meal
osmolality, meal temperature and exercise conditions [30]. There are several indications
suggesting that light to moderate exercise accelerates gastric emptying of either liquid and
solid meals [30-33]. Intestinal absorption per se has not been evaluated in great detail during
exercise, but probably changes little [34]. All together, this explains the results of the five
56
The 13C-MTG breath test in healthy adults
mentioned individuals quite well. Unexpectedly, two subjects responded differently. At
present, no clear explanation could be obtained for the particular results in these two subjects;
both were healthy according to medical histories and, like the others, showed no symptoms of
lipid malabsorption, such as diarrhea or gastrointestinal complaints. This observation does
underline, however, the importance of standardizing the resting conditions during the MTG
breath test.
In summary, we have found that the repeatability of the mixed triglyceride test in
healthy adults is low. The results also suggest that two distinct test meals (cream versus bread
and butter) give a similar cumulative 13CO2 response and that stringency on continuous fasting
during the test is unnecessary, which is in favor of the applicability of the test in pediatric
patients. Standardization of resting conditions still seems preferable. In future patient studies it
remains to be established whether MTG-test results obtained under conditions of an impaired
intestinal lipolysis allow the sensitive and specific discrimination of affected from unaffected
individuals.
References
1.
Carey MC, Hernell O. Digestion and absorption of fat. Sem Gastrointest Dis 1992;3:189208.
2. Shiau Y-F. Lipid digestion and absorption. In: Johnson LR, ed. Physiology of the
Gastrointestinal Tract. 2nd Ed. New York: Raven Press, 1987:1527-1556.
3. Schmidt E, Schmidt FW. Advances in the enzyme diagnosis of pancreatic diseases. Clin
Biochem 1990;23:383-394.
4. Goldberg DM, Durie PR. Biochemical tests in the diagnosis of chronic pancreatitis and in
the evaluation of pancreatic insufficiency. Clin Biochem 1993;26:253-275.
5. Vantrappen GR, Rutgeerts PJ, Ghoos YF, Hiele MI. Mixed triglyceride breath test: A
noninvasive test of pancreatic lipase activity in the duodenum. Gastroenterology
1989;96:1126-1134.
6. Bach AC, Babayan VK. Medium-chain triglycerides: an update. Am J Clin Nutr
1982;36:950-962.
7. Schwabe AD, Bennett LR, Bowman LP. Octanoic acid absorption and oxidation in
humans. J Appl Physiol 1964;19:335-337.
8. Ghoos YF, Vantrappen GR, Rutgeerts PJ, Schurmans PC. A mixed-triglyceride breath
test for intraluminal fat digestive activity. Digestion 1981;22:239-247.
9. Maes BD, Ghoos YF, Geypens BJ, Hiele MI, Rutgeerts PJ. Relation between gastric
emptying rate and rate of intraluminal lipolysis. Gut 1996;38:23-27.
10. Van Aalst K, Veereman-Wauters G, Ghoos YF, Schiffelers S, Van 't Westeinde T,
Eggermont E. The 13C mixed triglyceride breath test in children. Gastroenterology
1995;108:A759(Abstract).
11. Pedersen NT, Jorgensen BB, Rannem T. The [14C]-triolein breath test is not valid as a test
of fat absorption. Scand J Clin Lab Invest 1991;51:699-703.
57
Chapter 3
12. Watkins JB, Schoeller DA, Klein PD, Ott DG, Newcomer AD, Hofmann AF. 13Ctrioctanoin: a nonradioactive breath test to detect fat malabsorption. J Lab Clin Med
1977;90:422-430.
13. Watkins JB, Tercyak AM, Szczepanik P, Klein PD. Bile salt kinetics in cystic fibrosis:
influence of pancreatic enzyme replacement. Gastroenterology 1977;73:1023-1028.
14. Murphy MS, Eastham EJ, Nelson R, Aynsley-Green A. Non-invasive assessment of
intraluminal lipolysis using a 13CO2 breath test. Arch Dis Child 1990;65:574-578.
15. Amarri S, Coward WA, Harding M, Weaver LT. Importance of measuring CO2
production rate in 13C breath tests. Proc Nutr Soc 1994;54:111A(Abstract).
16. Watkins JB, Klein PD, Schoeller DA, Kirschner BS, Park R, Perman JA. Diagnosis and
differentiation of fat malabsorption in children using 13C-labeled lipids: trioctanoin, triolein
an palmitic acid breath tests. Gastroenterology 1982;82:911-917.
17. Murphy JL, Jones A, Brookes S, Wootton SA. The gastrointestinal handling and
metabolism of [1-13C]palmitic acid in healthy women. Lipids 1995;30:291-298.
18. Brooks GA, Mercier J. Balance of carbohydrate and lipid utilization during exercise: the
"crossover" concept. J Appl Physiol 1994;76:2253-2261.
19. Coyle EF. Substrate utilization during exercise in active people. Am J Clin Nutr
1995;61:968S-979S.
20. Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E, Wolfe RR.
Regulation of endogenous fat and carbohydrate metabolism in relation to exercise
intensity and duration. Am J Physiol 1993;265:E380-E391.
21. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods
of clinical measurement. Lancet 1986;307-310.
22. British Standards Institution. Precision of test methods I: Guide for the determination and
reproducibility for a standard test method (BS 5497, part I), 1979. (Abstract).
23. Matthews JNS, Altman DG, Campbell MJ, Royston P. Analysis of serial measurements in
medical research. Br Med J 1990;300:230-235.
24. Bland JM, Altman DG. Correlation, regression, and repeated data. Br Med J
1994;308:896.
25. Maes BD, Ghoos YF, Geypens BJ, Hiele MI, Rutgeerts PJ. Relation between gastric
emptying rate and energy intake in children compared with adults. Gut 1995;36:183-188.
26. Hölzer HH, Turkelson CM, Solomon TE, Raybould HE. Intestinal lipid inhibits gastric
emptying via CCK and a vagal capsaicin-sensitive afferent pathway in rats. Am J Physiol
1994;267:G625-G629.
27. Edelbroek M, Horowitz M, Maddox A, Bellen J. Gastric emptying and intragastric
distribution of oil in the presence of a liquid or a solid meal. J Nucl Med 1992;33:12831290.
28. Hunt JN, Stubbs DF. The volume and energy content of meals as determinants of gastric
emptying. J Physiol London 1975;245:209-225.
29. Hunt JN, Knox MT. A relation between the chain length of fatty acids and the slowing of
gastric emptying. J Physiol 1968;194:327-336.
30. Moses FM. The effect of exercise on the gastrointestinal tract. Sports Med 1990;9:159172.
58
The 13C-MTG breath test in healthy adults
31. Moore JG, Datz FL, Christian PE. Exercise increases solid meal gastric emptying rates in
men. Dig Dis Sci 1990;35:428-432.
32. Cammack J, Read NW, Cann PA, Greenwood B, Holgate AM. Effect of prolonged
exercise on the passage of a solid meal through the stomach and small intestine. Gut
1982;23:957-961.
33. Marzio L, Formica P, Fabiani F, LaPenna D, Vecchiett L, Cuccurullo F. Influence of
physical activity on gastric emptying of liquids in normal human subjects. Am J
Gastroenterol 1991;86:1433-1436.
34. Feldman M, Nixon JV. Effect of exercise on postprandial gastric secretion and emptying
in humans. J Appl Physiol 1982;53:851-854.
59
CHAPTER 4
The 13C-palmitic acid test with plasma sampling
detects fat malabsorption in bile-diverted rats
M. Kalivianakis, D.M. Minich, R. Havinga, F. Kuipers,
F. Stellaard, R.J. Vonk , H.J. Verkade
Chapter 4
CHAPTER 4
The 13C-palmitic acid test with plasma sampling
detects fat malabsorption in bile-diverted rats
Abstract
Background & Aims: The fecal fat balance does not discriminate between the distinct intestinal
processes, such as lipolysis, solubilization, chylomicron formation, as causes of fat
malabsorption. In the present study we characterized a rat model with fat malabsorption due
to bile diversion and we investigated the diagnostic potency of the [1-13C]palmitic acid test.
Methods: Bile-diverted and control rats were fed standard (14 en% fat) or high-fat chow (35
en% fat) for 2 weeks. After intraduodenal administration of [1-13C]palmitic acid (33 mg kg-1
BW) blood samples were taken for measurements of 13C-palmitic acid enrichment. Food
intake was quantified and feces was collected for balance studies. Intestinal histology was
determined. Results: Total fat absorption was highly efficient in control rats (mean ± SEM:
standard chow 96.7 ± 0.2%; high-fat chow 93.2 ± 0.4%), but was significantly decreased in
bile-diverted rats (standard chow 87.2 ± 0.9%, P<0.001; high-fat chow 53.9 ± 3.9%,
P<0.001). Plasma 13C-palmitate concentrations allowed discrimination between normal
(>91%) and decreased fat absorption due to bile diversion. Conclusion: The [1-13C]palmitic
acid absorption test detects fat malabsorption due to bile diversion in rats. Application of this
test in clinical states with fat malabsorption may allow design of specific treatment strategies.
62
The 13C-palmitic acid test in bile-diverted rats
Introduction
Adequate absorption of dietary fats by the intestine is required for supply of energy, membrane
constituents, and precursors for the formation of hormones and inflammatory mediators [1,2].
In Western diets, triacylglycerols composed of long-chain fatty acids constitute 92 to 96% of
dietary fats [1]. The absorption of these fats involves several specific processes. Firstly,
lipolysis, by lipolytic enzymes originating predominantly from the pancreas, leads to hydrolysis
of triacylglycerols into fatty acids and 2-monoacylglycerols. Secondly, during the process of
solubilization, mixed micelles are formed consisting of bile acids, phospholipids and
cholesterol [3]. The mixed micelles are thought to act as the physiological transport vehicles of
lipolytic products from dietary fats in the intestinal lumen. Finally, the fatty acids and
monoacylglycerols are translocated across the intestinal epithelium, converted back to
triacylglycerols, assembled into chylomicrons and secreted into the lymph [1,4,5].
The efficiency of intestinal fat absorption in patients is routinely determined by means
of a fat balance, requiring detailed analysis of daily fat intake and the complete recovery of
feces for 72 h. However, in the case of fat malabsorption, this method does not discriminate
between the potential causes, such as impaired intestinal lipolysis, disturbed intestinal
solubilization of long-chain fatty acids or chylomicron formation. Stable isotope techniques
have been introduced in the development of novel diagnostic strategies. Several 13C-labeled
fats, such as tri-[1-13C]octanoin and 1,3-distearoyl, 2[1-13C]octanoyl glycerol, have been
successfully applied for the rather specific detection of impaired lipolysis [6-8]. Attempts to
develop a specific test for the detection of impaired solubilization have been less successful.
Watkins et al. [9] administered [1-13C]palmitic acid, tri-[1-13C]octanoin or tri-[1-13C]olein to
patients with impaired lipolysis, bile salt deficiency or mucosal disease and measured breath
13
CO2 excretion.
So far, the use of 13C-labeled fats for quantitative studies on defective fat absorption
has been limited to breath and feces analysis [9-11]. The excretion rate of 13C in the form of
exhaled 13CO2, however, does not necessarily reflect quantitative differences in the absorption
of the 13C-labeled parent compound, e.g., due to variations in the post-absorptive metabolism
[12,13]. The availability of gas chromatography isotope ratio mass spectrometry allows for
accurate determination of 13C enrichments in plasma fatty acids [14]. The determination of
plasma concentrations of absorbed 13C-labeled fats as a measure of their absorption offers a
theoretical advantage over breath 13CO2 analysis, since numerous steps are involved in the
(post-absorptive) metabolism of the tracer prior to exhalation of 13CO2 [15].
In order to determine the potency of a novel test, the availability of an animal model
is almost essential. Manipulation of the enterohepatic circulation of bile components has been
repeatedly and successfully used in rat studies on fat (mal)absorption and metabolism [16-19].
During bile diversion, no bile components are available in the intestine and fat malabsorption
appears to be mainly due to impaired solubilization of long-chain fatty acids [20]. However,
recent observations have suggested that bile is also essential for efficient chylomicron
formation [21-23]. Bile-diverted rats regain normal feeding behavior and normal growth
curves within several days after surgical interruption of the enterohepatic circulation [24].
63
Chapter 4
In the present study, firstly, we aimed to characterize the rat model of fat
malabsorption due to bile diversion. For this purpose, total fat absorption and [1-13C]palmitic
acid absorption was determined in rats that received either standard (14 en% fat) or high-fat
chow (35 en% fat). Secondly, we investigated the potency of the [1-13C]palmitic acid test with
plasma 13C-palmitic acid concentrations to detect fat malabsorption due to bile diversion.
Control experiments, in which tri-[1-13C]palmitoylglycerol was administered, were performed
to check whether lipolysis is unaffected in our animal model.
Materials and Methods
Animals
Male Wistar rats (Harlan, Zeist, The Netherlands), weighing 300 to 400 g, were housed in an
environmentally controlled facility with diurnal light cycling and free access to food and tap
water (and additional saline, 0.9% NaCl w/v, in the case of bile-diverted rats). Experimental
protocols were approved by the Ethical Committee for Animal Experiments, Faculty of
Medical Sciences, University of Groningen.
13
C-labeled substrates
[1-13C]-labeled palmitic acid was purchased from Isotec Inc. (Matheson, USA) and was 99%
13
C-enriched. Tri-[1-13C]palmitoylglycerol was purchased from ICN Biomedicals Inc.
(Cambridge, United Kingdom) and was 99% 13C-enriched.
Study protocol
Rats were assigned to either standard chow (14 en% fat; 4.575 kcal kg-1 food; fatty acid
composition measured by GC analysis: C8-C12, 0.9 mol%; C16:0, 25.2%; C18:0, 5.5%;
C18:1n-9, 30.3%; C18:2n-6, 33.9%; C18:3n-3, 3.6%) or to 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). After 1 week, rats were equipped with permanent catheters in
jugular vein, bile duct and duodenum as described by Kuipers et al. [24]. This experimental
model allows for physiological studies in unanesthetized rats with long term bile diversion
without the interference of stress or restraint. One day after surgery, catheters in bile duct and
duodenum were either connected at time of surgery to restore the enterohepatic circulation
(control rats) or catheters were chronically interrupted (bile-diverted rats). Animals were
allowed to recover from surgery for 6 days.
On day 7, 1.67 mL 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 mol%; 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 mol% ; C8:0, 50-65% max.; C10:0, 30-45%; C12:0, 3% max.) and contained either 33 mg
kg-1 body weight [1-13C]palmitic acid or 33 mg kg-1 body weight tri-[1-13C]palmitoylglycerol.
The fat bolus represented approximately 25 and 15% of the daily fat intake in the standard and
64
The 13C-palmitic acid test in bile-diverted rats
the high-fat group, respectively. 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, 2000 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 fat bolus and ending 2 days afterwards. Feces samples were stored
at -20°C prior to analysis. Food intake was determined for 3 days by daily weighing of the
food container.
Analytical techniques
Plasma fats. Total plasma fats (triacylglycerols, phospholipids, etc.) were extracted,
hydrolyzed and methylated according to Lepage and Roy [25]. 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 13Cenrichment of palmitic acid. The concentration of 13C-palmitic acid in plasma was expressed as
the percentage of the dose administered per liter plasma (% dose L-1).
Rat chow and fecal fats. Feces was freeze-dried and mechanically homogenized.
Aliquots of rat chow and freeze-dried feces were extracted, hydrolyzed and methylated [25].
Resulting fatty acid methyl esters were analyzed by gas chromatography to calculate total fat
intake, total fecal fat excretion, and total palmitic acid concentration in food and feces. Fatty
acid methyl esters were analyzed by gas chromatography combustion isotope ratio mass
spectrometry to calculate the 13C-enrichment of palmitic acid. 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 output (g day -1 )
Percentage of total fat absorption =
× 100%
Fat intake (g day -1 )
A similar calculation was performed to measure the absorption of [1-13C]palmitic acid and tri[1-13C]palmitoylglycerol. The absorption of the label was determined from the intake and
excretion of 13C-palmitic acid. Values were expressed as percentage of the dose administered
(% dose).
Gas liquid chromatography. Fatty acid methyl esters were separated and quantified
by gas liquid chromatography on a Hewlett Packard gas chromatograph Model 5880 equipped
with a CP-SIL 88 capillary column (Chrompack; 50 m x 0.32 mm) and an FID detector
[26,27]. 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
achieved 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) [28]. Separation of
the methyl esters was achieved on a CP-SIL 88 capillary column (Chrompack; 50 m x 0.32
65
Chapter 4
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. Significance of differences was
calculated with the use of the two-tailed Student’s t-test for unpaired data. For correlating two
variables, linear 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.
Results
Fecal fat balance
In Table 4.1 nutritional data of control and bile-diverted rats on standard chow (14 en% fat)
and high-fat chow (35 en% fat) are shown.
Standard chow (14 en% fat). Mean food intake, and thus fat intake, over the 3-day
period was significantly increased in bile-diverted rats on standard chow when compared to
control rats (P<0.05). Bile-diverted rats excreted significantly more fat into the feces when
compared to control rats (P<0.001). Although the percentage of total fat absorption was
lower in bile-diverted rats when compared to control rats (87.2 ± 0.9% vs. 96.7 ± 0.2%,
respectively, P<0.001), net fat uptake in both control and bile-diverted rats was similar
(P=0.91).
High-fat chow (35 en% fat). Both food and fat intake were significantly increased in
bile-diverted rats when compared to control rats (P<0.01). Similarly, fecal fat excretion was
significantly increased in bile-diverted rats compared to control rats (P<0.001). Again, there
was no significant difference in the net fat uptake of either control and bile-diverted rats
(P=0.10), although percentage of total fat absorption was considerably decreased in bilediverted rats when compared to control rats (53.9 ± 3.9% vs. 93.2 ± 0.4%, respectively,
P<0.001).
Table 4.1
Nutritional data (mean ± SEM) of control and bile-diverted rats on standard chow (14 en% fat) and high-fat chow (35
en% fat).
Category
n Food intake
(g day-1)
Standard chow
Control rats 9 18.9 ± 0.7
Bile-diverted 11 21.2 ± 0.7#
High-fat chow
Control rats 9 15.7 ± 0.7
Bile-diverted 6 21.9 ± 1.6§
Fat intake
(g day-1)
Fecal fat
(g day-1)
Net fat uptake Fat absorption
(g day-1)
(% intake)
1.04 ± 0.04 0.04 ± 0.00
1.17 ± 0.03# 0.15 ± 0.01*
1.00 ± 0.04
1.01 ± 0.04
96.7 ± 0.2
87.2 ± 0.9*
1.92 ± 0.10 0.13 ± 0.01
2.67 ± 0.20§ 1.15 ± 0.12*
1.80 ± 0.11
1.51 ± 0.10
93.2 ± 0.4
53.9 ± 3.9*
Symbols indicate significant difference within the same dietary group: #, P<0.05; §, P<0.01; * P<0.001.
66
The 13C-palmitic acid test in bile-diverted rats
[1-13C]palmitic acid
absorption (% dose)
100
r=0.89, P<0.001
90
80
70
0
0
50
60
70
80
90
100
% Total fat absorption
13
-1
Figure 4.1 Correlation between total fat absorption and the absorption of [1- C]palmitic acid (33 mg kg body weight). Results of
both control and bile-diverted rats, standard (14 en% fat) and high-fat chow (35 en% fat) are combined. Equation of the line:
y=0.44x+49; r=0.89, P<0.001.
