Lactose intolerance: the role of
Copyright © 2006 Tao He
All rights reserved. No part of this book may be reproduced or transmitted in any
form or by any means without written permission of the author and the publisher
holding the copyright of the published articles.
Printed by: Drukkerij C. Regenboog, Groningen, The Netherlands
Front cover: a Chinese knot with the chemical structure of lactose
Back cover: a ‘Lakenvelder’ cow and fecal bacteria (Gram staining)
Lactose intolerance: the role of
ter verkrijging van het doctoraat in de
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
woensdag 8 november 2006
om 13.15 uur
geboren op 26 augustus 1969
te Kunming, China
Prof. dr. R.J. Vonk
dr. G.W. Welling
Prof. dr. R-J.M. Brummer
Prof. dr. J.E. Degener
Prof. dr. J.H. Kleibeuker
dr. E.H.H.M. Rings
The research described in this dissertation was supervised by Prof. dr. R.J. Vonk,
dr. G.W. Welling of the University of Groningen, and dr. K. Venema of the
Wageningen Centre for Food Sciences (WCFS), the project leader of the WCFS C-
012 project ‘Microbe-mediated gut metabolism’ under the programme ‘Microbial
Functionality and Safety’.
Microbial Functionality and Safety
The programme Microbial Functionality and Safety studies the metabolic and enzymatic activities of
microorganisms. The aim is to improve the health-promoting and organoleptic properties of foods and
to maintain food safety by destroying food pathogens or increasing shelf life by inhibiting spoilage
Microbe-mediated gut metabolism
WCFS aims to identify global and specific relevant metabolic conversions of dietary components by
the microbiota in the colon, link these to the microbial diversity and its spatial distribution, and study
their effect on intestinal health, in order to gain insight in and to optimize intestinal functionality by
addressing the effects of diets on this. Various relatively unrelated disciplines such as microbial
physiology, human physiology and gastroenterology are combined, and incorporated with
developments in the biomedical and nanotechnology fields, using a unique combination of stable
isotopes and ~omics approaches. These include in vitro experiments, interventional strategies in
human volunteers, and investigational tools, including stable isotope probing, spectroscopic analysis
and metabolomics, transcriptomics and proteomics. Another part is focused on the development and
application a sampling device for in situ sampling in the large intestine for the determination of short-
chain fatty acids produced in the colon. The research described here relates to the study of lactose
metabolism in the colon and its effect on lactose-intolerance and has made use of the various research
tools describe above. The research was carried out in the Department of Medical Microbiology and
Center for Medical Biomics, University Medical Center Groningen, University of Groningen, as part
of the WCFS project C-012 ‘Microbe-mediated gut metabolism’.
Gerwin C. Raangs
The printing of this thesis was financially supported by the following organizations.
Their contribution is gratefully acknowledged:
Groningen University Institute for Drug Exploration (GUIDE)
University of Groningen
Wageningen Centre for Food Sciences (WCFS)
Scope of the thesis?
Results and discussion? ?
Effect of lactose on oro-cecal transit in
lactose digesters and maldigesters
Identification of bacteria with ß-
galactosidase activity in faeces from lactase
Colonic fermentation may play a role in
lactose intolerance in humans
Effects of yogurt and bifidobacteria
supplementation on the colonic microbiota
and lactose-induced symptoms in lactose
Differential analysis of protein expression of
Bifidobacterium grown on different
The research protocol for an in vivo study
on colonic fermentation of lactose using
Brucella blood agar
denaturing gradient gel electrophoresis
fluorescent in situ hybridization
irritable bowel syndrome
liquid chromatography tandem mass spectrometry
lactose digestion index
oro-cecal transit time
short-chain fatty acid
sodium dodecyl sulfate polyacrylamide gel electrophoresis
surface-enhanced laser desorption ionization - time of
6-h symptom score
single strand conformation polymorphism
temperature gradient gel electrophoresis
two-dimensional polyacrylamide gel electrophoresis
terminal-restriction fragment length polymorphism
temporal temperature gradient gel electrophoresis
Dairy products provide us with calcium and other valuable nutrients. However,
they also contain lactose which is maldigested by a large part of the world adult
population. The mechanisms by which lactose maldigestion causes symptoms of
lactose intolerance are not fully understood. Studies on the pathophysiology of
lactose intolerance may aid to design strategies for dietary management of lactose
intolerance. Limited evidence suggests that colonic metabolism of lactose, in
addition to the small-intestinal lactase activity and transit, might be involved in the
development of symptoms.