Excretion of 13C-palmitic acid into feces
[1-13C]palmitic acid experiments. Table 4.2 shows the percentage absorption of [113
C]palmitic acid, assessed by fecal 13C-palmitic acid concentration. The amount of 13Cpalmitic acid excreted into the feces was calculated for the 48-h period following
administration of [1-13C]palmitic acid. The highest levels of 13C-palmitic acid in the feces were
observed in the first 24 h after bolus administration, which accounted for 81% of the amount
of label excreted in 48 h in the case of control rats. 13C-palmitic acid excretion in bile diverted
rats was significantly retarded with 62% excreted in the first 24 h (P<0.005). Control and bilediverted rats on standard chow absorbed similar amounts of [1-13C]palmitic acid over the 48-h
period studied (P=0.95). In bile-diverted rats on high-fat chow, the apparent absorption of [113
C]palmitic acid was significantly lower than in their control counterparts (P<0.001).
Table 4.2
13
-1
Absorption of [1- C]palmitic acid (33 mg kg body weight) by control and bile-diverted
rats on standard chow (14 en% fat) and high-fat chow (35 en% fat).
Chow
[1-13C]palmitic acid absorption
(% dose)
Category
n
Control rats
Bile-diverted
4
5
88.8 ± 2.6
89.0 ± 1.8
Control rats
Bile-diverted
5
3
91.3 ± 0.5
73.7 ± 2.1*
Standard
High-fat
* indicates a significant difference within the same dietary group (P<0.001).
Relationship between fecal fat balance and absorption of [1-13C]palmitic acid. To
compare whether [1-13C]palmitic acid was handled by the intestine similarly as dietary fats, the
relationship between the percentage of total fat absorption and absorption of [1-13C]palmitic
acid after 48 h was determined. Figure 4.1 shows a linear relationship between the absorption
67
Chapter 4
of total fat and the absorption of [1-13C]palmitic acid (r=0.89, P<0.001; equation of the line: y
= 0.44 x + 49).
Tri-[1-13C]palmitoylglycerol experiments. In control experiments with tri-[113
C]palmitoylglycerol we verified if lipolyis in bile-diverted rats was not impaired. In bilediverted rats on standard chow, the absorption of tri-[1-13C]palmitoylglycerol was virtually
identical to that of [1-13C]palmitic acid (88.3 ± 1.7% and 89.0 ± 1.8%, respectively). In bilediverted rats on high fat chow, again, the absorption of tri-[1-13C]palmitoylglycerol was not
significantly lower than the absorption of [1-13C]palmitic acid (80.8 ± 5.1% and 93.6 ± 6.4%,
respectively). These results indicate that lipolysis per se is not impaired in the bile-diverted rat.
Plasma 13C-palmitic acid concentrations
[1-13C]palmitic acid experiments. Figure 4.2A shows the time course patterns of 13C-palmitic
acid appearance in plasma after intraduodenal administration of [1-13C]palmitic acid to rats on
stardard chow (14 en% fat). After administration of [1-13C]palmitic acid to control rats,
plasma 13C-palmitic acid concentrations increased within 1 h, reaching a maximum value of 58
± 21% dose L-1 plasma at 2 h after bolus administration (Figure 4.2A). Upon bile diversion,
plasma 13C-palmitic acid concentrations were significantly lower than in controls (P<0.05). A
maximum value of 10 ± 2% dose L-1 plasma was obtained at 6 h (Figure 4.2A). Figure 4.2B
shows the time course patterns of 13C-palmitic acid appearance in plasma after intraduodenal
administration of [1-13C]palmitic acid to rats on high-fat chow (35 en% fat). After
administration of [1-13C]palmitic acid to control rats on high-fat chow, plasma 13C-palmitic
acid concentrations increased within 1 h after administration of the bolus and reached a
maximum value of 55 ± 7% dose L-1 plasma at 3 h (Figure 4.2B). Upon bile diversion, plasma
13
C-palmitic acid concentrations were significantly lower (P<0.05) when compared to the
values obtained in the controls rats (Figure 4.2B) and a maximum value of 16 ± 6% dose L-1
plasma was obtained after 6 h.
80
13C16:0
40
20
*
*
*
#
#
Time (h)
60
#
#
#
*
13C16:0
*
*
B
40
Plasma
60
(% dose L -1)
A
Plasma
(% dose L -1)
80
20
*
Time (h)
0
0
0
1
2
3
4
5
6
24
0
1
2
Time (h)
Figure 4.2 Time courses of
13
3
4
5
6
24
Time (h)
C-palmitic acid concentration in plasma of rats fed (A) standard chow (14 en% fat) and (B) high-fat
13
-1
chow (35 en% fat) after intraduodenal administration of [1- C]palmitic acid (33 mg kg body weight). (Ž) Control rats, (±) bile13
diverted rats. Symbols indicate significant difference between control and bile-diverted rats after [1- C]palmitic acid administration
(* P<0.05, # P<0.01).
68
The 13C-palmitic acid test in bile-diverted rats
(% dose L -1)
T=1 h
60
40
20
0
C
80
40
20
90
100
90
100
0
50
60
70
80
% Total fat absorption
90
100
T=3 h
13 C16:0
60
40
Plasma
(% dose L -1)
50
60
70
80
% Total fat absorption
T=2 h
60
0
0
B
80
13C16:0
A
80
Plasma
Plasma
13C16:0
(% dose L -1)
Relationship between fecal fat balance and plasma 13C-palmitic acid concentrations.
To compare the results of the 3-day fecal fat balance with the results of the 13C-palmitic acid
test, total fat absorption was related to plasma 13C-palmitic acid concentrations at 1 h, 2 h, and
3 h (Figure 4.3) after administration of [1-13C]palmitic acid. Already at 1 h and 2 h after label
administration, a clear distinction between control and bile-diverted rats was observed with
respect to plasma 13C-palmitic acid concentrations. Yet, plasma 13C-palmitic acid
concentrations at 3 h were most discriminative. Plasma 13C-palmitic acid concentrations at 3 h
were at least 3-fold lower in bile-diverted rats compared to controls, irrespective of the type of
chow. If we would regard 91% as the lower limit for normal fat absorption and 10 to 20%
dose L-1 plasma as the lower limit for normal plasma values, the test has a sensitivity and
specificity of 100%, under the conditions employed.
20
0
0
50
60
70
80
% Total fat absorption
13
Figure 4.3 Correlation between the results of the 72-h fecal fat balance and plasma C-palmitic acid concentrations at (A) 1 h, (B)
13
-1
2 h, and (C) 3 h after intraduodenal administration of [1- C]palmitic acid (33 mg kg body weight). Results of all experimental
groups are combined: control rats, standard chow (~); control rats, high-fat chow (€); bile-diverted rats, standard chow (±); bilediverted rats, high-fat chow (Ž).
Tri-[1-13C]palmitoylglycerol experiments. After administration of tri-[113
C]palmitoylglycerol to control rats and bile-diverted rats on standard chow, the 13C-palmitic
acid concentrations in plasma were not significantly different when compared with
administration of [1-13C]palmitic acid. Maximum values of 13C-palmitic acid concentration in
control and bile-diverted rats were 35 ± 5% dose L-1 plasma and 12 ± 1% dose L-1 plasma,
respectively. When the rats were fed high-fat chow, again, similar results were obtained.
Maximum values of 13C-palmitic acid concentration in control and bile-diverted rats were 50 ±
69
Chapter 4
20% dose L-1 plasma and 6 ± 4% dose L-1 plasma, respectively. These results are in
accordance with results on fecal excretion of 13C-palmitic acid reported above, and further
underline that lipolysis is not impaired in the bile-diverted rat.
Discussion
In the present study we characterized a rat model with a defined cause of fat malabsorption
due to bile diversion and we investigated the potency of the [1-13C]palmitic acid test to detect
this fat malabsorption. As no bile is available in the intestinal tract of bile-diverted rats, this
animal model is useful for determining the contribution of bile (components) to the process of
intestinal fat absorption. Although the role of bile for efficient fat absorption is wellestablished, its quantitative importance has been a matter of debate and is likely dependent on
the amount and composition of dietary fats [16-18].
Total dietary fat absorption was examined in chronically bile-diverted rats on
standard chow (14 en% fat) and on high-fat chow (35 en% fat). In control rats on either
standard or high-fat chow, the absorption of dietary fats was very efficient and varied from
92% to 97%. This percentage fat absorption is in accordance to that found in healthy humans
[20]. Total fat absorption was significantly decreased in bile-diverted rats. Bile-diverted rats
on standard chow still absorbed 87% of their dietary fats. An explanation could be the
formation of liquid crystalline vesicles in the intestinal lumen, as proposed by Carey et al. [20].
They suggested that liquid crystalline vesicles are formed, when the amount of fat in the
aqueous intestinal phase is relatively high compared to the amount of bile. These vesicles may
play an important role in in the uptake of fats by enterocytes in certain disease states [29].
However, bile-diverted rats on high-fat chow absorbed only 54% of their dietary fats,
indicating that at relatively high dietary fat intake, surface increase and formation of vesicles
are not sufficient to restore fat absorption completely.
Yet, in spite of the decrease in percentage of fat absorption in bile-diverted rats, the
rats managed to maintain a similar net fat uptake by increasing their food intake. This effect
was observed on either of the two diets. It has been observed previously that after bile
diversion rats increase their food intake in order to maintain an adequate energy balance [24].
It is intriguing to speculate which physiological stimulus mediates the adaptation of food
intake. A possible candidate is apolipoprotein A-IV. Synthesis of apo A-IV is stimulated upon
transport of absorbed lipid via chylomicrons in lymph [30-32]. Evidence of decreased
chylomicron assembly due to interference of biliary phospholipid availability by manipulation
with cholestyramine or dietary zinc was presented previously [21,22]. Impaired chylomicron
assembly in bile-diverted rats would decrease concentrations of apo A-IV in plasma, resulting
in enhanced food intake [33].
Using this animal model, we found that at 1 h, 2 h, and 3 h after administration of [113
C]palmitic acid, plasma 13C-palmitic acid concentrations clearly differentiated between
control rats and chronically bile-diverted rats (Figure 4.3). Using 10% dose L-1 plasma as the
lower limit of normal plasma values and 91% as the lower limit of normal fat absorption, the
test had a sensitivity and specificity of 100% under the test conditions used. The results were
70
The 13C-palmitic acid test in bile-diverted rats
essentially similar on standard and high-fat chow, emphasizing the potency of the 13C-palmitic
acid absorption test. It is interesting to hypothesize on the low plasma 13C-palmitic acid
concentrations in bile-diverted rats when compared to control rats. It is not likely that the
whole effect is solely due to impaired solubilization, because fat absorption in bile-diverted
rats on standard chow is still rather efficient. However, the effect of dimished solubilization
could be enhanced by the absence of stimulatory effects of biliary phospholipids on assembly
of intestinal chylomicrons [21-23]. If chylomicron assembly is impaired, 13C-palmitic acid will
appear in plasma to a lesser extent and on a slower time scale.
Time course patterns of plasma 13C-palmitic acid concentrations in control and bilediverted rats were considerably different (Figure 4.2). In control rats plasma 13C-palmitic acid
concentrations increased rapidly and peak values were observed after 1 to 3 h. In bile-diverted
rats plasma 13C-palmitic acid concentrations continuously increased up to 5 or 6 h but did not
reach the high values obtained in the control rats. Brand & Morgan [34] showed that fat
absorption occurs largely from the upper small intestine in control rats, whereas, in the
absence of bile lower small intestine is also involved. Presumably, the absorptive reserve of the
distal small intestine is called upon in the case of bile diversion and much of the fat which
failed to enter the proximal intestinal mucosa is absorbed more distally [35]. The delayed time
course patterns of plasma 13C-palmitate concentrations upon bile diversion could also be due
to decreased intestinal motility. In support of this explanation, fecal 13C-palmitate excretion
was significantly retarded in bile-diverted rats compared to controls in the first 24 h after
administration of [1-13C]palmitate. A cyclic pattern of motor activity known as the migrating
motor complex occurs in dogs, humans, and most other mammals during fasting [36-38].
Feeding interrupts the migrating motor complex and induces a different pattern of intermittent
contractile activity. However, the migrating motor complex activity does not seem to be
affected by bile diversion in rats [34] and dogs [39].
The fat bolus containing the [1-13C]palmitic acid was administered to the rats
intraduodenally. Although oral administration would have been more physiological,
intraduodenal administration has the benefit that the results are not influenced by gastric
emptying. Intra-individual and inter-individual variation of gastric emptying have been shown
to vary widely in humans [40,41] and would probably affect the outcomes of the study. This is
also a likely explanation for the high specificity and sensitivity of the test in our experiments.
An adventitious circumstance is that experiments run for a shorter period when the bolus is
administered intraduodenally, as gastric emptying of oils delays the process of fat absorption
with approximately 2 to 3 h [42,43].
In order to determine if stable isotopically-labeled fats are handled within the body
similar as those of dietary origin, the percentage of total fat absorption was compared to the
absorption of [1-13C]palmitic acid. Under physiological circumstances, palmitic acid is
consumed in the diet in the form of mixed triacylglycerols predominantly esterified at the sn-1
and sn-2 positions [44]. Hydrolysis of dietary triacylglycerols in the intestinal lumen by
pancreatic lipase and other enzymes, such as carboxyl ester hydrolase, results in the generation
of 2-monoacylglycerols and fatty acids. After interaction with bile components and the
formation of mixed micelles, the hydrolyzed fats can be absorbed. The absorption of dietary
fats was significantly correlated with the absorption of [1-13C]palmitic acid, assessed by fecal
71
Chapter 4
13
C-palmitic acid concentration (Figure 4.1). Thus, it seems that the fate of [1-13C]palmitic
acid with respect to absorption reflects the fate of total mass of dietary fats under the
experimental circumstances of this study. It has to be noted that the line of correlation does
not cut the origin of the plot, and that the slope of the line is smaller than unity. Apparently,
[1-13C]palmitic acid is preferentially absorbed when compared to dietary fats, which may be
due to the fact that only tracer amounts of [1-13C]palmitic acid were administered to the rats in
a specific, soluble form.
Absorption of tri-[1-13C]palmitoylglycerol and plasma 13C-palmitic acid
concentrations were determined to ascertain that lipolysis was not affected in the bile-diverted
rat model. In case of impaired lipolysis, tri-[1-13C]palmitoylglycerol could only be hydrolyzed
partially, resulting in decreased plasma 13C-palmitic acid concentrations when compared to
administration of [1-13C]palmitic acid. In addition, fecal 13C-palmitic acid concentrations
would be expected to be increased in analogy to studies involving drug-induced impairment in
lipolysis [45]. In both control and bile-diverted rats on standard chow, the plasma 13C-palmitic
acid concentration curves after administration of either [1-13C]palmitic acid or tri-[113
C]palmitoylglycerol were not significantly different (Figure 4.2). After administration of tri[1-13C]palmitoylglycerol, fecal excretion of 13C-palmitic acid was not significantly increased
compared with administration of [1-13C]palmitic acid. Based on these observations, we
conclude that lipolysis is not a rate-limiting step in this experimental model, in accordance to
previous observations [18,29]. Hamilton et al. [18] reported that in bile-diverted rats
absorption of free fatty acids was equal to the absorption of triacylglycerols with respect to
both palmitic acid and stearic acid. Similarly, Porter et al. [29] reported that lipolysis was
unimpaired in bile fistula man.
In summary, we show in a rat model of fat malabsorption due to bile deficiency that
percentage of dietary fat absorption depends on the presence of bile in the intestinal lumen.
With the use of this rat model, the [1-13C]palmitic acid absorption test, based on the
quantification of plasma 13C-palmitic acid concentrations, was sensitive enough to discriminate
between total fat absorption above 91% (control rats) and below 91% (bile-diverted rats).
These observations underline the potency of the 13C-palmitic acid absorption test in
combination with the technique of gas chromatography combustion isotope ratio mass
spectrometry to detect disorders in intestinal solubilization. Application of the test in clinical
absorption studies may allow a differentiated diagnosis and subsequent specific treatment.
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27. Gutnikov G. Fatty acid profiles of lipid samples. J Chromatogr B 1995;671:71-89.
28. Guo Z, Nielsen S, Burguera B, Jensen MD. Free fatty acid turnover measured using
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32. Kalogeris TJ, Monroe F, Demichele SJ, Tso P. Intestinal synthesis and lymphatic
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34. Brand SJ, Morgan RG. The movement of an unemulsified oil test meal and aqueous- and
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35. Lin HC, Zhao X-T, Wang L. Fat absorption is not complete by midgut but is dependent
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36. Szurszewski JH. A migrating electric complex of the canine small intestine. Am J Physiol
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38. Zenilman ME, Parodi JE, Becker JM. Preservation and propagation of cyclic myoelectric
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The 13C-palmitic acid test in bile-diverted rats
40. Maes BD. Measurement of gastric emptying using dynamic breath analysis. 1994;1133.(Abstract)
41. Brophy CM, Moore JG, Christian PE, Egger MJ, Taylor AT. Variability of gastric
emptying measurements in man employing standardized radiolabeled meals. Dig Dis Sci
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42. Carney BI, Jones KL, Horowitz M, Sun WM, Penagini R, Meyer JH. Gastric emptying of
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43. Meyer JH, Hlinka M, Kao D, Lake R, MacLauglin E, Graham LS, Elashoff JD. Gastric
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lipid handbook. London: Chapman Hall, 1986:49-170.
45. Isler D, Moeglen C, Gains N, Meier MK. Effect of the lipase inhibitor orlistat and of
dietary lipid on the absorption of radiolabelled triolein, tri-gamma-linolenin and tripalmitin
in mice. Br J Nutr 1995;73:851-862.
75
CHAPTER 5
The 13C-palmitic acid test for detection of mild fat
malabsorption in healthy adults on calcium
supplementation
M. Kalivianakis, F. Stellaard, R. Havinga, H. Elzinga,
R.J. Vonk, E.T.H.G.J. Oremus, H.J. Verkade
Chapter 5
CHAPTER 5
The 13C-palmitic acid test for detection of mild fat
malabsorption in healthy adults on calcium
supplementation
Abstract
Background & Aims: Recently we developed a [1-13C]palmitic acid absorption test for the
detection of fat malabsorption in rats with chronic bile diversion. In the present study in
healthy human adults we investigated whether this test was sensitive enough to detect mild fat
malabsorption induced by dietary supplementation of calcium carbonate. Methods: After oral
supplementation of [1-13C]palmitic acid (10 mg kg-1) to 10 healthy adults, breath and plasma
samples were obtained for 8 h and feces was collected for 72 h. Dietary fat intake was
assessed on the basis of a 4-day dietary record. After collection of feces, volunteers were
supplemented with 1000 mg calcium twice daily for 1 week, after which the [1-13C]palmitic
acid experiment was repeated. Results: Percentage of total fat absorption in healthy volunteers
on their habitual diets was (mean ± SEM) 96.6 ± 0.6%. Daily calcium supplementation led to a
slight but significant decrease in total fat absorption (94.9 ± 0.9%, P<0.05). The 8-h
cumulative percentage of 13CO2 expiration decreased from 11.4 ± 1.2% under control
conditions to 10.3 ± 1.1% upon calcium supplementation (P<0.05). Yet, plasma 13C-palmitic
acid concentrations were significantly higher after calcium supplementation when compared to
the control experiment. Conclusion: Dietary calcium supplementation to healthy adults leads
to a slight impairment of fat absorption. Although calcium supplementation clearly affects the
outcomes of the [1-13C]palmitic acid test, present data do not indicate that the test is sensitive
enough to reliably quantitate this degree of fat malabsorption in human adults.