Data on digestion and transit of lactose in the small intestine are needed when
clarifying possible involvement of the colon in lactose intolerance. The oro-cecal
transit time (OCTT) and the degree of lactose digestion were determined using
newly developed stable isotope-based methods. Lactose triggered faster oro-cecal
transit in lactose maldigesters, but not in digesters. This could not be explained by
intestinal distention resulting from the osmotic load posed by maldigested lactose,
and thus suggested a direct effect of lactose on intrinsic factors regulating intestinal
motility. The lactose tolerant and intolerant subjects did not differ in OCTT or the
degree of lactose digestion, which indicates the involvement of other
pathophysiological mechanisms in lactose intolerance. We hypothesize that colonic
metabolism of lactose is one of these mechanisms.
Two in vitro studies were carried out to investigate the possible role of
fermentation of lactose by the colonic microbiota in lactose intolerance. During
colonic fermentation, lactose is first hydrolyzed to glucose and galactose, catalyzed
by ß-galactosidase. Glucose and galactose are subsequently fermented, leading to
production of short-chain fatty acids (SCFA) and gases. When incubated with
lactose in vitro, fecal bacteria from the lactose intolerant subjects produced more
lactate, acetate, propionate and butyrate at a higher rate than the tolerant subjects.
The results suggest that a faster and higher production of microbial intermediate
and end metabolites during colonic fermentation of lactose, may be related to the
development of lactose-induced symptoms. It is unlikely that the degree and rate of
lactose hydrolysis are involved. Bacterial ß-galactosidase activity is abundant in
the colon as more than 80% of the cultured fecal bacteria were found to possess
this activity. The lactose tolerant and intolerant subjects did not differ in the
relative amount or composition of the fecal bacteria with ß-galactosidase activity,
ß-galactosidase activity in feces, or hydrolysis of lactose in vitro. We hypothesize
that accumulation of SCFA and/or other metabolites resulting from fast
fermentation of lactose in combination with insufficient removal plays a role in
onset of symptoms.
As the in vitro study indicates that colonic fermentation may be involved in
lactose intolerance, we continued to: (i) investigate whether the colonic microbiota
could be modulated by dietary supplementation for the purpose of alleviating
symptoms; (ii) explore proteomics techniques to study metabolic pathways of
lactose fermentation by the colonic microbiota; (iii) design an in vivo study to
verify the observations of the in vitro study.
A 2-w supplementation of probiotic bacteria (Bifidobacterium longum) and a
yogurt enriched with Bifidobacterium animalis modified the amount and probably
the metabolic activities of the colonic microbiota of lactose intolerance subjects.
Lactose-induced symptoms decreased
supplementation did not increase the endogenous lactase activity in the small
intestine. Results indicate that the changes in the colonic microbiota may play a
role in alleviation of intolerant symptoms.
Bifidobacteria were used as a model system to explore proteomic methodology
which can be used to study metabolic pathways of lactose by the colonic
microbiota. Differential protein expression profiles of Bifidobacterium grown on
lactose, glucose or galactose were obtained by using surface-enhanced laser
desorption ionization - time of flight MS and sodium dodecyl sulfate
polyacrylamide gel electrophoresis. LC-MS/MS was used for identification of
An in vivo study is proposed to verify our observations in the in vitro study that
fast colonic fermentation of lactose plays a role in lactose intolerance. Stable
after the supplementation. The
isotope-labeled lactose will be delivered to the cecum via a catheter and kinetics of
13C-acetate in the peripheral blood will be monitored.
The results presented in this dissertation contribute to the understanding of the
colonic metabolism of lactose in the context of lactose intolerance. Colonic
fermentation of lactose may play a role in lactose intolerance. Dietary modulation
of the colonic microbiota provides a promising strategy for management of lactose
Dairy products are important sources of many nutrients including calcium, high-
quality protein, potassium and riboflavin. It is not clear why there has to be a
special carbohydrate, lactose, in milk. Lactose is the principal carbohydrate in
human and animal milk. Human milk contains an average of 7% lactose, while
whole cow’s milk contains 4.8%. During infancy, all human and mammals possess
high levels of the enzyme lactase in their small intestine, which enables digestion
of lactose. After weaning, a large part (~75%) (1) of the world population
undergoes a genetically-determined decline in lactase activity, which can lead to
maldigestion of lactose. Lactose maldigestion can, but not necessarily, cause
unpleasant gastrointestinal symptoms, termed lactose intolerance.