78
The 13C-palmitic acid test in humans on calcium supplementation
Introduction
Adequate absorption of dietary fats by the intestine is required for supply of energy, membrane
constituents, and precursors for the formation of hormones or inflammatory mediators [1-4].
In Western diets, triacylglycerols composed of long-chain fatty acids constitute 92 to 96% of
dietary fats [2]. The absorption of these fats involves several processes. Firstly, lipolysis, by
lipolytic enzymes originating predominantly from the pancreas, leads to hydrolysis of
triacylglycerols into fatty acids and 2-monoacylglycerols. Secondly, during the process of
solubilization, mixed micelles are formed, consisting of bile acids, phospholipids, cholesterol
and the products of lipolysis (free fatty acids and monoacylglycerol) [5]. The mixed micelles
are thought to act as the physiological transport vehicles of lipolytic products from dietary fats
in the intestinal lumen. Finally, the fatty acids and monoacylglycerols are translocated across
the intestinal epithelium, reassembled into chylomicrons and secreted into the lymph [2,6,7].
The efficiency of intestinal fat absorption in patients is routinely determined by means
of a fat balance, requiring detailed analysis of daily fat intake and the complete recovery of
feces for 72 h. However, the fat balance does not provide information on the etiology of fat
malabsorption, i.e. impaired lipolysis or solubilization. This feature limits its use for defining
optimal treatments for patients. One approach to address specifically the causes of fat
malabsorption involves the determination of (mal)absorption of stable isotopically-labeled fats.
Recently, we described the results of the application of 13C-labeled linoleic acid [8] and of
palmitic acid [9] for detection of fat malabsorption due to chronic bile diversion in rats. In
these studies we observed that, after intraduodenal administration of the label, plasma 13C-lipid
concentrations reflect the absorption efficiency of dietary lipids. The present study was
designed to determine whether the [1-13C]palmitic acid test is sensitive enough to detect a
slight fat malabsorption in healthy adults.
Under physiological conditions, healthy individuals excrete approximately 4-6 g day-1
of fat via the feces [10], which generally means that over 96% of the dietary fats entering the
intestinal lumen is absorbed [10]. Even under physiological circumstances, non-lipid
components in the diet, such as calcium, have been demonstrated to interfere with the efficient
absorption of the lipids [11-14]. Oral calcium supplementation in healthy subjects has been
reported to increase fat excretion via the feces in a dose-dependent fashion, presumably due to
intestinal precipitation of bile salts and/or formation of insoluble calcium-fatty acid complexes,
leading to impaired solubilization [13-17]. Thus, oral calcium supplementation in humans
seems to be a reproducible method to induce a slight fat malabsorption due to impaired
solubilization of long-chain fatty acids.
The aim of the present study was to investigate whether the [1-13C]palmitic acid
absorption test could detect a mild degree of fat malabsorption in humans. Fat malabsorption
was achieved by supplementation of calcium. We investigated whether the absorption
efficiency of dietary fats was mildly decreased upon calcium supplementation for 1 week to
healthy adults and whether this possible effect could be quantified after oral ingestion of
administered [1-13C]palmitic acid. Quantification was achieved by determination of plasma
13
C-palmitic acid concentrations and breath 13CO2 concentrations.
79
Chapter 5
Materials and methods
Human volunteers
10 healthy students (7 females, 3 males) with a mean ± SEM age of 22 ± 0.3 y and a body
mass index of 20.5 ± 0.1 kg m-2 participated in the studies. The volunteers were healthy
according to medical histories and showed no symptoms of diarrhea, fat malabsorption, or
gastrointestinal complaints. The study protocol was approved by the Medical Ethics
Committee of the University Hospital Groningen.
Study protocol
Each subject completed two tests with [1-13C]-labeled palmitic acid separated by an interval of
one week. [1-13C]palmitic acid was purchased from Isotec Inc. (Matheson, USA) and was
99% 13C-enriched. The subjects were asked to maintain their usual dietary habits during the
total experimental period of 11 days. The subjects were instructed to avoid consuming
naturally 13C-enriched foods (e.g. corn products, pineapple, cane sugar) for at least two days
prior to and during each three-day study period. The same pre-selected test meal was
consumed throughout both tests to limit any effect that diet may have on the metabolism of [113
C]palmitic acid. During each test, intake of nutrients was calculated from 4-day consecutive
food diaries by a clinical dietitian using The Netherlands Nutrients Table “NEVO” 1993.
Subjects started with a control experiment during which they had their habitual
dietary calcium intake. After an overnight fast, the subjects consumed [1-13C]palmitic acid at a
dose of 10 mg kg-1 body weight as part of a controlled standard test meal consisting of 2 slices
of wheat bread, 20 g butter, 1 boiled chicken egg, 25 g cheese or liver-pie according to their
personal preference, 150 ml orange juice, and 150 g full fat yogurt (3500 kJ; 37 g fats, 94 g
carbohydrates, 32 g proteins). Butter was used as a vehicle to administer the [1-13C]palmitic
acid. Before consuming the test meal, breath samples were collected in duplicates to provide a
measure of baseline 13C-excretion in expired CO2. After ingestion of the test meal, breath
samples were collected periodically at 30-min intervals for a period of 8 hours. A baseline
blood sample (5 ml, EDTA) was collected before consuming the test meal, and was then
collected hourly during the 8-h study period. Plasma was isolated and stored frozen (-20ºC)
until further analysis. A baseline feces sample was collected on the day before administration
of [1-13C]palmitic acid. Thereafter, all stools passed were collected for three days,
homogenized, and frozen at -20ºC until further analysis. All the subjects were rested for the
duration of the test. No additional food or liquids were permitted during the test except for
non-calorie drinks such as water and tea.
From 3 days after the first study, calcium intake was increased for 7 days by oral
supplementation of 2000 mg calcium per day in the form of calcium carbonate, divided over
two doses (before breakfast and before dinner). At day 5 after the start of the calcium
supplementation, the [1-13C]palmitic acid test was repeated identical to the procedures
described above. Before ingestion of the test meal (breakfast), calcium supplementation was
administered.
80
The 13C-palmitic acid test in humans on calcium supplementation
The influence of the test meal on 13C-enrichment in breath 13CO2 was examined by
having one subject completing the study, using unlabeled palmitic acid instead of [113
C]palmitic acid.
Analytical techniques
Breath sample analysis. End expiratory breath was collected via a straw into a 10 mL tube
(Exetainers; Labco Limited, High Wycombe, United Kingdom), from which aliquots were
taken to determine 13C-enrichment by means of continuous flow isotope ratio mass
spectrometry (Finnigan Breath MAT, Finnigan MAT GmbH, Bremen, Germany). The 13Cabundance 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 study
period (cum % 13C).
Mean values of whole body CO2 excretion were measured by indirect calorimetry
(Oxycon, model ox-4, Dräger, Breda, The Netherlands) at 2 separate periods of 5 minutes
during both test days. As a control, this sampling method was compared to sampling every 30
min (results not shown). These results indicated that, under the test conditions chosen, the
mean values of the CO2 production obtained from 2 randomly chosen periods were within the
95% confidence interval of the mean values obtained when sampling occurred every 30 min.
Plasma fats. Plasma fats were extracted, hydrolyzed and methylated according to
Lepage and Roy [18]. Resulting fatty acid methyl esters were analyzed both by gas
chromatography to measure the total amount of palmitic acid and by gas chromatography
combustion isotope ratio mass spectrometry to measure the enrichment of palmitic acid. The
concentration of 13C-palmitate in plasma was expressed as the molar percentage of the dose
per liter plasma (% dose L-1).
Fecal fats. Total fecal fat excretion in human subjects was measured according to the
method of Van de Kamer et al. [19]. Feces was partly freeze-dried and mechanically
homogenized. Aliquots of freeze-dried feces were extracted according to the method of Bligh
and Dyer [20], and subsequently hydrolyzed and methylated [18]. Resulting fatty acid methyl
esters were analyzed by gas chromatography to calculate both total fecal fat excretion and
total palmitic acid concentration. Fatty acid methyl esters were analyzed by gas
chromatography combustion isotope ratio mass spectrometry to calculate the isotopic
enrichment of palmitic acid. Total fecal fat excretion was expressed as g fat day-1 and the
percentage of total fat absorption was calculated from the daily dietary intake and the daily
fecal fat output and expressed as a percentage of the daily fat intake.
Fat intake (g day -1 ) − Fat output (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.
Gas liquid chromatography. Fatty acid methyl esters were separated and quantified
by gas liquid chromatography on a Hewlett Packard gas chromatograph Model 5880 equipped
81
Chapter 5
with a CP-SIL 88 capillary column (50 m x 0.32 mm) and an FID detector [21,22]. 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 achieved 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 by a gas chromatography combustion isotope
ratio mass spectrometer (Delta S/GC Finnigan MAT, Bremen, Germany) [23]. Separation of
the methyl esters was achieved on a CP-SIL 88 capillary column (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 using the two-tailed Student’s t-test for paired data. For correlating two variables,
linear 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.
Results
Total fat absorption
In Table 5.1 the nutritional data of the control and the calcium supplementation experiments
are shown. After 1 week calcium supplementation, the percentage of total fat absorption
showed a small but significant decrease: 94.9 ± 0.9% compared to 96.6 ± 0.6% in the control
situation (P<0.01). Habitual calcium intake of the subjects was approximately 1000 mg per
day. Upon calcium supplementation, the calcium intake increased 3-fold. Yet, no correlation
was found between total calcium intake and percentage of total fat absorption (r=0.44,
P=0.06).
Table 5.1
Subject
Nutritional data (mean ± SEM) during the control and the calcium supplementation experiment.
Calcium intake
(mg day-1)
Control
951 ± 133
Calcium 2909 ± 103
Total fat absorption
(% intake)
96.6 ± 0.6
94.9 ± 0.9#
Breath 8-h 13CO2
recovery (% dose)
11.4 ± 3.8
10.3 ± 1.1*
[1-13C]palmitic acid
absorption (% dose)
77.4 ± 4.9
82.6 ± 4.3
A symbol indicates a significant difference compared to the control situation: * P<0.05, # P<0.01.
Excretion of 13C-palmitic acid into feces
Table 5.1 shows the percentage absorption of [1-13C]palmitic acid, assessed by fecal 13Cpalmitate concentration. The amount of 13C-palmitic acid excreted into the feces was
calculated for the 72-h period following administration of [1-13C]palmitic acid. In the control
82
The 13C-palmitic acid test in humans on calcium supplementation
experiment and in the calcium experiment the absorption of [1-13C]palmitic acid was similar:
77.4 ± 4.9% and 82.6 ± 4.3% dose, respectively (P=0.39). No correlation was found between
calcium intake and percentage absorption of [1-13C]palmitic acid (r=0.17). A significant
relationship was observed between percentage of total fat absorption and absorption of [113
C]palmitic acid (Figure 5.1; r=0.47, P<0.05), indicating that [1-13C]palmitic acid was
handled by the intestine in a similar fashion as the mass of unlabeled dietary fats.
[1-13C]palmitic acid
absorption (% dose)
100
r=0.47, P<0.05
90
80
70
60
50
0
00
1 85 2
390
4
95 5
6100
% Total fat absorption
13
-1
Figure 5.1 Correlation between total fat absorption and the absorption of [1- C]palmitic acid (10 mg kg
body weight) of 10
-1
healthy adults in the control situation (€) and upon calcium supplementation (~) (2000 mg day ; CaCO3). r=0.47, P<0.05.
Plasma 13C-palmitate concentration
In the control experiment, analysis of the 13C-palmitic acid concentration in plasma samples
showed a slow increase over the time and a maximum of 0.44 ± 0.10% dose L-1 plasma was
obtained after 5 h in the control experiment (Figure 5.2). Upon calcium supplementation the
13
C-palmitate concentrations in plasma were initially similar, yet after 4 h significantly
increased when compared with the controls (P<0.05, Figure 5.2), with a maximum of 0.81 ±
0.21% dose L-1 plasma obtained after 6 h, respectively.
Plasma
13C16:0
(% dose L -1)
1.2
1.0
0.8
*
*
0.6
*
0.4
0.2
0.0
00
11
2 2 3
34
54
6 5 7
68
Time (h)
Figure 5.2 Time courses of
13
C-palmitate concentration in plasma of 10 healthy adults in the control situation (€) and upon
-1
13
-1
calcium supplementation (~) (2000 mg day ; CaCO3) after a single oral dose of [1- C]palmitic acid (10 mg kg body weight) (*
P<0.05).
83
Chapter 5
Breath 13CO2 excretion measurements
The 13C excretion rate in breath in the control experiment increased slowly and reached a
maximum value of 2.6 ± 0.3% 13C dose h-1 between 7 and 8 h after administration of the label
(Figure 5.3A). In most subjects no decay of 13C was observed. The 13C expiration rate during
calcium supplementation rose more slowly, and reached a similar level after 8 h (3.2 ± 0.3%
13
C dose h-1) when compared with the control experiment (Figure 5.3A).
Expiration
14
3.0
2.5
10
13CO
2
2.0
12
1.5
% Cumul
% Dose h -1 13CO2 Expiration
3.5
1.0
0.5
0.0
*
8
#
6
#
*
*
*
*
*
*
4
2
0
0
1
2
3
4
5
6
7
8
0
1
2
3
Time (h)
Figure 5.3 Time courses for the (A)
4
5
6
7
8
Time (h)
13
CO2 excretion rate and (B) cumulative
13
13
CO2 excretion in breath over the 8-h study period
-1
following oral ingestion of [1- C]palmitic acid (10 mg kg body weight) to 10 healthy adults in the control situation (€) and upon
-1
#
calcium supplementation (~) (2000 mg day ; CaCO3) (* P<0.05, P<0.01, ** P<0.001).
The cumulative 13CO2 excretion data are summarized in Figure 5.3B and Table 5.1.
At 5 h after [1-13C]palmitic acid administration, the cumulative 13CO2 excretion was
significantly lower upon calcium supplementation compared to the control situation and this
difference persisted until the end of the experiment (Figure 5.3B). At 8 h after [1-13C]palmitic
acid administration, the cumulative 13CO2 excretion amounted to 11.4 ± 1.2% in the control
experiment, and to 10.3 ± 1.1% upon calcium supplementation.
Plasma 13C16:0 (% dose L -1)
1.5
1.0
0.5
0.0
0
1
2
3
4
5
% Dose h-1 13CO2 expiration
Figure 5.4 Correlation between the
13
13
CO2 excretion rate and plasma
13
C-palmitic acid at all time points following oral ingestion of
-1
[1- C]palmitic acid to 10 healthy adults in the control situation (€) and upon calcium supplementation (~) (2000 mg day ; CaCO3).
r=0.63, P<0.001.
84
The 13C-palmitic acid test in humans on calcium supplementation
In order to compare whether 13CO2 excretion in breath may be extrapolated directly to
absorption efficiency, the relationship between the breath 13CO2 excretion rates and plasma
13
C-palmitic acid concentrations was determined. A significant relationship was observed
between breath 13CO2 excretion rates and plasma 13C-palmitic acid concentrations (Figure 5.4;
r=0.63, P<0.001).
Background 13C-enrichment after an unlabeled test meal
The background enrichment of 13C was examined in breath, plasma and feces in 1 subject by
performing the control experiment and the experiment during the calcium supplementation
without administration of the label. There was no detectable change in the plasma 13Cpalmitate, breath 13CO2 and fecal 13C-palmitate over the 8-h study period in either of the
experimental settings (data not shown).
Discussion
Recently we characterized the [1-13C]palmitic acid absorption test for the detection of fat
malabsorption due to long-term bile diversion in rats [9]. In the present study we investigated
whether this test was sensitive enough to detect mild fat malabsorption in healthy volunteers,
induced by oral supplementation of 2000 mg calcium carbonate per day. It has been
demonstrated that an increased calcium intake leads to modestly increased amounts of fat in
the feces, leading to decreased percentages of total fat absorption [15,16]. Also in our study,
percentage of total fat absorption was slightly but significantly decreased upon calcium
supplementation.
The absorption of dietary fats was significantly correlated with the absorption of [113
C]palmitic acid, assessed by fecal 13C-palmitate concentrations (Figure 5.1), indicating that
the fate of [1-13C]palmitic acid parallels the fate of total mass of dietary fats with respect to
absorption under the experimental circumstances of this study. Correlations between total fat
absorption and absorption of labeled fats have been reported before with outcomes varying
from a strong correlation [9,24] to absence of a correlation [25,26]. It could be that the
presentation of [1-13C]palmitic acid to the absorptive site may not always be the same, as
palmitic acid is normally hydrolyzed from dietary triacylglycerols in the gastrointestinal tract,
indicating that analysis of fecal 13C-palmitate concentrations is not a representative method to
determine dietary fat absorption.
Theoretically, based on the positive correlation between total fat absorption and
absorption of [1-13C]palmitic acid, one would expect that, upon calcium supplementation, not
only total fat absorption would be decreased but also the absorption of [1-13C]palmitic acid,
resulting in reduced amounts of 13C-palmitic acid in blood plasma and decreased amounts of
13
CO2 in breath. Indeed, in breath a small but significantly decreased amount of 13CO2 was
recovered after 8 h upon calcium supplementation when compared with controls. Our results
with respect to breath 13CO2 excretion during the control experiment are rather similar to what
other scientists report [25,27]: peak excretion rate of 13CO2 appears rather late (after
approximately 6 h) and does not exceed 3% dose h-1. Only a few studies have appeared in
which the [1-13C]palmitic acid breath test was studied in patients with disturbed fat absorption
85
Chapter 5
[26,27]. Watkins et al. [27] reported data on the [1-13C]palmitic acid breath test in patients
with known bile salt deficiency, and found a significantly lower 6-h cumulative 13CO2
expiration when compared with healthy controls.
In contrast to the decrease observed in breath 13CO2 excretion upon calcium
supplementation, plasma 13C-palmitic acid concentrations after 4 h were significantly increased
upon calcium supplementation when compared with controls. The apparent contradiction
between plasma 13C-palmitic acid concentrations and breath 13CO2 recovery suggests that
post-absorptive metabolic changes take place. Previously, it has been reported that plasma
triacylglycerol concentrations were increased upon dietary calcium fortification of 1800 mg
day-1 in humans [16]. However, it is not known whether this observation is related to the
results we obtained.
In summary, we show in healthy humans that percentage of total fat absorption can
be manipulated to a minor extent with the use of calcium administration. Calcium
supplementation resulted in a small but significant decrease of percentage of total fat
absorption due to impaired bile solubilization. After oral ingestion of [1-13C]palmitic acid, the
calcium-induced fat malabsorption was associated with a decreased cumulative expiration of
13
CO2 in breath but with increased 13C-palmitic acid concentrations in plasma. Present data
indicate that calcium supplementation does not only affect the overall quantity of fat
absorption, but also leads to alterations in post-absorptive metabolism. Finally, the present
data indicate that the [1-13C]palmitic acid test is not sensitive enough to detect mild fat
malabsorption induced by calcium supplementation in human adults.
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Beylot M. The use of stable isotopes and mass spectrometry in studying lipid metabolism.