Lactose intolerance is one of the factors that may influence milk consumption.
Studies suggest that lactose maldigesters consume less milk than digesters,
possibly as a result of experiences of unpleasant symptoms after ingestion of
lactose-containing dairy products (2-5). Persons who consume less milk as a result
of lactose intolerance generally have lower intake of calcium and other nutrients
supplied by milk. Several studies have indicated an increased frequency of lactose
maldigestion in patients with osteoporosis (6,7). A connection between lactose
maldigestion and decreased absorption of calcium has not been proven. The reason
for the high incidence of lactase deficiency in people with osteoporosis could be a
lower calcium intake in this group because of lactose intolerance (2).
The causes of the symptoms of lactose intolerance are not well understood.
Several factors are considered to be involved in the occurrence of symptoms,
including the amount of lactose ingested, lactase activity, intestinal transit time,
and other factors such as visceral sensitivity or bowel motor abnormalities (8).
Recently the possible involvement of colonic factors has been suggested (9).
However, little knowledge is available concerning the role of the colon in lactose
intolerance. Understanding the pathophysiology of lactose intolerance will aid to
design strategies for dietary management of lactose intolerance. The dietary
management of lactose intolerance would help lactose intolerant subjects to
consume dairy products, without or with less complaints.
1. Lactose digestion and maldigestion, and lactose intolerance
1.1 Lactose metabolism in normal-lactasia
Lactose is a disaccharide composed of the two monosaccharides, glucose and
galactose. To be absorbed, lactose needs to be hydrolyzed into glucose and
galactose. The hydrolysis is catalyzed by lactase, lactase-phloritzin hydrolase (EC
18.104.22.168/26), a ß-galactosidase. Lactase is located in the brush border of the
intestinal epithelium and has its highest activity in the jejunum. Of all the dietary
sugars, lactose is hydrolyzed the most slowly. Hydrolysis of lactose proceeds at
approximately half the rate of sucrose hydrolysis (10). After hydrolysis, glucose
and galactose are absorbed from the intestine by active transport. Galactose is
metabolized mainly in the liver via the Leloir pathway to glucose. This pathway is
very efficient, almost half of the galactose administered enters the body glucose
pool within 30 min (11,12).
1.2 Hypolactasia, lactose maldigestion and lactose intolerance
Hypolactasia refers to a very low activity of lactase in the jejunal mucosa (13). It
can be primary (genetic) or secondary. Primary hypolactasia is genetically
determined and occurs soon after weaning in almost all animals and in many
human ethnic groups. The lactase activity drops to about one tenth or less of the
suckling level. Primary hypolactasis is also referred to as adult-type hypolactasia
or lactase non-persistence. Secondary hypolactasia results from damages to the
intestinal mucosa which can be caused by intestinal resections, gastrectomy or
some intestinal disease. Congenital lactase deficiency is extremely rare. Lactase
activity is decreased or absent at birth, and this deficiency persists throughout life
Hypolactasia leads to lactose maldigestion, which in turn can cause lactose
intolerance, but not in all cases. Lactose intolerance refers to the gastrointestinal
symptoms associated with the incomplete digestion of lactose (1). The symptoms
include abdominal pain, cramps, flatulence, nausea, or diarrhea. Lactose
maldigestion correlates poorly with symptoms of lactose intolerance (15). This is
supported by the following observations (15): (i) not all lactose maldigesters will
develop symptoms after lactose ingestion, some maldigesters can be lactose
tolerant (9); (ii) the decline of lactase activity starts much earlier than does the
manifestation of clinical symptoms (16); (iii) not all lactose intolerant subjects are
symptom-free after ingestion of lactose-free diets. Therefore, lactose intolerance
can be referred to as symptomatic lactose maldigestion. In the literature the term
“lactose intolerance” is sometimes wrongly used to mean lactose maldigestion (13).
1.3 Prevalence and genetics of adult-type hypolactasia
In general, hypolactasia is more common in populations outside Europe than inside
Europe. Prevalence of hypolactasia in European countries around the North Sea is
as low as less than 10% and rises in Central and Southern Europe to 70% in Sicily.
The highest prevalences of hypolactasia have been reported from the countries in
Far East Asia, a prevalence of 100% was found in Northern Thailand and Vietnam.
The prevalences in different Chinese groups range from 43 to 92%. In the United
States, the prevalences are 6% to 19% in whites, 53% in Mexican Americans and
80% in African Americans. Prevalences ranging from 13% to 90% were reported
for South Africa (17).
The inter-individual differences in lactase activity are due to a genetic
polymorphism. The lactase non-persistent people are homozygous for an autosomal
recessive allele, while lactase persistent people are heterozygous or homozygous
for a dominant allele LCT*P. Lactase persistence behaves as a dominant trait
because half the levels of the normal lactase activity are sufficient to show
significant digestion of lactose. The different lactase phenotypes are controlled by a
polymorphic element cis-acting to the lactase gene. A putative causal nucleotide
change has been identified and occurs on the background of a very extended
haplotype that is frequent in Northern Europeans, where lactase persistence is
frequent. This single nucleotide polymorphism is located 14 kb upstream from the
start of transcription of lactase in an intron of the adjacent gene MCM6. This
change does not, however, explain all the variation in lactase expression. There is
no evidence for adaptive alteration in lactase expression (18). Genotyping of single
nucleotide polymorphism C/T(-13910) (19,20)and c.1993+327C (21) has been
suggested as a first stage screening test for adult-type hypolactasia. During et al.
reported peroral gene therapy of lactose intolerance using an adeno-associated
virus vector (22).
1.4 Factors that may influence lactose digestion and lactose intolerance
In persons with hypolactasia, besides the lactase activity in the small intestine,
other factors may influence lactose digestion and occurrence of lactose intolerance
as well. These factors include gastrointestinal transit, the amount of lactose
ingested, etc. These are discussed in short below.
1.4.1 Gastrointestinal transit
Prolonged gastric emptying and intestinal transit enable longer contact between the
residual brush-border lactase and lactose, and thus may improve lactose digestion
and alleviate symptoms. This can be the mechanism behind the observations in
many studies which show pasteurized yogurt improves lactose digestion (23-25)
and improved lactose tolerance after ingestion of chocolate milk (26,27) and full-
fat milk compared with skimmed milk or ingestion of milk with a meal instead of
milk on its own (28,29). Several studies show that a longer oro-cecal transit time
(OCTT) contributes to less symptoms in lactose maldigesters (9,30-32). However,
Roggero et al. did not observe differences in the small bowel transit times between
symptomatic and asymptomatic malabsorbing subjects (33).
1.4.2 Amount of lactose ingested
Most of the lactose maldigesters can ingest a certain amount of lactose without
developing any symptoms. The term lactose intolerance should be used when
referring to the symptomatic response to a defined amount of lactose load. A small
amount of lactose (6-7 g) does not induce symptoms of lactose intolerance (34,35).
The amount of lactose (12 g) in one cup of milk (240 ml) can be tolerated by most
maldigesters (36-38). Ingestion of 50 g of lactose causes symptoms in 80% to
100% of lactose maldigesters (39,40). Even after ingestion of a large amount of
lactose, a small percentage of maldigesters remained symptom-free (14). The
reason for this is not known.
Men and women do not differ in the prevalence of hypolactasia (41,42). However,
women seem to report higher symptom scores than men, while it is not clear
whether there is difference in hydrogen production between the two genders (42-
Villar et al. demonstrated that 44% of women who maldigested 360 ml of milk (18
g of lactose) before the 15th week of gestation, were able to digest that amount of
lactose by the end of their pregnancy (45). The mechanism is unknown. Some
researchers hypothesize that slower intestinal transit during pregnancy improves
digestion of lactose (46,47).
The age at which manifestation of hypolactasia occurs is generally earlier in a
population with a high prevalence of hypolactasia (more than 80%) than in a
population with low prevalence. The former starts at the age of 2 to 7 y and the
latter starts after 4-5 y and continues until 20 y (17).
In animal studies, lactase activity was found to decrease with age (48,49). But
lactase activity in human duodenal biopsies did not change significantly with age
(50,51). Gastric emptying was prolonged in the elderly (52-54). The small bowel
transit time, OCTT or whole gut transit time did not change with age (52-55).
However, Pilotto et al. found that OCTT increased in healthy aging (56). There
might be differences in hydrogen production after ingestion of lactose, but the
findings are not consistent (41,42,57,58). Results of the experience of symptoms of
lactose intolerance according to age are also contradictory (58,59).