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Sardesai VM. The essential fatty acids. Nutr Clin Pract 1992;7:179-186.
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Shiau Y-F. Lipid digestion and absorption. In: Johnson LR, ed. Physiology of the
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Staggers JE, Hernell O, Stafford RJ, Carey MC. Physical-chemical behavior of dietary and
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Hernell O, Staggers JE, Carey MC. Physical-chemical behavior of dietary and biliary lipids
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Minich DM, Kalivianakis M, Havinga R, Kuipers F, Stellaard F, Vonk RJ, Verkade HJ. A
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Carey MC, Small DM, Bliss CM. Lipid digestion and absorption. Ann Rev Physiol
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Gallaher D, Olds Schneeman B. Intestinal interaction of bile acids, phospholipids, dietary
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Lairon D, Lafont H, Vigne J-L, Nalbone G, Léonardi J, Hauton JC. Effects of dietary
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Saunders D, Sillery J, Chapman R. Effect of calcium carbonate and aluminium hydroxide
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Govers MJAP, Termont DSML, Lapré JA, Kleibeuker JH, Vonk RJ, Meer Rv. Calcium in
milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits
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Welberg JWM, Monkelbaan JF, De Vries EGE, Muskiet FAJ, Cats A, Oremus ETHGJ,
Boersma-van Ek W, Van Rijsbergen H, Meer Rv, Mulder NH, Kleibeuker JH. Effects of
supplemental dietary calcium on quantitative and qualitative fecal fat excretion in man.
Ann Nutr Metab 1994;38:185-191.
Denke MA, Fox MM, Schulte MC. Short-term dietary calcium fortification increases fecal
saturated fat content and reduces serum lipids in men. J Nutr 1996;123:1047-1053.
Potter SM, Kies CV, Rojhani A. Protein and fat utilization by humans as affected by
calcium phosphate, calcium carbonate, and manganese gluconate supplements. Nutrition
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Lepage G, Roy CC. Direct transesterification of all classes of lipids in a one-step reaction.
J Lipid Res 1986;27:114-120.
Van de Kamer JH, Ten Bokkel Huinink H, Weyers HA. Rapid method for the
determination of fat in feces. J Biol Chem 1949;177:347-355.
Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 1959;37:911-917.
Eder K. Gas chromatography analysis of fatty acid methyl esters. J Chromatogr B
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Gutnikov G. Fatty acid profiles of lipid samples. J Chromatogr B 1995;671:71-89.
Guo Z, Nielsen S, Burguera B, Jensen MD. Free fatty acid turnover measured using
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Pedersen NT, Halgreen H. Simultaneous assessment of fat maldigestion and fat
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Murphy JL, Jones A, Brookes S, Wootton SA. The gastrointestinal handling and
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Murphy JL, Jones AE, Stolinski M, Wootton SA. Gastrointestinal handling of [113
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Watkins JB, Klein PD, Schoeller DA, Kirschner BS, Park R, Perman JA. Diagnosis and
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87
CHAPTER 6
Fat malabsorption in cystic fibrosis patients on
enzyme replacement therapy is due to impaired
intestinal uptake of long-chain fatty acids
M. Kalivianakis, D.M. Minich, C.M.A. Bijleveld, W.M.C. van Aalderen,
F. Stellaard, M. Laseur, R.J. Vonk, H.J. Verkade
Am J Clin Nutr (1998) in press
Chapter 6
CHAPTER 6
Fat malabsorption in cystic fibrosis patients on
enzyme replacement therapy is due to impaired
intestinal uptake of long-chain fatty acids
Abstract
Background & Aim: Pancreatic enzyme replacement therapy frequently fails to correct
intestinal fat malabsorption completely in cystic fibrosis (CF) patients. The reason behind
therapy failure in these patients is unknown. We investigated whether fat malabsorption in CF
patients treated with pancreatic enzymes is caused by insufficient lipolysis of triacylglycerols
or by defective intestinal uptake of long-chain fatty acids. Methods: In 10 CF patients
receiving their habitual pancreatic enzymes, lipolysis was determined by analysis of breath
13
CO2 recovery after oral ingestion of 1,3-distearoyl, 2[1-13C]octanoyl glycerol (13C-MTG).
Intestinal uptake of long-chain fatty acids was determined by analysis of plasma 13C-linoleic
acid concentrations after oral ingestion of 13C-linoleic acid (13C-LA). For 3 days, dietary intake
was recorded and feces was collected. Results: Fecal fat excretion ranged from 5.1 to 27.8 g
day-1 (mean ± SD: 11.1 ± 7.0 g day-1) and fat absorption ranged from 79 to 93% (89 ± 5%).
After ingestion of 13C-MTG no relationship was observed between breath 13CO2 recovery and
dietary fat absorption (r=0.04). In contrast, a strong relationship was observed between 8-h
plasma 13C-LA concentrations and dietary fat absorption (r=0.88, P<0.001). Conclusion: Our
results suggest that continuing fat malabsorption in CF patients on enzyme replacement
therapy is not likely due to insufficient lipolytic enzyme activity, but rather due to either
incomplete intraluminal solubilization and/or reduced mucosal uptake of long-chain fatty acids.
90
Impaired uptake of fats in CF patients
Introduction
In humans, triacylglycerols composed of long-chain fatty acids constitute 92 to 96% of dietary
fats [1]. Absorption of these fats comprises two main processes. Firstly, lipolysis, by lipolytic
enzymes originating predominantly from the pancreas, leads to hydrolysis of triacylglycerols
into fatty acids and 2-monoacylglycerols. And secondly, intestinal uptake involves the
formation of mixed micelles composed of bile components and lipolytic products, followed by
the desintegration of the mixed micelles in the unstirred water layer, and the translocation of
the lipolytic products across the intestinal epithelium [1-4].
Most CF patients have a considerable malabsorption of dietary fats due to pancreatic
insufficiency leading to impaired lipolysis [5,6]. The symptoms of pancreatic insufficiency,
such as steatorrhea and poor growth, can be alleviated by oral supplementation of pancreatic
enzymes. However, despite recent improvements in the pharmacokinetics of the supplements,
many patients continue to experience a certain degree of steatorrhea [7-9], with fat absorption
reaching 80 to 90% of their dietary fat intake. It has not been elucidated if the remaining fat
malabsorption is due to an insufficient dosage of pancreatic enzyme replacement therapy. This
possibility is not unlikely because a decreased pancreatic bicarbonate secretion may negatively
affect enzyme activity by sustaining a low pH in the duodenum [10,11]. At a low duodenal
pH, the release of the enzymes from the (micro)capsules is inhibited and the denaturation of
the enzymes is stimulated [11,12]. However, it has been demonstrated that increasing the
pancreatic enzyme dosages does not completely correct fat malabsorption [13]. In addition,
attempts to increase lipolysis by high-strength pancreatic enzyme supplements has led to the
reported association with fibrosing colonopathy [14-16].
An alternative explanation for the continuing fat malabsorption in CF patients on
pancreatic enzyme replacement therapy may involve inefficient intestinal uptake of fatty acids
[7,17]. Impaired uptake in CF patients can be due to an altered bile composition, decreased
bile salt secretion by the liver, bile salt precipitation, a decreased bile salt pool size, and/or bile
salt inactivation at low intestinal pH [9,17-20]. Furthermore, small bowel mucosal dysfunction
or alterations in the mucus layer contribute to inefficient intestinal uptake of long-chain fatty
acids in CF patients [5,21].
The gold standard for monitoring enzyme replacement therapy is the fat balance. A
drawback of the fat balance is that it does not provide insight into the pathophysiology of fat
malabsorption. Insight into the adequacy of these separate processes (lipolysis, intestinal
uptake) would enable treatment in individual patients by modulating diet therapy, pancreatic
enzyme replacement therapy and supplementation of antacids and bile salts. So far, it has not
been possible to determine whether fat malabsorption in CF patients is due to impaired
lipolysis or due to impaired uptake of long-chain fatty acids. Therapeutic improvements of fat
absorption may be of benefit for CF patients, as a positive correlation has been observed
between a good nutritional status and long-term survival or well-being of CF patients [22].
The aim of the present study was to determine whether continued fat malabsorption
encountered in pediatric CF patients on their habitual pancreatic enzyme replacement therapy
results from either insufficient lipolysis or from defective intestinal uptake of long-chain fatty
acids in the lumen. We choose to measure lipolysis and uptake by two indepedent tests in CF
91
Chapter 6
patients in vivo. Previously, a test to determine lipase activity was described and validated in
CF patients, based on oral ingestion of a 13C-labeled mixed triglyceride (13C-MTG, 1,3distearoyl, 2[1-13C]octanoyl glycerol) and excretion of 13C in breath [23-26]. Inadequate
intestinal uptake can be measured by oral ingestion of long-chain fatty acids, e.g. 13C-labeled
linoleic acid (13C-LA) [27,28]. The concentration of 13C-LA in plasma and the expiration of
13
CO2 could then serve as parameters to quantify uptake of 13C-LA.
Subjects and Methods
Patient characteristics
The study protocol was approved by the Medical Ethics Committee of the University Hospital
Groningen, and included informed consent obtained from the parents and the children.
Patients. The study group included 10 pediatric CF patients, three male and seven female,
ranging in age from 7 to 18 y. The diagnosis of CF had been established by the sweat test and
a DNA genotype analysis [29]. The ∆F508/∆F508 genotype was present in six patients, and
the ∆F508/other in four (subjects 1, 4, 8, 9; Table 6.1). All patients were pancreatic
insufficient and therefore received enteric coated pancreatic enzymes. None of the patients
received antacids.
Table 6.1
Comparison of energy intake, ingested lipase enzymes, and fasting plasma concentrations of bile salts in individual CF
patients and mean ± SD.
Patient Age Weight Energy Carbohydr.
Fats
Proteins
Lipase Plasma bile
(y) (kg) (% RDA) (% energy) (% energy) (% energy) (IU/g fat) (µmol L -1)
1 F 18
55
66
52
33
15
560
13.8
2 F 18
58
104
52
31
17
710
13.5
3 M 16
53
115
48
39
13
1820
20.6
4 M 15
56
113
48
38
15
680
12.8
5 F
9
34
91
52
35
13
440
23.4
6 F
9
26
102
57
30
13
1520
13.5
7 M 8
23
110
50
37
13
830
11.8
8 F
7
27
111
52
35
13
590
18.2
9 F
7
24
92
56
32
12
860
11.6
10 F
7
23
121
54
35
11
460
30.3
103 ± 16
52 ± 3
35 ± 3
14 ± 2
850 ± 460 16.6 ± 5.9
F, female; M, male. Normal range fasting plasma bile salts, 1-10 µmol L -1.
Anthropometry. Anthropometric evaluation consisted of weight, height, midarm
circumference, and skinfold thickness measurements at 4 sites (biceps, triceps, subscapula, and
suprailiac), done by one pediatrician. The Z-scores of all these anthropometric parameters
were calculated based on the reference data for Dutch children described by Gerver and De
Bruin [30]. The Z-score is defined as X- x /S where X is the patient’s measurement, x is the
92
Impaired uptake of fats in CF patients
median value for age and sex, and S is the standard deviation of x . A negative value indicates
a value under the median reference value.
Pulmonary function. Pulmonary function was assessed by standard spirometric
techniques and was characterized by the parameters forced vital capacity, and forced
expiratory volume in one second. For each patient, results were expressed as percentage of
predicted (control) values for sex and height [31].
Liver function tests. Liver function had been screened during a standard routine
control at the time of the study using serum enzyme activities: g-glutamyl transpeptidase,
aspartate transaminase, and alanine transaminase.
Diet evaluation. Intake of nutrients was calculated from 3-day consecutive food
diaries by a clinical dietitian using The Netherlands Nutrients Table “NEVO” 1993. Intakes
were expressed as the recommended dietary allowance (RDA) for weight, age and sex (Table
6.1).
13
C-labeled substrates
The mixed triglyceride (1,3-distearoyl, 2[1-13C]octanoyl glycerol; S*OS) was purchased from
Euriso-Top (Saint Aubin Cedex, France) and was 99% 13C-enriched. In the original literature,
the breath test performed with the use of this molecule has been named the mixed-triglyceride
breath test or the 13C-MTG breath test [23,25]. For reasons of consistency, we adhered to this
nomenclature. Uniformly labeled 13C-linoleic acid (13C-LA), obtained from Campro Scientific
B.V. (Veenendaal, The Netherlands), had an enrichment exceeding 97%. 13C-LA was included
into a gelatin capsule coated with an acid-resistant layer consisting of 4.8% cellulose acetate
hydrogen phthalate in acetone.
Study protocol
The subjects were instructed to avoid consumption of naturally 13C-enriched foods (e.g. corn
or corn products, pineapple, cane sugar) for at least two days prior to the study. The 13C-LA
test and the 13C-MTG test were performed on two subsequent days. On day 1, after an
overnight fast, the patients received a capsule with 13C-LA (1 mg kg-1 BW), together with
their habitual breakfast (bread, butter, ham, cheese, etc.) and pancreatic enzymes. A baseline
blood sample (EDTA) was collected before consumption of breakfast, every 2 h for 8 h, and at
24 h. Immediately after sampling, plasma was isolated and stored frozen (-20ºC) until further
analysis. Breath samples were collected in duplicate at baseline and every 30 min for 6 h. On
day 2, the patients received 13C-MTG (4 mg kg-1 BW) mixed with their habitual breakfast and
pancreatic enzymes. Breath samples were collected in duplicate at baseline and every 30 min
for 6 h. The fecal fat balance and both breath tests were performed in the same 3-day period.
On the day before the 13C-LA test, a feces sample was collected for baseline 13Cmeasurements. After consumption of the breakfast on the first day, all feces passed was
collected for three days (72 h) to determine the presence of fat malabsorption and the amount
of 13C-LA excretion into the feces. Collected feces was stored at -20°C. During this period,
intake of nutrients was determined from food diaries also. During the first six hours of both
tests, no additional food or liquids were permitted except for non-caloric drinks such as water
93
Chapter 6
and tea (without milk and sugar). After 6 hours, patients were allowed to have their habitual
lunch, including pancreatic enzymes.
Analytical techniques
Breath sample analysis. End expiratory breath was collected via a straw into a 10 ml tube
(Exetainers; Labco Limited, High Wycombe, United Kingdom), from which aliquots were
taken to determine 13C-enrichment by means of continuous flow isotope ratio mass
spectrometry (Finnigan Breath MAT, Finnigan MAT GmbH, Bremen, Germany), conform
previous experiments [24]. The 13C-abundance of breath CO2 was expressed as the difference
per mil from the reference standard Pee Dee Belemnite limestone (δ13CPDB, ‰).
Mean values of whole body CO2 excretion were measured by indirect calorimetry
(Oxycon, model ox-4, Dräger, Breda, The Netherlands) at 2 separate periods of 5 minutes
during both test days. This sampling method was compared to sampling every 30 min (results
not shown). The results indicated that, under the test conditions chosen, the mean values of
the CO2 production obtained from 2 randomly chosen periods were within the 95% confidence
interval of the mean values obtained when sampling occurred every 30 min.
Plasma fats. Plasma fats were extracted, hydrolyzed and methylated according to
Lepage and Roy [32]. Resulting fatty acid methyl esters were analyzed both by gas
chromatography and by gas chromatography combustion isotope ratio mass spectrometry.
Quantification of the resulting fatty acid methyl esters was performed with the use of
heptadecanoic acid (C17:0) as an internal standard.
Fecal fats. After thawing, feces was weighed and homogenized. Fecal fat was
determined according to the method of Van de Kamer et al. [33] and expressed as g fat day-1.
The percentage of total fat absorption was calculated from the daily dietary fat intake and the
daily fecal fat output and expressed as a percentage of the daily fat intake.
Fat intake (g day -1 ) − Fecal fat output (g day -1 )
Percentage of total fat absorption =
× 100%
Fat intake (g day -1 )
Aliquots of freeze-dried feces were extracted according to the method of Bligh and Dyer [34],
and subsequently hydrolyzed and methylated [32]. Resulting fatty acid methyl esters were
analyzed by both gas chromatography and gas chromatography combustion isotope ratio mass
spectrometry.
Plasma and fecal bile salts. Fasting and postprandial plasma bile salts up to 8 h were
determined by an enzymatic fluorimetric assay [35]. Results were expressed as µmol L -1
plasma. Fecal bile salts were extracted from an aliquot of dried homogenate of a 24-h feces
fraction [36] and fluorimetrically measured [35].
Gas liquid chromatography. Fatty acid methyl esters were separated and quantified
by gas liquid chromatography on a Hewlett Packard gas chromatograph Model 5880 equipped
with a CP-SIL 88 capillary column (Chrompack; 50 m x 0.32 mm) and an FID detector
[37,38]. 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). Adequate separation of linoleic acid could be
94
Impaired uptake of fats in CF patients
achieved in this way. 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) [39]. 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). Adequate separation of linoleic acid
could be achieved in this way.
Statistics
The experimental data are reported as means ± SD. Corresponding to the literature [40-42],
relationships between the percentage of total fat absorption and either plasma 13C-LA
concentrations or breath 13CO2 expiration were considered exponential. All other correlations
were assumed to be linear. Correlations between variables were calculated with the least
square method and are expressed as Pearson’s coefficient of variation r. Differences between
means were considered statistically significant at the level of P<0.05.
Table 6.2
Results of the fecal bile salt concentrations and fecal fat balance in 10 individual CF patients.
Patient
1
2
3
4
5
6
7
8
9
10
Mean ± SD
Fecal bile salts
(mmol/kg wet weight)
30.2
10.6
0.7
8.7
22.1
20.5
5.3
6.8
16.1
16.7
13.8 ± 9.0
Fat intake
(g day-1)
54
85
124
133
92
66
85
93
64
88
84 ± 22
Fecal fat
(g day-1)
4.9
7.0
14.8
27.8
9.6
5.1
6.1
16.7
9.2
7.1
11.1 ± 7.0
Total fat
absorption (%)
91
92
88
79
90
92
93
82
86
92
89 ± 5
Normal range fecal bile salt: 0.1-1 mmol kg-1 fecal wet weight.
Results
Patient characteristics
Z-scores for all anthropometric parameters in CF patients were low to normal. For all
parameters, the 95% confidence interval does include the reference 50th centile line (Z-score
0), indicating that there is no significant difference between our study group and the healthy
reference population. Most patients had some degree of lung disease. Subjects 1 and 6-10 had
95
Chapter 6
normal liver biochemistry. Previously, subject 3 was diagnosed as having liver cirrhosis with
portal hypertension. This patient receives ursodeoxycholic acid (750 mg day-1) and the
condition of this patient has been stable for the past few years. The bile salt concentration in
plasma of this subject is in the same range as that of the other patients (Table 6.1). Analysis of
3-day dietary food records is shown in Table 6.1. Energy intake of 7 patients exceeded the
recommended dietary allowance. In all patients approximately 50% of the energy was derived
from carbohydrates, 35% from fat, and 15% from protein. Patients took pancreatic enzyme
supplements in a dosage of approximately 440 - 1820 IU lipase per gram fat ingested (Table
6.1).
Table 6.3
13
13
Results of the C-LA test, and C-MTG test in 10 individual CF patients.
13
Patient
1
2
3
4
5
6
7
8
9
10
Mean ± SD
Breath
6-h cum 13CO2
(% dose)
11.0
1.6
2.2
1.7
0.2
3.6
1.1
3.1
2.4
0.5
2.7 ± 3.1
C-LA test
Plasma
13
C-LA at 8 h
(% dose L-1)
1.5
1.9
0.9
0.6
1.3
2.0
1.3
0.5
0.5
1.2
1.2 ± 0.5
13
Feces
C-LA
(% dose)
0.6
0.2
1.8
0.6
0.0
0.7
0.2
1.3
0.3
0.2
0.6 ± 0.6
13
C-MTG test
Breath
Breath 6-h cum
13
CO2 (% dose)
2.4
9.7
15.1
5.8
30.8
11.3
40.2
28.9
11.5
8.7
16.4 ± 12.5
LA, linoleic acid; MTG, mixed triglyceride. Normal range fecal bile salt: 0.1-1 mmol kg-1 fecal wet weight.
Fat balance
In the studied CF patients, dietary intake of fat over the 3-day period ranged from 54 to 130 g
day-1, and the excretion of fat in feces ranged from 4.9 to 27.8 g day-1 (Table 6.2). The
percentage of total fat absorption ranged from 79 to 93% (Table 6.2). Under physiological
conditions, healthy individuals excrete approximately 4-6 g day-1 of fat via the feces [43],
which generally means that over 96% of the dietary fats entering the intestinal lumen is
absorbed [43]. These observations were confirmed by experiments performed in our own
laboratory with dietary records and feces of healthy human adults (n=13, fecal fat excretion:
3.0 ± 0.9 g day-1, total fat absorption: 97 ± 2%, data not shown). Despite standard pancreatic
enzyme replacement therapy, fecal fat excretion in 8 out of 10 patients was higher than 6 g fat
per day, and the percentage of total fat absorption was below 96% in all patients studied.
According to the prevailing reference values [43], all patients but 2 have fat malabsorption.
In studies in infants between 0 and 6 months, Fomon et al. [44] found that fecal fat
excretion per kg body weight correlated with fat intake per kg body weight. In our study we
observed a similar curvilinear correlation (r=0.71, P<0.05) despite a considerably lower intake
96
Impaired uptake of fats in CF patients
of fat per kg BW compared to infants [44]. However, when we compared fat intake per kg
body weight with percentage of total fat absorption, no correlation was observed (r=-0.06),
indicating that fat malabsorption in our study was not due to high fat intake. In addition, no
correlation was observed between the percentage of total fat absorption and the amount of
pancreatic enzymes ingested (r=0.12).
13
C-MTG test
The baseline 13C-abundance in breath prior to consumption of the 13C-MTG label was -23.2 ±
2.6‰ (range -25.5 to -17.1‰). After ingestion of the 13C-MTG label, different time-course
patterns were observed for the excretion of 13C-label in breath over the 6-h study period
(Figure 6.1A). When expressed as a proportion of administered 13C, the excretion rate reached
a mean maximum value of 4.9 ± 3.1% per hour between 3 and 6 h after administration of the
label (range 0.7 to 10.7%). Over the 6-h study period the cumulative excretion of 13C in breath
was 16.4 ± 12.5% of that administered, ranging between 2.4 and 40.2% (Figure 6.1B, Table
6.3). If defective lipolysis would be responsible for the continuing fat malabsorption in CF
patients, then a low percentage of fat absorption would be expected to correlate with low
expiration of 13CO2 after 13C-MTG ingestion. However, no significant relationship was
observed between 6-h cumulative 13CO2 expiration and either daily fecal fat excretion (r=0.02) or the percentage of total fat absorption (r=0.04).
12
Excretion
A
B
40
30
13CO
2
8
6
% Cumul
% 13C Dose h -1
10
4
2
20
10
0
0
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Time (h)
Time (h)
13
Figure 6.1 Time courses for the excretion of C in breath over the 6-h study period following oral ingestion of
13
C-MTG (4 mg per
kg body weight) at time 0 in 10 CF patients. Each symbol represents a patient. Figure (A) represents the excretion rate, whereas
13
figure (B) represents the cumulative CO2 excretion.
13
C-LA test
The baseline 13C-LA abundance in plasma prior to consumption of the 13C-LA label was -29.1
± 2.2‰ (range -32.6 to -25.5‰). 13C-LA concentration in plasma samples, expressed as
percentage of the dose per liter plasma, increased steeply after approximately 6 h (Figure 6.2).
Peak values of 13C-LA concentrations in plasma after administration occurred between 8 and
24 h. At 24 h after ingestion of the label, the enrichment of 13C-LA in plasma had not yet
returned to the level of baseline 13C-abundance. Plasma 8-h 13C-LA concentrations varied from
0.5 to 2.0% dose L-1 plasma (Table 6.3).
97
Chapter 6
13C-LA
Conc. (% Dose L -1)
2.5
2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
24
Time (h)
13
Figure 6.2 Time courses of
C-LA appearance in plasma of 10 CF patients after a single oral dose of
13
-1
C-LA (2 mg kg body
weight) at time 0. Each symbol represents a patient.
If defective intestinal uptake of long-chain fatty acids would be responsible for the
continuing fat malabsorption in CF patients, then a low percentage of fat absorption would be
expected to correlate with low concentrations of 13C-LA in plasma after 13C-LA ingestion.
Figure 6.3 shows the relationship between the 8-h plasma 13C-LA concentrations and either
fecal fat excretion or the percentage of total fat absorption. A strong, negative relationship
was observed between fecal fat excretion and 8-h plasma 13C-LA concentrations (Figure 6.3A;
r=-0.75, P<0.01) and, correspondingly, a strong, positive relationship was observed between
the percentage of total fat absorption and 8-h plasma 13C-LA concentrations (Figure 6.3B;
r=0.88, P<0.001).
2.5
A
Conc. (% Dose L -1)
r=-0.75, P<0.01
2.0
1.5
1.0
13C-LA
13C-LA
Conc. (% Dose L -1)
2.5
0.5
0.0
0
5
10
15
20
Fecal Fat Excretion (g day-1)
25
30
B
r=0.88, P<0.001
2.0
1.5
1.0
0.5
0.0
0
80
Figure 6.3 Relationship between the results of the 72-h fecal fat balance and the 8-h plasma
oral dose of
13
85
90
95
Total Fat Absorption (% intake)
13
C-LA concentration after a single
-1
C-LA (1 mg kg body weight) at time 0 in 10 CF patients. Figure (A) represents the relationship between daily fecal
fat excretion and 8-h plasma
13
C-LA concentration and figure (B) the relationship between the percentage of total fat absorption
13
and 8-h plasma C-LA concentration.
Since a breath test would be more convenient to the patient than a test requiring
blood sampling, we investigated whether similar information on intestinal uptake of long-chain
fatty acids could become available using breath 13CO2 analysis after 13C-LA ingestion. The
baseline 13C-abundance in breath prior to consuming the 13C-LA label was -24.3 ± 2.3‰,
(range -27.2 to -20.9‰). In most subjects, the 13C excretion rate in breath was low during the
98
Impaired uptake of fats in CF patients
first hours, then increased rapidly and reached a possible maximum value at 6 h after
administration of the label (Figure 6.4A). In most subjects no decay was observed during the
time course of the study. This time course pattern was very similar to the pattern obtained for
13
C-LA concentrations in plasma, except for subject 1, whose 13C excretion rate in breath
already increased after 90 min. The 6-h cumulative 13CO2 expiration (Table 6.3) amounted to
2.7 ± 3.1% dose. In Figure 6.4B the 6-h cumulative 13CO2 expiration for all patients is plotted.
In contrast to plasma values, no significant relationship between 6-h cumulative 13CO2
expiration and either fecal fat excretion (r=0.00) or the percentage of total fat absorption (r=0.13) was observed. In addition, there was no correlation between plasma 13C-LA
concentrations and cumulative breath 13CO2 expiration (r=0.32), indicating that the multitude
of metabolic processes limits the utility of breath samples to measure uptake of long-chain
fatty acids [45]. The results indicate that for the measuring intestinal uptake of long-chain fatty
acids, plasma sampling cannot be easily replaced by breath sampling.
Finally, we investigated the excretion of 13C-LA in feces. The apparent absorption of
13
C-label was determined from the difference between the amount of 13C-LA administered and
that excreted in feces. 13C-LA excretion in feces over the 3-day period was very low and
varied between 0.0 and 1.8% of the dose administered (Table 6.3). No metabolites of 13C-LA
were observed in the feces. There was no significant correlation observed between the
excretion of 13C-LA and of total fat in feces (r=0.22, P=0.54).
3.5
12
% Cumul 13CO2 Excretion
A
% 13C Dose h -1
3.0
2.5
2.0
1.5
1.0
0.5
B
10
8
6
4
2
0
0.0
0
1
2
3
4
5
Time (h)
Figure 6.4 Time courses for the excretion of
6
0
1
2
3
4
5
6
Time (h)
13
C in breath over the 6-h study period following oral ingestion of
13
-1
C-LA (1 mg kg
body weight) at time 0 in 10 CF patients. Figure (A) represents the excretion rate, whereas figure (B) represents the cumulative
13
CO2 excretion.
Total bile salt concentrations in plasma and feces
Total bile salt concentrations were determined in plasma and feces. Fasting plasma total bile
salt concentrations in CF patients were high when compared with normal healthy control
values and ranged from 11.6 to 30.3 µmol L -1 (mean 17.2 µmol L -1) (Table 6.1). Following a
meal there was no significant change in total plasma bile salts (data not shown). Fecal total bile
salt concentrations in most CF patients were elevated (range 0.7-30.2; mean 13.8 mmol per kg
fecal wet weight) when compared with healthy control values, indicating that they had bile salt
malabsorption (Table 6.2). Bile salt malabsorption could result in a decreased amount of bile
salts available for the formation of mixed micelles, leading to fat malabsorption. However, no
99
Chapter 6
significant correlation was found between percentage of dietary fat absorption and fecal bile
salt concentrations (r=0.26).
Discussion
In CF patients, pancreatic enzyme replacement therapy frequently does not correct disordered
fat absorption to values obtained in controls. Our results of the 3-day fat balance confirm the
presence of mild to moderate fat malabsorption (percentage of total fat absorption: 79-93%) in
a group of pediatric CF patients on enzyme replacement therapy despite good clinical
conditions. The aim of the present study was to elucidate whether fat malabsorption in CF
patients receiving habitual pancreatic enzyme replacement therapy is due to deficient lipolysis
of triacylglycerols or due to impaired intestinal uptake of fatty acids.
We applied two fat substrates with different physical and chemical properties, i.e.
13
C-MTG and 13C-LA. The principle of the 13C-MTG breath test is based on lipolysisdependent 13CO2 excretion via the breath. Efficient absorption of the 13C label from the mixed
triglyceride is limited primarily by lipolysis [23], and the 13C-MTG test therefore distinguishes
pancreatic insufficiency from deficient intestinal uptake of long-chain fatty acids. After 13CMTG ingestion, no relationship was observed between recovery of 13CO2 in breath and
percentage of total fat absorption, indicating that fat malabsorption in CF patients on their
habitual enzyme replacement therapy is probably not related to defective lipolysis. The
recovery of expired 13CO2 obtained in the present study was similar to those obtained in other
studies, indicating sufficient supplementation of pancreatic enzymes to the CF patients in this
study. In healthy adults the 6-h cumulative percentage of 13C expired via the breath after
ingestion of 13C-MTG varied between 23 and 52% of the dose in one study [23] and between
3 and 48% in another study [24]. The recovery of expired 13CO2 in CF patients receiving
regular amounts of pancreatic enzymes varied between 0 and 45% [23,25]. In neither of these
studies total fat absorption was related to the percentage of 13C recovered in the breath.
Efficient absorption of 13C-LA, a long-chain unesterified fatty acid, differs
predominantly from 13C-MTG in its dependence on adequate intestinal uptake [27]. Minich et
al. [28] showed in a rat model for fat malabsorption (permanently interrupted enterohepatic
circulation) that measuring plasma 13C-LA concentrations is a valuable method to assess the
intestinal uptake of long-chain fatty acids and correlates with fat absorption. The 13C-LA test
therefore distinguishes deficient intestinal uptake of long-chain fatty acids from pancreatic
insufficiency [28]. After ingestion of 13C-LA, a strong relationship was observed between 8-h
plasma 13C-LA concentrations and total fat absorption, indicating that the observed fat
malabsorption in CF patients on their habitual enzyme replacement therapy is due to defective
intestinal uptake of long-chain fatty acids.
Impaired intestinal uptake of long-chain fatty acids may result from several processes.
In the absence of adequate bicarbonate secretion, gastric acid entering the duodenum may
lower intestinal pH until well into the jejunum [11]. Bile salts are readily precipitated in an acid
milieu [17], and duodenal bile salt concentration may fall below the critical micellar
concentration, thereby exacerbating fat malabsorption. Precipitated bile salts also appear to be
100
Impaired uptake of fats in CF patients
lost from the enterohepatic circulation in greater quantities, thus reducing the total bile salt
pool and decreasing the fraction of bile salts conjugated with glycine [20]. Intracellullar events
may also contribute to impaired uptake of long-chain fatty acids in CF patients, e.g. due to
absent fatty acid binding proteins or impaired chylomicron assembly and secretion [46].
Viscous, thick intestinal mucus, with altered physical properties, may have a deleterious effect
on the thickness of the intestinal unstirred water layer, limiting translocation of long-chain
fatty acids over the intestinal epithelium [5,21]. Our data on increased fecal bile salt losses are
in agreement with several other studies [47-49] and could be in agreement with a diminished
bile salt pool in CF patients. Watkins et al. [18] showed that bile acid pool size was nearly
doubled upon treatment with pancreatic enzymes in a group of CF patients with normal fecal
bile salt losses. Although the present data suggest that the problem is related to insufficient
long-chain fatty acid uptake, they do not allow a clear identification of the individual process
responsible for impaired uptake.
The 13C-LA bolus was administered in an acid-resistant coated capsule, preventing
the capsule from being opened at a low pH environment (gastric or intestinal). In patients with
a low intestinal pH, e.g. due to inadequate bicarbonate secretion [10,11], the bioavailability of
13
C-LA was hypothesized to be impaired, resulting in a decreased amount of 13C-LA
incorporated into plasma linoleic acid. Since low intestinal pH affects uptake of long-chain
fatty acids, we reasoned that the acid-resistant capsule probably enhances the effect of the 13CLA test in correctly diagnosing solubilization disorders. The release of the substrate may be
delayed in some patients, which can explain the differences in timing for the onset of the
individual 13C-LA curves. In addition, delayed time courses for the onset of 13CO2 in breath
have been observed before and may be explained by, e.g., delayed gastric emptying [24,50,51].
The study was designed such that the patients served as their own controls. Thus, in
each individual patient we calculated the percentage of total fat absorption and related these
results to the measurements of the 13C-MTG breath test and the 13C-LA test. We reasoned that
these controls would be the most appropriate, given the fact that neither the optimal positive
control group (pancreatic sufficient CF patients with known impaired intestinal uptake) nor the
optimal negative control group (pancreatic sufficient CF patients without intestinal uptake
disorder) exists or is available. The present approach allowed us to relate the results of total
fat absorption to the results of lipolysis and intestinal uptake in the individual patient.
In conclusion, fat balance data indicate that, despite enzyme replacement therapy,
pediatric CF patients have increased fecal fat excretion and, correspondingly, decreased
percentage of fat absorption. The results of the 13C-MTG test and 13C-LA test indicate that
continuing fat malabsorption is not likely due to insufficient enzyme replacement therapy, but
rather due to either incomplete intraluminal solubilization and/or reduced mucosal uptake of
long-chain fatty acids. Indirect indications exist that an increased bile salt loss leading to a
diminished bile salt pool may contribute to this problem. Therapeutic attempts to normalize fat
absorption in pediatric CF patients need to include a strategy to improve intestinal uptake of
long-chain fatty acids.
101
Chapter 6
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104
CHAPTER 7
Increased fecal bile salt excretion is independent of
the presence of dietary fat malabsorption in two
mouse models for cystic fibrosis
M. Kalivianakis, I. Bronsveld, H.R. de Jonge, M. Sinaasappel,
R. Havinga, F. Kuipers, B.J. Scholte, H.J. Verkade
Chapter 7
CHAPTER 7
Increased fecal bile salt excretion is independent of
the presence of dietary fat malabsorption in two
mouse models for cystic fibrosis
Abstract
Background & Aim: Recent studies from our laboratory suggested that fat malabsorption in
cystic fibrosis (CF) patients on pancreatic enzyme replacement therapy is partially due to
impaired intestinal uptake of long-chain fatty acids [1], which may be due to bile-related
processes. To obtain more insight into the effects of CF on fat absorption and bile formation,
we studied two mouse models for CF: mice homozygous for the ∆F508 mutation in the cftr
gene (∆F508/∆F508), and mice in which the cftr gene is disrupted (cftr -/-). Methods: Fat
absorption was studied by means of a 3-day fat balance, after feeding a standard (14 en% fat)
or a high-fat (35 en% fat) diet for 2 weeks. Biliary bile salt secretion was determined during
80 min after cannulation of the gallbladder. Fecal bile salts were determined for 3 days.
Results: In ∆F508/∆F508 mice, dietary fat absorption was not significantly different from
controls, and above 94% in all groups. However, dietary fat absorption in cftr -/- mice was
significantly decreased compared to controls: standard diet: 82.8 ± 3.0% (mean ± SEM) and
93.9 ± 1.3% (P<0.01); high-fat diet: 88.8 ± 1.6% and 95.0 ± 1.4% (P<0.01), respectively.
Biliary bile salt secretion rates were similar for the CF mouse models and their respective
controls on either diet. The contribution of cholic acid to the biliary bile salt pool was slightly
increased in both CF mice at the expense of deoxycholic acid. Primary bile salts were slightly
increased, whereas secondary bile salts were slightly decreased in CF mice. Fecal bile salt
excretion was increased in ∆F508/∆F508 and in cftr -/- mice when compared with their
respective controls (10 versus 5 µmol g -1 feces, respectively, P<0.01). No significant
correlation was observed between fecal excretion of bile salts and of fats. Conclusion: Cftr -/mice, but not ∆F508/∆F508 mice, have an impaired dietary fat absorption, which does not
result from alterations in bile production. In both CF mouse models, fecal bile salt excretion
was increased, but this was not related to increased fecal fat excretion. Bile composition data
indicate that the increased fecal loss of bile salts is compensated for by an increased bile salt
neosynthesis.
106
Fat (mal)absorption in CF mice
Introduction
Cystic fibrosis (CF), the most common recessive disorder in Caucasian populations, is caused
by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR).
The CFTR gene encodes for a phosphorylation-regulated Cl- channel and is expressed in the
apical membrane of various epithelial cells [2-4]. Malfunction of this chloride channel in CF
patients is associated with obstruction and inflammation of airways, pancreatic ducts, intestine
and bile ducts, frequently resulting in intestinal fat malabsorption [5,6]. The most common
mutation in the CFTR gene in Caucasian populations is a deletion of a phenylalanine residue at
amino acid position 508 of the protein (∆F508), which is found in 90% of the CF patients in
Northern Europe [3,4]. The ∆F508 mutation disrupts the biosynthetic processing of CFTR to
its mature glycosylated form [7], so that the protein is retained in the endoplasmic reticulum
and subsequently degraded [8]. The protein does not reach the apical plasma membrane, and
as a result, affected epithelia lack CFTR in the apical membrane and are deficient in cAMPstimulated Cl- permeability [8].