1.4.6 Subjective factors
Lactose maldigestion can be diagnosed by objective testing. The classification of
lactose intolerance, however, is based on the individual’s perception of symptoms
except when diarrhea is prominent. Symptom reporting by individuals seems to be
influenced by other factors besides lactose maldigestion. Some individuals,
digesters or maldigesters, reported intolerance symptoms to whatever placebos
used in double-blinded studies (38,60-62). Familiarization with the test procedure
also influences symptom recording (63). These observations suggest the possible
involvement of psychological factors and that some gastrointestinal complaints are
often mistakenly attributed to the consumption of lactose or milk. Well-designed
and double-blinded clinical trials are recommended for the studies of lactose
1.4.7 Functional gastrointestinal disorders
Functional gastrointestinal disorders are a variable combination of chronic or
recurrent gastrointestinal symptoms not explained by structural or biochemical
abnormalities. The symptoms of functional bowel disorders (for instance, irritable
bowel syndrome (IBS)) and dysmotility-type dyspepsia resemble those of lactose
intolerance (8). In several studies, a relation between lactose intolerance and IBS
was observed (64-66), whereas data from other studies do not support this
observation (67,68). Visceral hypersensitivity and hyperalgesia (69) and small
bowel dysmotility (70) have been documented in IBS. Motor-sensory interactions
is also suggested for IBS, i.e. altered motility potentiates the sensory response to
relatively physiological levels of intraluminal stimulation (71). Hammer et al. (72)
investigated the role of symptom perception in lactose intolerance and suggested
that subjective symptoms of lactose intolerance are not due to the amount of
malabsorbed lactose or to the volume or rate of gas accumulation per se, but are
related to increased perception of gas.
1.4.8 Colonic processing of lactose
(see below, “2. Colonic processing of lactose and lactose intolerance”)
2. Colonic processing of lactose and lactose intolerance
2.1 Lactose metabolism in hypolactasia
When the lactase activity in the small intestine is not enough to hydrolyze all the
ingested lactose, maldigested lactose enters the colon where it is fermented by the
colonic microbiota. Lactose is first hydrolyzed by bacterial ß-galactosidase into
glucose and galactose. Galactose will be converted into glucose via the Leloir
pathway, glucose will be subsequently fermented (73,74). Short-chain fatty acids
(SCFA) (acetate, propionate and butyrate) and gases (CO2, H2 and CH4) are the
end-metabolites of bacterial fermentation of lactose (Figure 1). Some intermediates,
for instance, lactate, ethanol and succinate, are produced and then further
metabolized to SCFA. SCFA and gases are thought to be readily absorbed from the
colon (10,11,75). Acetate is the principal SCFA produced (~50%). It passes
through the liver and is finally metabolized in the peripheral tissues (76). Butyrate
serves as an important fuel for colonocytes (77). Absorbed propionate and butyrate
are metabolized in the liver (76). Gases are partially absorbed from the intestine
into the blood and partially excreted through the lung and partially excreted as
flatus or used for synthesis of other metabolites (78,79).
2.2 Colonic processing of lactose might play a role in lactose intolerance
Colonic fermentation of lactose might be involved in lactose intolerance, which is
supported by the following observations:
(i) the colonic microbiota is involved in metabolism of maldigested lactose
(ii) Subjects with similar OCTT and degree of lactose digestion in the small
intestine developed symptoms of different severity (9).
(iii) Adaptation of long-term lactose ingestion may be related to adaptation of the
colonic microbiota and colonic function. Continuous lactose consumption
reduces breath hydrogen excretion, increases fecal ß-galactosidase activity
and improves lactose intolerant symptoms (63,80,81). Adaptive changes in
colonic functions (motility, transit, and pH (82,83)) and the colonic
Figure 1. Fermentation of lactose by the colonic microbiota. (Modified based on Reilly &
Rombeau (75) and Morrison et al. (161))
microbiota (84), less bacterial hydrogen production (85), decreased
perception of symptoms by the subjects, and placebo effects have been
suggested as explanations for these observations (15).
However, few studies have been directed to investigate the possible role of
colonic fermentation of lactose in lactose intolerance.
2.3 Aspects in colonic processing of lactose that may influence lactose
The following aspects of the colon might affect the symptoms of lactose
intolerance (8,86): (i) the composition and metabolic activities of the colonic
microbiota; (ii) the ability of the colon to remove fermentation metabolites; (iii)
visceral sensitivity (symptom perception) (72).