In an attempt to further elucidate the pathophysiology of CF, mouse models were
developed [9-11]. The initial excitement generated by the emergence of these mouse models
was somewhat tempered by the finding that none of the models showed spontaneous airway
disease, which is primarily responsible for most of the morbidity and mortality in the human
CF population. However, the various CF mouse models are remarkably similar to their human
counterparts with respect to intestinal pathophysiology [11-14]. Most importantly, the
intestinal tract of the CF mouse models demonstrates the absence or decrease of cAMPmediated chloride transport which often results in intestinal obstructions, a hallmark of CF.
Most CF patients display a considerable malabsorption of dietary fats due to
pancreatic insufficiency resulting in impaired lipolysis [5,15]. Pancreatic insufficiency can be
alleviated by oral supplementation of pancreatic enzymes, however, many patients continue to
experience a degree of steatorrhoea, with fat absorption ranging from 80 to 90% of their
dietary fat intake [16-18]. Recently, we reported strong indications that fat malabsorption in
CF patients on pancreatic enzyme replacement therapy is partially due to impaired intestinal
uptake of long-chain fatty acids [1]. Impaired uptake may involve bile-related processes such
as altered bile composition, decreased bile salt secretion, or bile salt inactivation at low
intestinal pH [18-22]. Furthermore, small bowel mucosal dysfunction or alterations in the
mucus layer have been suggested to contribute to inefficient intestinal uptake of long-chain
fatty acids in CF patients [5,23]. So far it has been difficult to differentiate between these
processes, partially due to the relative inaccessibility of the processes for mechanistic
investigations in humans. Yet, a more detailed insight into the processes causing impaired
uptake of long-chain fatty acids would allow the development of improved nutritional
therapies. This will likely benefit CF patients, because the positive correlation between a good
nutritional status and long-term survival or well-being of CF patients is well documented [24].
To obtain more insight into the effects of CF on fat absorption and bile formation we
studied two recently generated CF mouse models: (i) Mice homozygous for the ∆F508
mutation in the cftr gene [11]. This mouse model was chosen because the ∆F508 mutation is
the most frequently observed mutation in cystic fibrosis patients in Caucasian population; (ii)
107
Chapter 7
Mice with complete inactivation of the cftr gene, cftr -/- “null” mice [14], expected to result in
complete inactivation of the cftr gene, the most severe phenotype.
Materials and Methods
Animals
The male and female mice with the ∆F508 mutation (∆F508/∆F508) used in this study were
generated by Van Doorninck et al. and are described in reference [11]. Mice with a targeted
disruption in the cftr gene (cftrm1cam knockout mice, cftr -/-), resulting in complete loss of
CFTR function, and their controls (cftr +/+) were described by Ratcliff et al. [14].
Experiments involving ∆F508/∆F508 mice and their controls (N/N) were performed with the
strain in 129/FVB genetic background [11], whereas experiments involving cftr -/- and cftr
+/+ mice were performed with the strain in 129/C57/Bl6 genetic background [14]. All mice
were obtained from the breeding colony at the Erasmus University Rotterdam, The
Netherlands. The animals used for the experiments reported here were approximately 2-3
months old, had no obvious signs of disease or discomfort, and an average weight of 29 ± 1 g.
The genotype of each individual animal was tested by Southern blotting of tail DNA [4]. For
two weeks prior to the experiments, animals were kept on a semi-synthetic diet with standard
amounts of fat (14 en% fat; 4.538 kcal kg-1 food; fatty acid composition: C8-C12, 2.6%;
C16:0, 13.5%; C18:0, 4.2%; C18:1n-9, 21.3%; C18:2n-6, 48.4%; C18:3n-3, 1.1%) or an
isocaloric high fat diet (35 en% fat; 4.538 kcal kg-1 food; fatty acid composition: C8-C12,
4.4%; 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). The high-fat diet was applied to challenge the
absorptive system for fats in the mouse intestine. Mice were housed in an environmentally
controlled facility with diurnal light cycling and had free access to chow and tap water.
Experimental protocols were approved by the Ethical Committee for Animal Experiments,
Erasmus University Rotterdam.
Study protocol
For fat balance measurements, feces was collected and food intake was recorded for 3 days.
The gallbladder of mice was cannulated under Hypnorm (fentanyl/fluanisone, 1 mL kg-1 body
weight) and Diazepam anesthesia (10 mg kg-1 body weight) and bile was collected in 20minutes fractions for 80 minutes. Bile production was assessed by weight assuming that 1 mL
of bile corresponds to 1 g of bile. After bile collection, a large blood sample (0.5-1 mL) was
collected by cardiac puncture.
Analytical techniques
Lipids. Rat chow and feces were freeze-dried and mechanically homogenized. Aliquots of
chow and feces were extracted, hydrolyzed and methylated [25]. Resulting fatty acid methyl
esters were analyzed by gas chromatography to calculate fat intake and fecal fat excretion, as
detailed below. 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.
108
Fat (mal)absorption in CF mice
Percentage of total fat absorption =
Fat intake (g day -1 ) − Fecal fat output (g day -1 )
× 100%
Fat intake (g day -1 )
Bile salts. Total bile salt concentrations in bile and plasma were determined by an
enzymatic fluorimetric assay [26]. Individual bile salts in bile were analyzed by gas
chromatography after extraction with commercially available Sep-Pak-C18 cartridges (Waters
Associates, Milford, MA, USA) [27]. Total fecal bile salt concentrations were extracted from
an aliquot of freeze-dried homogenate [28] and fluorimetrically measured [26].
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 capillary column (Hewlett Packard - Ultra 1, crosslinked methyl silicone gum; 50 m x
0.2 mm) and an FID detector. The gas chromatograph oven was programmed from an initial
temperature of 160°C to 290°C in 2 temperature steps (160°C held 2 min; 160-240°C, ramp
2°C min-1, held 1 min; 240-290°C, ramp 10°C min-1, held 10 min). Quantification of the fatty
acid methyl esters was performed by adding heptadecanoic acid (C17:0) as internal standard.
Bile salts were separated and quantified with a CP-SIL 19CB capillary column (25 m
x 0.25 mm; Chrompack, Middelburg, The Netherlands). The gas chromatograph oven was
programmed from an initial temperature of 240°C to 280°C in 1 temperature step (240°C held
4 min; ramp 10°C min-1; held 26 min). Quantification of bile salts was performed by adding
5β-cholestane-3βol (coprostanol) as internal standard.
Calculations and statistics
The experimental data are reported as means ± SEM. Differences between sample means of
CF mice and their controls were analyzed by the two-tailed Student’s t-test for unpaired data
or one-way ANOVA followed by post-hoc analysis (Student-Newman-Keuls). Differences
between means were considered statistically significant at the level of P<0.05. Analysis was
performed with SPSS for Windows software (SPSS, Chicago, IL, USA).
Results
Fecal fat balance
Nutritional data of ∆F508/∆F508 mice, cftr -/- mice and their respective controls on standard
chow and high fat chow are shown in Table 7.1. No differences were observed between
∆F508/∆F508 and their controls with respect to fat intake, fecal fat excretion, net fat uptake
or percentage of dietary fat absorption on either standard or high-fat diet. In cftr -/- mice on
standard diet, fecal fat excretion was significantly increased when compared with controls (82
± 12 vs. 31 ± 4 µmol day -1, respectively, P<0.01). Accordingly, percentage of dietary fat
absorption was significantly decreased from 93.9 ± 1.3% in cftr +/+ mice to 82.8 ± 3.0% in
cftr -/- mice (P<0.01). Similar results were obtained when cftr mice were fed the high-fat diet,
although net fat uptake was approximately three-fold higher when compared with the standard
diet: fecal fat excretion was significantly increased and percentage of dietary fat absorption
109
Chapter 7
was significantly reduced in cftr -/- mice when compared with their control counterparts
(Table 7.1).
Bile salt concentrations
Plasma. Plasma total bile salt concentrations of ∆F508/∆F508 mice, cftr -/- mice and their
respective controls on standard and high-fat chow are shown in Table 7.2. In CF patients,
elevated plasma total bile salt concentrations are often an indication of liver disease or
cholestasis [29]. No significant differences in plasma bile salt concentrations were observed
between CF mice and their controls indicating that hepatic secretion of bile salts was not likely
to be inhibited in either group.
STANDARD DIET
∆F508/∆
∆F508 and controls
500
400
300
200
100
0
0
20
40
60
HIGH-FAT DIET
∆F508/∆
∆F508 and controls
600
Bile salts (nmol/min/100 g BW)
Bile salts (nmol/min/100 g BW)
600
500
400
300
200
100
0
80
0
Time periods of bile collection (min)
Bile salts (nmol/min/100 g BW)
Bile salts (nmol/min/100 g BW)
400
300
200
100
0
20
40
60
60
80
HIGH-FAT DIET
cftr -/- and controls
600
500
0
40
Time periods of bile collection (min)
STANDARD DIET
cftr -/- and controls
600
20
500
400
300
200
100
80
Time periods of bile collection (min)
0
0
20
40
60
80
Time periods of bile collection (min)
Figure 7.1 Bile salt output during 20-minute fractions of bile collection after gallbladder cannulation for 80 minutes in ∆F508/∆F508
mice (129/FVB genetic background) (€) and their controls (~), and in cftr -/- mice (129/C57/Bl6 genetic background) (Ž) and their
controls (±) on a standard diet (14 en% fat) and a high fat diet (35 en% fat).
Bile. Total biliary bile salt secretion in the 80-min period of bile collection was not
significantly different between the CF mouse models and their respective controls on either
diet (Table 7.2). In the ∆F508/∆F508 mice and their controls biliary bile salt secretion
appeared to be slightly increased when they were fed the high-fat diet, however, the difference
110
Fat (mal)absorption in CF mice
was not significant. Bile salt secretion during the 80 minutes of bile collection decreased in all
mice, indicating that the bile salt pool was depleting (Figure 7.1). In the 20-min period
immediately after cannulation of the gallbladder, biliary bile salt profiles show that cholic acid
and β-muricholic acid are the most predominant bile salts present in bile, together accounting
for approximately 90% of the biliary bile salts (Table 7.2). The percentual contribution of
cholic acid to the bile salt pool in cftr -/- mice on standard diet was significantly increased
when compared with their controls (P<0.01). The percentual contribution of deoxycholic acid
to the bile salt pool was decreased in ∆F508/∆F508 mice on standard and high-fat diet, and in
cftr -/- mice on a high-fat diet, compared with their controls (P<0.01). This tendency could
also be observed in the other groups although the differences did not reach significance
(P=0.07 and P=0.08). No differences were observed between CF mice and their controls with
respect to concentrations of chenodeoxycholic acid, ursodeoxycholic acid, and β-muricholic
acid on either diet.
STANDARD DIET
12
10
*
*
8
6
4
2
0
N/N ∆F/∆F
HIGH-FAT DIET
14
Fecal bile salts (µmol/g dry weight)
Fecal bile salts (µmol/g dry weight)
14
**
12
10
8
h
6
4
2
0
cftr(+/+) cftr(-/-)
N/N ∆F/∆F
cftr(+/+)
cftr(-/-)
Figure 7.2 Fecal bile salt excretion in ∆F508/∆F508 mice (129/FVB genetic background), cftr -/- mice (129/C57/Bl6 genetic
background) and their respective controls (N/N and cftr +/+) on a standard diet (14 en% fat) and a high-fat diet (35 en% fat).
r=0.27, P=0.07
Fecal fat excretion (µmol/day)
250
200
150
100
50
0
0
5
10
15
Fecal bile salts (µmol/g dry weight)
Figure 7.3 Correlation between fecal fat excretion and fecal bile salt excretion in ∆F508/∆F508 mice (129/FVB genetic background)
(€), cftr -/- mice (129/C57/Bl6 genetic background) (~) and their respective controls (N/N (€) and cftr +/+ (~)) on a standard diet
(14 en% fat) and a high fat diet (35 en% fat).
111
Table 7.1
Nutritional data of ∆F508/∆F508 mice (129/FVB genetic background), cftr -/- mice (129/C57/Bl6 genetic background) and their respective controls (N/N and cftr +/+) on standard chow (14
en% fat) and high-fat diet (35 en% fat) (mean ± SEM).
Category
Diet
n
6 (6M)
5 (4M/1F)
6 (3M/3F)
5 (4M/1F)
Food intake
(g day-1)
3.3 ± 0.4
3.5 ± 0.4
4.6 ± 0.5
4.6 ± 0.3
Fat intake
(µmol day -1)
534 ± 60
565 ± 73
1990 ± 215
2024 ± 126
Fecal fat
(µmol day -1)
22 ± 2
29 ± 3
94 ± 31
123 ± 27
Net fat uptake
(µmol day -1)
513 ± 59
536 ± 72
1896 ± 202
1900 ± 115
N/N
∆F508/∆F508
N/N
∆F508/∆F508
Standard
Standard
High fat
High fat
cftr +/+
cftr -/cftr +/+
cftr -/-
Standard
Standard
High fat
High fat
6 (5M/1F)
6 (1M/5F)
6 (2M/4F)
5 (2M/3F)
3.5 ± 0.3
3.0 ± 0.1
3.5 ± 0.4
3.1 ± 0.4
562 ± 56
488 ± 22
1507 ± 185
1371 ± 171
31 ± 4
82 ± 12*
76 ± 23
151 ± 25#
531 ± 58
406 ± 30
1430 ± 178
1221 ± 162
Fat absorption
(% intake)
95.8 ± 0.4
94.8 ± 0.7
95.5 ± 1.2
94.0 ± 1.3
93.9 ± 1.3
82.8 ± 3.0*
95.0 ± 1.4
88.8 ± 1.6*
M, male; F, female. A symbol indicates a significant difference from controls; # P<0.05, * P<0.01.
Table 7.2 Plasma bile salt concentration, biliary bile salt output during the total 80-min period and bile salt composition during the first 20 minutes of bile cannulation in ∆F508/∆F508 mice (129/FVB
genetic background), cftr -/- mice (129/C57/Bl6 genetic background) and their respective controls (N/N and cftr +/+) on standard chow (14 en% fat) and high-fat diet (35 en% fat) (mean ± SEM).
Category
Diet
N/N
∆F508/∆F508
N/N
∆F508/∆F508
Standard
Standard
High fat
High fat
Plasma bile salts
(µM)
25.4 ± 5.5
16.2 ± 2.3
16.6 ± 1.3
12.4 ± 1.3
cftr +/+
cftr -/cftr +/+
cftr -/-
Standard
Standard
High fat
High fat
14.3 ± 3.0
19.9 ± 4.1
13.1 ± 1.6
18.5 ± 3.2
Biliary bile salts
(µmol/100 g BW)
8.2 ± 0.7
11.7 ± 2.2
18.7 ± 1.7
20.1 ± 4.5
11.2 ± 4.9
14.2 ± 2.6
11.3 ± 2.0
15.3 ± 0.9
C
(%)
61.0 ± 2.8
70.7 ± 3.7
61.8 ± 2.4
70.6 ± 2.6
BMC
(%)
29.4 ± 2.8
25.1 ± 3.4
18.2 ± 1.4
18.6 ± 2.3
CDC
(%)
2.2 ± 0.5
1.5 ± 0.1
4.8 ± 0.4
4.9 ± 0.7
DC
(%)
5.0 ± 0.9
0.1 ± 0.1*
7.6 ± 1.4
1.5 ± 0.3#
UDC
(%)
2.5 ± 0.3
2.6 ± 0.3
7.6 ± 1.4
4.3 ± 1.1
46.5 ± 3.8
61.3 ± 1.7*
53.5 ± 6.1
63.8 ± 5.9
42.6 ± 3.2
33.8 ± 1.4#
33.1 ± 5.8
27.3 ± 6.4
3.4 ± 0.4
2.0 ± 0.2#
4.5 ± 0.9
4.9 ± 0.6
1.7 ± 0.4
0.6 ± 0.4
3.8 ± 0.7
0.9 ± 0.3*
6.0 ± 0.5
2.3 ± 0.2*
5.1 ± 1.2
3.1 ± 0.7
Significantly different from controls: # P<0.05, * P<0.01. C, cholic acid; BMC, β-muricholic acid; CDC, ursodeoxycholic acid; DC, deoxycholic acid; UDC, ursodeoxycholic acid.
112
Fat (mal)absorption in CF mice
Feces. Fecal total bile salts were significantly increased in ∆F508/∆F508 mice and
cftr -/- mice when compared with their respective controls on a standard diet (∆F508/∆F508
mice: 9.1 ± 0.4 versus 5.9 ± 0.6 µmol per g dry weight of feces, respectively, P<0.01; cftr -/mice: 10.8 ± 1.0 versus 5.4 ± 0.3 µmol per g dry weight of feces, respectively, P<0.01, Figure
7.2). A significant increase was also obtained for ∆F508/∆F508 mice on a high-fat diet (10.8 ±
1.0 versus controls 5.4 ± 0.3 µmol per g dry weight P<0.001). There was no significant
correlation between fecal fat excretion and fecal bile salt excretion in the group as a whole
(r=0.27, P=0.07; Figure 7.3), nor when the mice were stratified according to their genotype.
Discussion
Recently, we reported strong indications suggesting that fat malabsorption in CF patients on
pancreatic enzyme replacement therapy is partially due to impaired intestinal uptake of longchain fatty acids [1], which may involve bile-related processes. The aim of the present study
was to investigate in more detail fat absorption and bile formation in two recently generated
CF mouse models: ∆F508/∆F508 mice (129/FVB genetic background) and cftr -/- mice
(129/C57/Bl6 genetic background) [11,14].
The current data show that cftr -/- mice exhibit fat malabsorption, in contrast to
∆F508/∆F508 mice. No differences were observed with respect to biliary bile salt output.
Hence, this is not likely the cause of the fat malabsorption. The observed fat malabsorption is
not likely due to differences in the composition of the bile salt pool either, since these
differences were observed in both ∆F508/∆F508 mice and cftr -/- mice, whereas only cftr -/mice exhibit fat malabsorption. It has been speculated that liver disease develops in patients
with CF as a consequence of the plugging of intrahepatic bile ducts [30-32]. The lack of
CFTR in the apical membrane of bile duct cells may lead to abnormalities in biliary drainage
with chronic cholestasis [30]. Cholestasis would result in increased amounts of bile salts in
plasma and decreased amounts of bile in the intestine, and absorption of dietary fats would be
impaired [29,33]. Our results show that intestinal bile salts were similar in all mice, indicating
that cholestasis is probably not a cause of fat malabsorption in cftr -/- mice. Plasma bile salts
were similar in all mice.
Apparently, the fat malabsorption in the cftr -/- mice can not be explained by
processes regarding bile formation in the liver. Other mechanisms that may contribute to
inefficient fat absorption are intestinal mucosal dysfunction or alterations in the mucus layer.
This would also be in concordance with the intestinal histologic abnormalities observed in
∆F508/∆F508 mice and in cftr -/- mice [11,14]. Finally, the difference of fat malabsorption
may be due to different pancreatic functioning. However, CF mice do not show major
histological abnormalities in the pancreas or pancreatic duct and the secretion of amylase is not
impaired [34]. This lack of pancreatic disease in CF mice is most plausibly due to a lower level
of CFTR expression and a relatively higher contribution of alternative Ca-activated chloride
channels as compared with human pancreas [35,36]. CF-related abnormalities in the intestine
rather than hepatobiliary or pancreatic disturbances are therefore the most probable cause of
fat malabsorption in cftr -/- mice.
113
Chapter 7
The observation that fat malabsorption is present in cftr -/- mice but absent in
∆F508/∆F508 mice, may be due to the fact that the “Rotterdam” ∆F508/∆F508 mice exhibit
residual cftr activity [11,37], whereas in cftr -/- mice, cftr function is completely abolished
[14]. Low apical cftr activity in ∆F508/∆F508 mice has been observed at physiological
temperatures in the gallbladder and in the ileum [11,37]. The level of residual cftr activity
could differ in various tissues and small variations in apical activity levels could have profound
effects on pathology. A careful analysis of cftr processing kinetics between distinct tissues is
necessary to confirm this hypothesis. Moreover, it can not be excluded that the different
genetic backgrounds (129/FVB and 129/C57/Bl6 for ∆F508/∆F508 and cftr -/- mice,
respectively) are in part responsible for the different phenotypes of the two CF mouse models.
Additionally, studies were performed with both male and female mice (Table 7.1), which may
also influence the results. However, it has not been reported before in the literature that fat
absorption, bile salt pool size or bile composition differ for males and females.
In the present study, bile salt secretion during an 80-min period decreased by
approximately 50%, showing that most of the bile salt pool was collected. Total biliary bile
salt output during this period was similar for CF mice and their respective controls, suggesting
that bile salt pools of CF and control mice were similar. Previous data on the bile salt pool size
in CF patients are conflicting; both a normal and a decreased bile salt pool size have been
reported in CF patients exhibiting fat malabsorption [19,38]. Our results indicate that bile salt
secretion rates and bile salt pool size in the two mouse models for CF are not affected.
The observation of a relative increase in the proportion of cholic acid at the expense
of deoxycholic acid is a well-known phenomenon in CF [38,39]. This result is consistent with
the finding of increased fecal bile salts in CF mice. Interruption of the enterohepatic circulation
by fecal bile salt loss is normally accompanied by an increase in bile salt synthesis in order to
maintain bile salt output [40]. The capacity to increase synthesis is estimated to equal three to
four times the pool size [41]. Apparently, the increased fecal loss of bile salts can be
compensated for by increased hepatic bile salt neosynthesis.
Both CF mouse models exhibited increased excretion of bile salts in the feces when
compared with their respective controls. Fecal bile salt loss is well recognized in patients with
CF and has been attributed to various intraluminal factors: 1. unhydrolyzed triacylglycerols
and phospholipids, 2. precipitation of bile salts in acidic duodenal content, 3. adsorption of bile
salts to non-absorbed dietary residues, 4. modification of bile salts by intestinal microflora and
5. defects in the ileal uptake of bile salts [42-44]. In the CF mouse models, we did not find a
significant correlation between fecal bile salt secretion and fecal fat excretion, indicating that
unhydrolyzed triacylglycerols and phospholipids do not contribute to the increased fecal bile
salt loss. The other factors can not be excluded. Since intestinal histologic abnormalities have
been observed in both ∆F508/∆F508 and in cftr -/- mice [11,14], we speculate that the most
logical explanation for bile salt malabsorption in these two CF mouse models would be defects
in the ileal uptake of bile salts due to intestinal mucosal dysfunction or alterations in the mucus
layer [45].
In conclusion, in this study we have shown that cftr -/- mice, but not ∆F508/∆F508
mice, have an impaired dietary fat absorption, which is not likely due to either decreased bile
salt pool size or altered bile composition. In both CF mouse models, fecal bile salt excretion
114
Fat (mal)absorption in CF mice
was increased, which was not secondary to increased fecal fat excretion. Bile composition data
indicate that the increased fecal loss of bile salts is compensated for by an increased bile salt
neosynthesis.
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Liver Dis 1994;14:259-269.
33. Robb TA, Davidson GP, Kirubakaran C. Conjugated bile acids in serum and secretion in
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1985;26:1246-1256.
34. Mills CL, Dorin JR, Davidson DJ, Porteus DJ, Alton EWFW, Dormer RL, McPherson
MA. Decreased beta-adrenergic stimulation of glycoprotein secretion in CF mice
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43. Weber AM, Roy CC, Morin CL, Lasalle R. Malabsorption of bile acid in children with
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pancreatic insufficiency in cystic fibrosis. Gut 1976;17:295-299.
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117
CHAPTER 8
General discussion
Chapter 8
CHAPTER 8
General discussion
The two main processes involved in fat absorption are lipolysis and solubilization. The first
part of this thesis deals with the process of lipolysis. Lipolysis is an important step in the
overall process of fat absorption and a shortage of lipase due to pancreatic insufficiency may
lead to severely reduced levels of fat absorption. A rat model in which fat malabsorption is
caused by impaired lipolysis of dietary triacylglycerols has been developed and characterized
(chapter 2). Impaired lipolysis was induced by feeding rats different doses of orlistat, an
inhibitor of gastric and pancreatic lipase. It has been demonstrated that orlistat inactivates
lipase enzymes by reacting covalently with serine (Ser-152) in the active site of the catalytic
subunit. Orlistat has been shown to reproducibly induce fat malabsorption in a dose-dependent
fashion [1,2]. The percentage of total dietary fat absorption was examined upon feeding the
rats 4 different doses of orlistat, i.e. 0, 50, 200, and 800 mg orlistat per kg rat chow. Fat
absorption decreased in a dose-dependent way from 80.2 ± 2.2% in control rats (mean ±
SEM) to 32.8 ± 3.7% when 800 mg orlistat per kg rat chow was added (P<0.001).
120
General discussion
A potential test for the diagnosis of impaired lipolysis would be the 13C-MTG breath
test, which was previously described by Vantrappen et al. [3]. The advantage of the 13C-MTG
is its sensitivity to pancreatic lipase activity, the most important enzyme with respect to
hydrolysis of triacylglycerols. The principle of the 13C-MTG breath test is based on lipolysisdependent 13CO2 excretion via the breath. The relationship between the extent of fat
malabsorption and the recovery of 13CO2 in breath after oral ingestion of 13C-MTG was
investigated in control and orlistat-fed rats. Percentage of 13CO2 in breath was examined upon
feeding the rats 0, 50, 200, and 800 mg orlistat per kg rat chow. A significant correlation
(r=0.88, P<0.001) was observed between percentage of total fat absorption and 6-h recovery
of 13CO2 in breath. The correlation was especially strong in rats having major fat
malabsorption, indicating that the 13C-MTG breath test can be used as a tool to detect
impaired lipolysis when fat malabsorption is severe. However, in rats with fat absorption
higher than 75% the coefficient of variation of cumulative breath 13CO2 excretion was large
(15%) compared to that of fat absorption (5%). Thus, even under controlled circumstances in
a homogeneous group of rats with similar genetic background and standard diet, a
considerable variation in 13CO2 expiration was observed. Potential causes of this large
variation may be differences in gastric emptying, hepatic clearance and metabolism, βoxidation, endogenous CO2 production and pulmonary excretion, since all these factors may
influence the recovery of 13CO2 in the breath. In summary, when fat malabsorption is severe,
impaired lipolysis is evidently the rate-limiting step, which can be identified by the 13C-MTG
breath test. However, upon rather mild fat malabsorption, the rate-limiting step of 13CO2
expiration is shifted from impaired lipolysis to one of the other mentioned factors, resulting in
large variations upon application of the 13C-MTG breath test. Although no direct extrapolation
from rats to humans can be performed, these results indicate the diagnostic limitations of the
13
C-MTG breath test in subjects with mild to moderate fat malabsorption.
The mechanistic studies were extended by characterization of the 13C-MTG breath
test in healthy adults (chapter 3). The effects of various test conditions on the 13CO2 response
have only partially been elucidated. Therefore, it was determined which factors, apart from
pancreatic insufficiency, may influence the quantitative recovery of 13CO2 in breath. The
physiological variation of the 13CO2 response in healthy human adults was examined by
performing the test twice under exactly the same test circumstances within 4 weeks. The
repeatability was calculated according to Bland and Altman [4] and it was found that when the
13
C-MTG breath test is repeated in the same individual, the results of both tests are
considerably different. In order to explore whether the application of the 13C-MTG breath test
could be simplified or the sensitivity improved, the following factors were studied: the effect
of two different test meals on the 13CO2 response, the effect of an additional meal during the
test, and the effect of physical exercise during the test. A variety of test meals has been
described for breath tests with a diversity of substrates [3,5-7]. So far, no standardized test
meal for clinical purpose of these breath tests has been proposed. A disadvantage of a test
meal containing bread and butter could be the extended time it would take for children to
consume it, and the risk of not consuming it quantitatively. Also, such a test meal is not
applicable to small infants. It was examined whether the 13CO2 response of a liquid test meal
(75 mL cream) is similar to the 13CO2 response of a solid test meal (2 slices of bread and 25 g
121
Chapter 8
butter). The results suggest that these two distinct test meals give a similar cumulative 13CO2
response. Since it may be cumbersome to keep patients, in particular infants, fasted for several
hours, the effect of consuming an extra meal during the test on the 13CO2 response was
examined. The extra meal was ingested 3 h after the start of the experiment and consisted of 2
slices of bread and 30 g of strawberry jam. It appeared that stringency on continuous fasting
during the test is unnecessary, which is in favor of the applicability of the test in pediatric
patients. It is known that physical activity considerably affects the production rate of CO2 and
nutrient oxidation [8-10], however, it is not established to what extent it influences the results
of the 13C-MTG test. Subjects were asked to perform moderate exercise (50 Watt) during the
first 5 h of the test on a bicycle ergometer. Most subjects showed a large increase in their
13
CO2 recovery in the breath, and thus standardization of resting conditions still seems
preferable.
A second important step in the overall process of fat absorption is solubilization of
the lipolytic products by bile components. A rat model has been developed and characterized
in which fat malabsorption is induced by long-term bile diversion (chapter 4). Bile diversion
was achieved by providing rats with a permanent catheter in the bile duct in order to interrupt
the enterohepatic circulation. This experimental model allows for physiological studies in
unanesthetized rats with long-term bile diversion without the interference of stress or restraint.
After 6 days of bile diversion, no bile is available in the intestine for the formation of mixed
micelles, and theoretically, lipid absorption is expected to be decreased. When rats were fed a
standard diet (14 en% fat), however, percentage of dietary fat absorption still appeared to be
efficient and decreased only from 96.7 ± 0.2% in control rats to 87.2 ± 0.9% (P<0.001) in
bile-diverted rats. In order to challenge the absorptive system of the rat, fat absorption was
also studied when rats were fed a high-fat diet (35 en% fat). Percentage of total dietary fat
absorption again was highly efficient in control rats (93.2 ± 0.4%), but was considerably
decreased in bile-diverted rats (53.9 ± 3.9%, P<0.001). The presently characterized rat model
allows the systematic evaluation of the quantitative role of the various bile components on
intestinal lipid absorption, in particular by reconstitution experiments involving the continuous
infusion of model bile solutions. In addition, this rat model will allow to evaluate the potency
of novel therapeutic approaches/drugs to improve lipid absorption under conditions of
impaired bile formation.
A potential substrate for the diagnosis of impaired solubilization would be a 13Clabeled long-chain fatty acid. A 13C-labeled long-chain fatty acid test measures the whole
process of uptake of long-chain fatty acids: solubilization of lipolytic products by bile
components and translocation of the fatty acids over the intestinal mucosa. In this thesis, the
substrate [1-13C]palmitic acid was selected, because palmitic acid is the most predominant
saturated fatty acid in the Western diet. The potency of the [1-13C]palmitic acid test to
quantify fat malabsorption due to impaired intestinal uptake of long-chain fatty acids was
investigated in chronically bile-diverted rats (chapter 4). So far, the use of 13C-labeled longchain fatty acids for quantitative studies on defective fat absorption has been limited to breath
and feces analysis [5,11,12]. The excretion rate of 13C in the form of exhaled 13CO2, however,
does not necessarily reflect quantitative differences in the absorption of the 13C-labeled parent
compound, e.g., due to variations in the post-absorptive metabolism [7,13]. The determination
122
General discussion
of plasma concentrations of absorbed 13C-labeled fats as a measure of their absorption offers a
theoretical advantage over breath 13CO2 analysis, since numerous steps are involved in the
post-absorptive metabolism of the tracer prior to exhalation of 13CO2 [14]. After intraduodenal
administration of [1-13C]palmitic acid, plasma 13C-palmitic acid concentrations clearly
differentiated between control rats and chronically bile-diverted rats. When 10% dose L-1
plasma was used as the lower limit of normal plasma values and 91% as the lower limit of
normal dietary fat absorption, the test had a sensitivity and specificity of 100% for detecting
fat malabsorption under the test conditions used. The results were essentially similar on the
standard and high-fat diet, emphasizing the potency of the [1-13C]palmitic acid absorption test
to detect impaired intestinal uptake of long-chain fatty acids.
In order to investigate the sensitivity of the [1-13C]palmitic acid test in humans, the
test was applied to a group of healthy volunteers in which fat absorption was slightly reduced
by dietary supplementation of calcium (chapter 5). It has been shown that calcium can form
insoluble precipitates, consisting of calcium, phosphate, bile acids and long-chain fatty acids
[15,16]. Oral calcium supplementation to healthy subjects has been reported to increase fat
excretion via the feces in a dose-dependent fashion [15,17-20]. Upon calcium administration
fat absorption significantly decreased from 96.6 ± 0.6% to 94.9 ± 0.9% (P<0.05). Thus, oral
calcium supplementation in humans seems to be a method to reduce fat absorption to a minor
extent. It was examined whether this effect could be quantified by means of orally
administered [1-13C]palmitic acid. Upon calcium administration, plasma 13C-palmitic acid
concentrations after 8 h were significantly increased when compared to control values, yet,
cumulative expiration of 13CO2 was significantly decreased. This discrepancy between the
results of the [1-13C]palmitic acid test in plasma and breath indicates that post-absorptive
metabolism is changed upon calcium supplementation. Percentage of dietary fat absorption did
not correlate to either breath 13CO2 recovery or plasma 13C-palmitic acid concentrations.
Although calcium supplementation clearly affects the outcomes of the [1-13C]palmitic acid
test, present data do not indicate that the test is sensitive enough to reliably quantify this small
degree of fat malabsorption in human adults.
A frequently encountered disorder in Caucasian populations associated with fat
malabsorption is cystic fibrosis. The pathophysiology of fat malabsorption in human cystic
fibrosis patients may involve both pancreatic insufficiency and bile acid deficiency. However,
so far it has not been possible to determine in the individual patient which of the processes is
rate-limiting, especially not when patients are supplemented with pancreatic enzymes. In order
to obtain more insight into the impaired processes of fat malabsorption in cystic fibrosis we
performed a study in pediatric cystic fibrosis patients treated with their usual pancreatic
enzyme replacement therapy (chapter 6). The substrates 13C-MTG and uniformly labeled 13Clinoleic acid were both applied to determine whether the rate-limiting step behind their fat
malabsorption was either impaired lipolysis or impaired intestinal uptake of long-chain fatty
acids, respectively. 13C-linoleic acid was selected as the substrate because, theoretically, it
could provide information on the essential fatty acid status and metabolism of the patients. The
13
C-linoleic acid test and the 13C-MTG breath test were both applied to 10 pediatric cystic
fibrosis patients receiving their habitual pancreatic enzymes. During the test days, a fat balance
was performed for 3 days to determine dietary fat absorption. Fecal fat excretion ranged from
123
Chapter 8
5.1 to 27.8 g day-1 and fat absorption ranged from 79 to 93%. After ingestion of 13C-MTG no
relationship was observed between breath 13CO2 recovery and dietary fat absorption (r=0.04).
In contrast, a strong relationship was observed between 8-h plasma 13C-linoleic acid
concentrations and dietary fat absorption after ingestion of 13C-linoleic acid (r=0.88,
P<0.001). Our results suggest that fat malabsorption in cystic fibrosis patients on enzyme
replacement therapy is not likely due to insufficient lipolytic enzyme activity, but rather due to
defective intestinal uptake of long-chain fatty acids. Therefore, therapeutic attempts to
normalize fat absorption in cystic fibrosis patients need to include a strategy to improve
intestinal uptake of long-chain fatty acids.
Impaired intestinal uptake of long-chain fatty acids may be caused by (combinations
of) processes such as altered bile composition, bile salt precipitation, decreased intestinal bile
salt concentration, small bowel mucosal dysfunction and/or alterations in the mucus layer [2125]. In order to obtain more mechanistic insight into the involved processes, we studied two
cystic fibrosis mouse models (chapter 7). The two cystic fibrosis mouse models are: 1. mice
with the ∆F508 mutation in the cftr gene, ∆F508/∆F508 (129/FVB genetic background), and
2. mice with complete inactivation of the cftr gene, cftr -/- (129/C57/Bl6 genetic background)
[26,27]. The basic defect in cystic fibrosis lies in the cystic fibrosis transmembrane regulator
(CFTR), a protein responsible for chloride ion transport. In ∆F508/∆F508 mice, the
biosynthetic processing of the cftr gene product to its mature glycosylated form is disrupted
[28], so that the protein is retained in the endoplasmic reticulum and is then degraded [29].
However, it has been observed that ∆F508/∆F508 mice exhibit residual cftr activity in the
gallbladder and ileum [26]. In cftr -/- mice, cftr function is completely abolished, and epithelia
lack cftr in the apical plasma membrane and, therefore, lack cAMP-stimulated Cl- permeability
[30]. Fat absorption was studied after feeding the mice a standard (14 en% fat) or a high-fat
(35 en% fat) diet for 2 weeks. In ∆F508/∆F508 mice, dietary fat absorption was similar
compared with controls on both diets (standard diet: 94.8 ± 0.7% compared to 95.5 ± 0.6%,
respectively; high-fat diet: 93.8 ± 2.2% compared to 95.3 ± 2.4 %, respectively). Absorption
of dietary fats by cftr -/- mice, however, was significantly less efficient when compared with
their control counterparts (standard diet: 82.8 ± 3.0% compared to 93.9 ± 1.3%, respectively,
P<0.01; high-fat diet: 88.8 ± 1.6% compared to 95.0 ± 1.4%, respectively, P<0.01). These
data indicate that the complete disruption of the cftr gene leads to moderate fat malabsorption,
in contrast to introduction of the ∆F508 mutation. However, it can not be completely excluded
that the observed difference in fat malabsorption is also influenced by the different genetic
background of the mice. In order to study the processes behind defective uptake of long-chain
fatty acids in more detail, biliary secretion of bile salts was determined after cannulation of the
gallbladder for 80 min and fecal bile salt excretion was determined. Biliary bile salt pool sizes
and biliary bile salt secretion rates were similar for the CF mouse models and their respective
controls on either diet, indicating that this is not the reason for fat malabsorption in cftr -/mice. Fecal bile salt excretion was increased in ∆F508/∆F508 and in cftr -/- mice when
compared with their respective controls (10 versus 5 µmol g -1 feces, respectively, P<0.01). No
significant correlation was observed between fecal bile salt excretion and fecal fat, indicating
that the increased excretion of fecal bile salts is not secondary to fat malabsorption. Biliary bile
salts in ∆F508/∆F508, cftr -/- and control mice were predominantly composed of cholate and
124
General discussion
ß-muricholate, with minor contributions of deoxycholate, ursodeoxycholate and
hyodeoxycholate. Significantly increased cholate and decreased deoxycholate concentrations
were observed in bile of ∆F508/∆F508 and cftr -/- mice when compared with their controls.