2.3.1 The balance between production and removal of the fermentation
metabolites decides the outcome of bacterial fermentation of lactose
Whether colonic fermentation of maldigested lactose would influence the
occurrence of lactose intolerance, either aggravate or alleviate it, depends on the
balance between the ability of the colonic microbiota to ferment lactose and the
ability of the colon to remove the fermentation metabolites. A low lactose-
fermenting capacity of the colonic microbiota, which leads to inefficient removal
of maldigested lactose (and/or its intermediate fermentation metabolites), or a low
absorption capacity of the colon which leads to inefficient removal of fermentation
metabolites, may contribute to development of symptoms. When lactose is
converted to SCFA by fermentation, the osmotic load is increased about 8-fold,
which makes the efficiency of the colon to absorb these fermentation metabolites
an important determinant for the outcome of the osmotic load caused by
malabsorbed lactose (86).
2.3.2 Removal of SCFA in the colon
SCFA produced by bacterial fermentation are removed from the colon through the
following routes: 1) absorption by the colon; 2) utilization by the colonocytes
(butyrate); 3) excretion in feces; 4) incorporation into the bacterial biomass: it has
been suggested that ~40% of carbon atoms produced from the fermented hexosyl
moiety may be used for bacterial growth (87).
SCFA are presumably absorbed by both ionic and non-ionic diffusion. It is
generally believed that the colon has a high capacity to absorb SCFA, with the
absorption rate being 6.1-12.6 µmol/(cm2.d) (88-91). SCFA absorption stimulates
sodium and chloride absorption and bicarbonate secretion (92,93). There are
differences among segments in colonic permeability for the three major SCFA.
Acetate is absorbed at the highest rate in the cecum and proximal colon, and
butyrate in the distal colon; propionate is absorbed at a similar rate in the proximal
and distal colon (94). Lactate is an intermediary organic acid in the bacterial
fermentation of carbohydrates and is further converted to SCFA and as a result, it
is rarely present in large amounts in feces (89,95,96).
Although the colon is believed to possess a high capacity to absorb SCFA, it
needs further investigation whether the absorption rate is sufficient to remove in a
short period in situ all the SCFA and lactate produced from rapid fermentation of
some carbohydrates, for instance, lactose.
2.4 Colonic metabolism of lactose and the pathophysiology of lactose
The mechanisms by which lactose maldigestion causes symptoms of lactose
intolerance are not fully understood yet. Generally speaking, the osmotic load of
maldigested lactose increases secretion of fluid and electrolytes to the lumen,
causing dilatation of the intestine. Intestinal dilatation induces acceleration of small
intestinal transit (31,97), which further aggravates maldigestion of lactose (8).
Distention caused by the additional water content in the lumen and the gaseous
products of fermentation, plus the possible effects of the SCFA on colonic motility,
lead to the characteristic signs and symptoms of lactose intolerance (10). In some
studies, the correlation between colonic fermentation and loose stool or diarrhea
and the correlation between bacterial production of gas and abdominal distention,
cramps and flatulence, were investigated.
2.4.1 Colonic fermentation and loose stool or diarrhea
Loose stool or diarrhea are generally believed to be results of the osmotic effect
exerted by maldigested lactose. The role of colonic fermentation of lactose in
diarrhea in lactose intolerance needs further clarification. Disordered peristalsis and
water absorption in the colon caused by products of lactose fermentation may be
involved in development of loose stool or diarrhea (98,99). However, as it is
generally believed that SCFA are rapidly absorbed from the colon, colonic
fermentation is suggested to help to reduce osmotic load in the colon
(10,92,93,100). Hammer et al. (101) investigated the influence of colonic
metabolism of malabsorbed carbohydrates on diarrhea by comparing diarrhea
induced by nonabsorbable, non-fermentable polyethylene glycol and by
nonabsorbable, fermentable lactulose. The results suggest that bacterial metabolism
affects diarrhea and the effect is dose-dependent. When the amount of malabsorbed
lactulose was within the metabolic capacity of the colonic microbiota (?45 g/d), the
osmolarity of lactulose was reduced by bacterial fermentation of lactulose to SCFA
and subsequent absorption of SCFA. Thus, diarrhea was attenuated. The mild
diarrhea observed after 45 g of lactulose was probably mainly due to unabsorbed
SCFA. When the amount of malabsorbed lactulose was beyond the metabolic
capacity of the colonic microbiota (>95 g/d), unfermented lactulose retains water in
the colon lumen and thus retards absorption of SCFA. The diarrhea was aggravated
and was due to unmetabolized lactulose and unabsorbed SCFA. However, a study
by Holtug et al. (102) does not fully support this conclusion. High intake of
lactulose caused a decrease in fecal pH to < 5, which inhibited colonic fermentation,
before the appearance of carbohydrate in feces. The osmotic drive due to the
unfermented carbohydrate, instead of SCFA, is interpreted to be the cause of
diarrhea. Nevertheless, this interpretation was only supported by results from half
the subjects in the study in whom diarrhea appeared suddenly and after appearance
of carbohydrate in feces. In the other half of the subjects, diarrhea developed
gradually and before appearance of carbohydrate in feces. Moreover, the drop of
fecal pH was likely the result of incomplete removal of fermentation products.
Clausen et al. (103) compared diarrhea induced by idolax and lactulose which are
fermentable but of different osmolarity. They concluded that difference in colonic
fermentation seem to play a determining role in the interindividual variability in
diarrhea associated with carbohydrate malabsorption. A high fermentation capacity
may help to abolish the laxative effect caused by the malabsorbed carbohydrates
Theoretically, the outcome of the colonic osmolar load of malabsorbed lactose is
determined by the relation between the ability of the fecal microbiota to ferment
lactose and the efficiency with which the colonic mucosa absorbs these
fermentation products (86).
2.4.2 Bacterial production of gas and abdominal distention, cramps and flatulence
Gas produced from bacterial fermentation of lactose is probably the cause of
abdominal bloating, flatulence and borborygmi and might be involved in
development of distention and cramps.
Abdominal distention and cramps were suggested to originate from the small
intestine (98). However, a recent study showed that symptoms seemed to originate
from the colon as lactulose either ingested orally or introduced directly to the colon
caused similar symptoms (104).
Theoretically, colonic fermentation of 50 g of lactose will produce ~17 L of
hydrogen (105). If allowed to accumulate, this volume would have major
implications for intestinal distention and gas problems. However, most of the gas is
consumed by other intestinal bacteria. Hammer et al. did not observe difference in
volume or rate of colonic hydrogen accumulation in lactose malabsobers with or
without symptoms after ingestion of 50 g of lactose. They suggested that symptoms
were related to increased perception of gas (72). Similar results were obtained in
studies on functional bowel disorders. Lasser et al. concluded that the functional
abdominal symptoms may result from disordered intestinal motility in combination
with an abnormal pain response to gut distention other than from increased gas
accumulation in the intestine (106). In IBS patients, increased intestinal gas content
results from impaired gas transit instead of from increased gas production. Gas
content and transit appear to conspire with the motor and sensory responses of the
gut and thus produce gas-related symptoms, both in normal individuals and
especially in IBS patients (107).
Intestinal gas tolerance is normally high as expeditious gas transit and
evacuation prevent gas accumulation. When gas transit and/or evacuation is
impaired, gas retention occurs, which causes abdominal symptoms and distention
(108). The perception of intestinal gas accumulation depends on the mechanism of
retention. Obstructed evacuation increased symptom perception, whereas gas
retention caused by defective propulsion was virtually unperceived (109).
Intraluminal gas distribution influences symptom perception. A similar
magnitude of gas retention produced significantly more abdominal symptoms with
jejunal or duodenal compared with rectal infusion (110,111).
3. The colonic microbiota
The human colon is the home for a complex consortium of microorganisms
(primarily obligatory anaerobic bacteria, but also fungi and protozoa), normally
referred to as the colonic microbiota. The total number of bacteria in the human gut
is ~1014, which outnumbers the total number of body cells by ~10-20 times. The
human gut microbiota plays an important role in human health and disease through
its involvement in nutrition, pathogenesis and immunology of the host (112).
3.1 The composition of the colonic microbiota
The number of microbial species present in the human colon is not clear yet. It has
been estimated statistically that 400 to 500 species are present (113). Sequence
analysis of 16S ribosomal RNA (rRNA) and 16S rDNA revealed that the majority
of intestinal microbiota clustered within 3 bacterial groups: Bacteroides,
Clostridium coccoides and Clostridium leptum (113,114).
The conventional method to quantify colonic bacteria is by cultivation.