This result is consistent with the finding of increased fecal bile salt loss, which upregulates
neosynthesis of bile salts thereby resulting in higher amounts of primary bile salts and
decreased amounts of secondary bile salts. In conclusion, in this study we have shown that cftr
-/- mice, but not ∆F508/∆F508 mice, have an impaired dietary fat absorption, which is not
likely due to either decreased bile salt pool size or altered bile composition. In both CF mouse
models, fecal bile salt excretion was increased, which was not secondary to increased fecal fat
excretion. Bile composition data indicate that the increased fecal loss of bile salts is
compensated for by an increased bile salt neosynthesis. Further investigations are needed to
establish whether impaired pancreatic function, intestinal mucosal dysfunction or alterations in
the intestinal mucosa are responsible for the observed fat malabsorption in cftr -/- mice.
In summary, in this thesis more information is obtained with respect to the various
pathophysiological processes involved in fat malabsorption. Various animal models for lipid
malabsorption were described and characterized: orlistat-fed rats to study impaired lipolysis,
bile-diverted rats to study bile deficiency, and transgenic and knock-out mice for the study of
cystic fibrosis. These animal models are expected to be of significant importance to investigate
the potency of novel diagnostic and therapeutic strategies in the near future. In addition, the
potency of diagnostic tests such as the 13C-MTG breath test and the 13C-palmitic acid test was
investigated to characterize the etiology behind fat malabsorption in animal models and in
humans. The present data in cystic fibrosis patients and mice do not only open the possibility
to determine pathophysiological mechanisms of lipid malabsorption in individual patients, but
they also give way for titration of therapy to individual patients.
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127
SAMENVATTING
Samenvatting
SAMENVATTING
Vetten in de voeding zijn belangrijk voor het menselijk lichaam als bronnen van energie en als
bouwstenen, vooral in periodes van groei en ontwikkeling. Gewoonlijk is het menselijk
lichaam in staat om voedingsvetten zeer efficiënt op te nemen; meer dan 96% van de
voedingsvetten wordt opgenomen in het lichaam. Er zijn echter verschillende aandoeningen
waarbij de absorptie van voedingsvetten verstoord is. Voor een goede behandeling van
patiënten met deze zogenaamde vetmalabsorptiesyndromen is inzicht in het vetabsorptieproces
essentieel. Het proces dat verantwoordelijk is voor de opname van vetten uit de voeding speelt
zich af in het maagdarmkanaal. Allereerst worden in de maag en in het duodenum de
voedingsvetten (voornamelijk triglyceriden) gesplitst in vetzuren en monoacylglyceriden. Dit
splitsingsproces, dat bekend is onder de naam lipolyse, wordt gekatalyseerd door lipaseenzymen afkomstig uit de maag en de pancreas. Vervolgens worden deze gesplitste vetdeeltjes
opgelost in de waterige omgeving van de dunne darm middels een proces dat bekend staat als
solubilisatie. Hiertoe worden micellen gevormd die bestaan uit galcomponenten, zoals
galzouten en fosfolipiden, en de vetdeeltjes. Vooral lange-keten vetzuren zijn sterk afhankelijk
van een efficiënte solubilisatie, in tegenstelling tot middellange-keten vetzuren, omdat langeketen vetzuren slecht oplosbaar zijn in water. Tenslotte worden de vetdeeltjes getransporteerd
over de celwand van de dunne darm, waarna ze terecht komen in de bloedbaan. De vetten die
niet worden opgenomen in het lichaam, worden uitgescheiden via de ontlasting. Tot nu toe
werd vooral aandacht besteed aan de efficiëntie van het totale proces van vetabsorptie,
waardoor het inzicht in de bijdrage van de onderliggende mechanismes aan de vetmalabsorptie
beperkt is. Een gedetailleerder inzicht in deze onderliggende mechanismes zou niet alleen
kunnen leiden tot verbeteringen in diagnostische methodes, maar ook tot verbeteringen in
behandelingsmethoden voor patiënten met vetmalabsorptie. Het doel van het huidige
onderzoek was dan ook om meer informatie te verkrijgen over de verschillende
pathofysiologische processen die betrokken zijn bij vetmalabsorptie en om de diagnostiek voor
vetmalabsorptie te verbeteren.
130
Samenvatting
Om deze doelstelling te verwezenlijken zijn studies uitgevoerd met proefdieren,
gezonde proefpersonen en patiënten. De studies werden zo opgezet dat, naast de totale
vetabsorptie, ook de processen lipolyse en solubilisatie werden onderzocht. De methode die in
de kliniek gebruikt wordt om de totale hoeveelheid van geabsorbeerde vetten te bepalen is de
vetbalans. Dit houdt in dat patiënten gedurende drie dagen in voedseldagboeken alles noteren
wat ze eten, waardoor de totale vetinname kan worden bepaald. In deze periode verzamelen
de patiënten ook al hun ontlasting, waarin de niet-geabsorbeerde hoeveelheid vetten kan
worden bepaald. Op deze manier kan dan de hoeveelheid geabsorbeerde vetten uitgedrukt
worden als percentage van de vetinname. Bijvoorbeeld, als de vetinname gedurende drie dagen
100 g per dag is en de hoeveelheid vet in de ontlasting gedurende deze periode 5 g per dag is,
dan betekent dit dat de vetabsorptie 95% is. Met behulp van deze methode kan echter geen
informatie over de achterliggende mechanismes (lipolyse en solubilisatie) worden verkregen.
Deze informatie kan wel verkregen worden door gebruik te maken van testen waarbij vetten
op een onschadelijke manier gelabeld worden met stabiele isotopen. In dit onderzoek zijn de
diagnostische capaciteiten van twee verschillende soorten testen beschreven, namelijk een test
voor het diagnosticeren van verstoorde lipolyse en een test voor het diagnosticeren van
problemen bij solubilisatie.
Verstoorde lipolyse kan worden aangetoond met behulp van de 13C-MTG ademtest.
13
C-MTG is de afkorting van het gelabelde vet waarvan de volledige chemische naam luidt:
1,3-distearoyl, 2[carboxyl-13C]octanoyl glycerol. Na inname van dit substraat splitsen lipaseenzymen, afkomstig van de pancreas, de twee vetzuurketens van het molekuul af (=lipolyse),
waarna het 13C-gelabelde restmolekuul opgenomen kan worden in het lichaam. Omdat
restmolekuul een middellange-keten vetzuur is, is de opname van het molekuul niet afhankelijk
van solubilisatie. Na opname wordt dit 13C-gelabelde restmolekuul grotendeels verbrand in het
lichaam en vervolgens uitgeademd als 13CO2. Dus, het principe van de 13C-MTG ademtest is
gebaseerd op het feit dat, na inname van het substraat, de hoeveelheid 13CO2 die uitgeademd
wordt, afhankelijk is van de activiteit van het pancreas lipase-enzym. Verstoorde solubilisatie
kan worden aangetoond met behulp van lange-keten vetzuren (bijv. 13C-palmitinezuur of 13Clinolzuur). Dit zijn molekulen die niet meer gesplitst hoeven te worden door lipase-enzymen en
de opname van deze molekulen in het lichaam is dus alleen afhankelijk van een goede
solubilisatie. Na inname van bijvoorbeeld het substraat 13C-palmitinezuur kan de door het
lichaam opgenomen hoeveelheid in het plasma gemeten worden als 13C-gelabeld palmitinezuur
en na verbranding in de adem als 13CO2. De hoeveelheid die niet wordt geabsorbeerd, wordt
uitgescheiden in de ontlasting en kan eveneens gemeten worden. Als het solubilisatieproces
goed werkt, zal er veel 13C-gelabeld palmitinezuur aanwezig zijn in plasma en veel 13CO2 in de
adem, maar weinig 13C-palmitinezuur in de ontlasting.
In het eerste deel van dit proefschrift werd het proces lipolyse onderzocht. Om dit
proces in detail te kunnen bestuderen werd allereerst een proefdiermodel ontwikkeld en
gekarakteriseerd voor vetmalabsorptie ten gevolge van verstoorde lipolyse (hoofdstuk 2).
Verstoorde lipolyse werd bewerkstelligd door ratten via hun gewone voer een stofje toe te
dienen, orlistat, dat de activiteit van lipase-enzymen vermindert. De mate van vetmalabsorptie
bleek afhankelijk te zijn van de toegediende hoeveelheid orlistat. Bij deze ratten werd
onderzocht of vetmalabsorptie kon worden aangetoond met behulp van de 13C-MTG
131
Samenvatting
ademtest. Er bleek een sterk verband te bestaan tussen het percentage vetabsorptie en de
totale hoeveelheid 13CO2 in de adem. Echter, als er sprake was van slechts een lichte
vetmalabsorptie was, dan bleek er toch een grote variatie in de hoeveelheid 13CO2 in de adem
te zijn. Dit zou kunnen komen doordat bij een milde vorm van verstoorde lipolyse de 13CO2
vorming niet alleen afhankelijk is van de lipolysesnelheid maar ook van factoren als
maagontlediging en verbrandingssnelheid. Dit betekent dat de 13C-MTG ademtest
waarschijnlijk minder geschikt is als diagnostisch middel indien vetmalabsorptie slechts in
geringe mate verstoord is. Om de toepassingsmogelijkheden van de 13C-MTG ademtest verder
te onderzoeken werd deze test vervolgens uitgevoerd bij gezonde volwassenen (hoofdstuk 3).
De volgende factoren werden bestudeerd: het effect van twee verschillende testmaaltijden op
de 13CO2 respons in de adem, het effect van een extra maaltijd tijdens de test, en het effect van
lichamelijke oefening tijdens de test. Uit de experimenten met twee verschillende
testmaaltijden bleek dat geen significante verschillen werden gevonden met betrekking tot de
13
CO2 respons in de adem na een vloeibare testmaaltijd (75 ml slagroom) of een vaste
testmaaltijd (2 sneeën tarwebrood en 25 g roomboter). Dit is vooral een voordeel voor
zuigelingen, die nog geen vast voedsel kunnen innemen. Een extra maaltijd drie uur na
aanvang van de test (2 sneeën tarwebrood en 25 g aardbeienjam), bleek de resultaten niet te
veranderen. Dit is een voordeel voor kinderen omdat die vaak moeite hebben met vasten
gedurende een langere periode. Ten derde bleek dat lichamelijk activiteit tijdens de test wel
degelijk de resultaten beïnvloedde en dat het dus belangrijk is om tijdens de test in rust te
blijven.
De tweede stap in het proces van vetabsorptie die onderzocht werd in dit proefschrift
is de solubilisatie van vetten. Om dit proces in detail te kunnen bestuderen werd ook hiervoor
een proefdiermodel ontwikkeld en gekarakteriseerd (hoofdstuk 4). Verstoorde solubilisatie
werd gerealiseerd door langdurige galonderbreking. Dit houdt in dat ratten werden voorzien
van een catheter in de galgang, waardoor de gal niet meer de dunne darm instroomt, maar
buiten het lichaam van de rat wordt opgevangen. De gal is dan niet meer beschikbaar voor
solubilisatie van vetten in de dunne darm met als gevolg dat de vetten niet goed meer
geabsorbeerd kunnen worden. In dit proefdiermodel werd onderzocht of verstoorde
solubilisatie kon worden aangetoond met behulp van de 13C-palmitinezuur test. De
concentraties 13C-palmitinezuur in plasma van gal-onderbroken ratten bleken duidelijk lager te
zijn dan de concentraties 13C-palmitinezuur in plasma van controle ratten. De test bleek zeer
geschikt te zijn voor het aantonen van een sterk solubilisatieprobleem bij ratten. Om de
gevoeligheid van de test verder te bepalen werden testen gedaan bij gezonde volwassenen
waarbij een zeer licht vetmalabsorptie was bewerkstelligd ten gevolge van verstoorde
solubilisatie (hoofdstuk 5). Dit werd gerealiseerd door de proefpersonen dagelijks 2 g calcium
toe te dienen in de vorm van calcium carbonaat. Calcium bindt aan galzouten, waardoor deze
niet meer beschikbaar zijn voor solubilisatie. Vervolgens werd onderzocht of deze milde
vetmalabsorptie kon worden aangetoond met behulp van de [1-13C]palmitinezuur test. Bij de
proefpersonen werden zowel adem- als plasmamonsters verzameld gedurende een periode van
8 uur. Er bleek echter een discrepantie te zijn tussen de resultaten in het plasma en in de adem
en de 13C-palmitinezuur test is dus blijkbaar niet gevoelig genoeg om een zeer lichte vorm van
vetmalabsorptie te detecteren.
132
Samenvatting
Vervolgens werden experimenten verricht om meer inzicht te verkrijgen in de
vetmalabsorptie die geassocieerd is met de genetische afwijking cystische fibrose. Cystische
fibrose patiënten lijden vaak aan een pancreas insufficiëntie wat betekent dat onvoldoende
pancreas lipase-enzymen in de dunne darm aanwezig zijn. Deze patiënten worden dan ook
behandeld door bij elke maaltijd lipase-enzymen in te nemen. Het is echter meerdere malen
aangetoond dat cystische fibrose patiënten, ondanks behandeling met pancreasenzymen, nog
steeds een bepaalde mate van vetmalabsorptie overhouden. Een hogere dosis
pancreasenzymen is echter niet gewenst omdat dit kan leiden tot aantasting van de dikke darm.
Het mechanisme van vetmalabsorptie in cystische fibrose patiënten behandeld met lipaseenzymen kan veroorzaakt worden door een inefficiënte enzymtherapie en/of een verstoorde
solubilisatie. Om meer inzicht te krijgen in deze processen werd een studie uitgevoerd bij
cystische fibrose patiënten die behandeld werden met pancreasenzymen (hoofdstuk 6). De
substraten 13C-MTG en 13C-linolzuur werden gebruikt om te bepalen wat de
snelheidsbepalende stap achter deze vetmalabsorptie was: respectievelijk een lipolysedefect of
verstoorde solubilisatie. Na inname van het 13C-MTG substraat werd geen verband gevonden
tussen de mate van vetabsorptie en de hoeveelheid 13CO2 in de adem. Dit betekent dat de
verminderde vetabsorptie in deze patiënten waarschijnlijk niet veroorzaakt wordt door een
verstoorde lipolyse. Er bleek echter wel een sterk verband te bestaan tussen de mate van
vetabsorptie en de concentratie 13C-linolzuur in plasma na inname van 13C-linolzuur. Dus,
patiënten met een relatief lage vetabsorptie hadden ook een lage hoeveelheid 13C-linolzuur in
hun plasma. Deze resultaten suggereren dat de vetmalabsorptie in cystische fibrose patiënten
die behandeld worden met pancreasenzymen waarschijnlijk veroorzaakt wordt door een
solubilisatiedefect. Om vetabsorptie in cystische fibrose patiënten te bevorderen is het dus
zinvol om de therapie aan te passen met betrekking tot het solubilisatiedefect. Een verstoring
in het solubilisatieproces kan echter verschillende oorzaken hebben, zoals verlaagde
galzoutconcentratie, andere galzoutsamenstelling, inactivatie van de galzouten door een lage
pH in de dunne darm. De volgende stap in dit onderzoek was dan ook om meer inzicht te
verkrijgen in dit proces bij cystische fibrose. Hiertoe werden studies uitgevoerd met muizen
die genetisch gemanipuleerd waren aan het gen voor cystische fibrose (hoofdstuk 7). Bij deze
muizen werd onderzocht of vetmalabsorptie werd veroorzaakt door een verminderde
galzoutpool, een verlaagde galzoutconcentratie, en/of een andere galzoutsamenstelling. Dit
bleek niet het geval te zijn. De muizen met cystische fibrose bleken wel verhoogde
concentraties galzouten in de ontlasting te vertonen, een verschijnsel dat ook bij patiënten met
cystische fibrose veel voorkomt. De oorzaak hiervan moet nog verder onderzocht worden.
Samenvattend kan worden gezegd dat diagnostische testen met stabiele isotopen, in
tegenstelling tot de klassieke vetbalans, gedetailleerde informatie geven over de processen
lipolyse en solubilisatie. Derhalve kan met behulp van deze testen gerichte farmacotherapie en
dieettherapie opgesteld worden voor patiënten met vetmalabsorptie. De 13C-MTG test kan bij
sterke verstoring van de lipolyse goed gebruikt worden; bij marginale verstoring spelen ook
andere experimentele condities een rol, die beter gekarakteriseerd dienen te worden. Met de
lange-keten vetzuurtest kunnen verstoringen in solubilisatie opgespoord worden. Deze
bevinding kan met name van belang zijn voor de verdere behandeling van patiënten met
cystische fibrose.
133
NAWOORD
Na ruim 4 jaar noeste arbeid sluit ik met het schrijven van deze allerlaatste pagina de periode
Groningen af. Alhoewel ik steeds heb geroepen dat ik Groningen een gezellige, knusse stad
vind (al is het dan een beetje ver verwijderd van de rest van Nederland) en ik een erg leuke tijd
in Groningen heb gehad, ben ik toch blij dat het nu dan eindelijk achter de rug is. In de
afgelopen periode heb ik veel mensen weten te waarderen voor hun steun en gezelligheid. Een
aantal mensen wil ik hier daarom persoonlijk noemen. Allereerst wil ik graag Henkjan Verkade
bedanken voor alle kennis die hij me heeft bijgebracht en voor zijn enthousiaste begeleiding.
Mijn promotor Roel Vonk wil ik bedanken voor zijn zinnige suggesties met betrekking tot de
lijn van mijn onderzoek. Frans Stellaard heeft me vooral in het eerste jaar van mijn onderzoek
geholpen met alles wat er over stabiele isotopen te leren valt. Daarnaast zijn er natuurlijk
ontzettend veel mensen die me wegwijs hebben gemaakt op het laboratorium, achter de
computer, in de proefdierruimte, en in de Groningse kroegen en eetcafés. Heel hartelijk
bedankt voor jullie professionele en emotionele bijdrage aan mijn promotieonderzoek. We
moeten nog maar eens een avondje wat gaan drinken, lachen en kletsen zodat ik jullie
persoonlijk kan bedanken.
135
Stellingen behorend bij het proefschrift
Mechanisms involved in malabsorption of dietary lipids
Mini Kalivianakis
Groningen, 23 september 1998
Severe fat malabsorption due to impaired lipolysis can be identified by the 13CMTG breath test. However, in situations of mild fat malabsorption, considerable
interindividual variation in the results of the 13C-MTG breath test occurs, which
may be explained by a shift in the rate-limiting step in the overall process of 13CO2
production.
(DIT PROEFSCHRIFT)
The continuing fat malabsorption in cystic fibrosis patients on enzyme replacement
therapy is not likely due to insufficient lipolytic enzyme activity, but rather due to
either incomplete intraluminal solubilization and/or reduced mucosal uptake of
long-chain fatty acids.
(DIT PROEFSCHRIFT)
Vrouwelijke wetenschappers moeten ongeveer 2½ maal beter presteren dan hun
mannelijke collega’s voor een vergelijkbaar resultaat.
(NATURE 1997;387:341)
Een promotieonderzoek is niet alleen een investering is in jezelf maar ook in de
universiteit. Dat dit besef ook begint door te dringen tot de universitaire wereld
blijkt uit het feit dat een aantal universiteiten bereid is de promovendi meer te
betalen.
Mini staat voor meer dan klein.
Ook voor stellingen geldt dat een hogere kwantiteit vaak niet ten goede komt aan
de kwaliteit.