Culturing techniques have long been suggested to be inadequate to quantify
intestinal bacteria as firstly, a large fraction of the microbiota cannot be cultured
yet (113,115) and secondly, specific culturing media may not be truly specific.
Therefore, recent years have seen a fast development in molecular techniques
(culture independent) to determine colonic bacteria. These molecular approaches
are based on the sequence diversity of the 16S rRNA. Frequently applied culture
independent approaches include sequencing of clone libraries of 16S rRNA
encoding genes, fingerprinting of 16S rRNA encoding genes (for instance,
denaturing gradient gel electrophoresis (DGGE)), fluorescent in situ hybridization
(FISH), diversity microarrays, etc. Zoetendal et al. summarized the currently used
molecular techniques to study complex microbial ecosystems (116) (Table 1).
For enumeration of the colonic microbiota, FISH is an extensively used culture-
independent technique (117-119). Fecal microbiota are suggested to be able to
reflect the colonic microbiota (120-122). Harmsen et al. (123) and He et al. (124)
investigated the composition of fecal microbiota of adults and the elderly (Table 2).
In adults, Bacteroides/Prevotella, the Eubacterium rectale/Clostridium group and
the Atopobium group are the most predominant groups in feces. Compared to
adults, the percentages of some bacterial groups to total microbiota in the elderly
were higher, i.e. the Ruminococcus group, Bifidobacterium, the Eubacterium
cylindroides group, Enterobacteriaceae and Lactobacillus/Enterococcus. The
percentages of other groups, such as Bacteroides/ Prevotella and the Eubacteriu.
rectale/Clostridium group, were lower in the elderly. As analyzed with FISH, in
breast-fed newborn infants, bifidobacteria dominate the colonic microbiota, with
lactobacilli and streptococci as the main minor groups. In formula-fed infants,
Bacteroides and bifidobacteria are equally predominant, with staphylococci,
Escherichia coli, and clostridia as the minor groups (125). Lay et al. (126) studied
the composition of the fecal microbiota of subjects from five Northern European
countries with FISH combined with flow cytometry. Clostridium coccoides and
Clostridium leptum were the dominant groups (28.0% and 25.2%), followed by
Bacteroides (8.5%). There were no significant differences in the bacterial
composition with respect to geographic origin, age, or gender.
3.2 Studying the metabolic activities of the colonic microbiota in vitro and in
Colonic bacteria ferment carbohydrates and proteins and produce SCFA (mainly
acetate, propionate and butyrate) and gases (H2, CO2, CH4). Most colonic bacteria
first ferment carbohydrates and switch to protein fermentation when carbohydrates
are used up (127). Carbohydrates and proteins available for microbial fermentation
are mostly of dietary origin but can also be host-derived, for instance, mucin and
pancreatic enzymes (128). Carbohydrate fermentation takes place in the proximal
part of the colon and protein fermentation occurs in the distal colon (129). In vitro
and in vivo models are used to study the metabolic activities of the colonic
3.2.1 In vitro
As sampling of the colonic content is difficult, in vitro models are often used in
studies on metabolic activities of the colonic microbiota. Fecal or cecal materials
are incubated in vitro in buffer or culture medium under anaerobic atmosphere.
There are two types of in vitro systems:
(i) static system: the culture system is sealed, there is no exchange of fluid
during incubation. Static systems are suitable for short-term studies as the
conditions in the cultures are constantly changing, for instance, pH and
(ii) continuous system: there is addition of fresh growth medium and removal
of used culture continuously or at intervals. The continuous system
simulates the in vivo gut to a certain degree. However, it ignores host input,
e.g. gut secretions, immunology and interaction with mucosal cells (130).
Table 1. A summary of current techniques used to study complex microbial ecosystems
Isolation; "the ideal"
Not representative; slow &
Laborious; subject to PCR bias
Subject to PCR bias; Semi-
requires clone library
Subject to PCR bias; semi-
requires clone library
Subject to PCR bias; semi-
requires clone library
Requires sequence information;
laborious at species level
Requires sequence information;
laborious at species level
In early stages of development;
additional 16S rRNA-based
16S rDNA sequencing
Monitoring of community/population
shifts; rapid comparative analysis
T-RFLP Monitoring of community shifts; rapid
comparative analysis; very sensitive;
potential for high throughput
Monitoring of community/population
shifts; rapid comparative analysis
FISH Detection; enumeration; comparative
analysis possible with automation
Detection; estimates relative abundance Dot-blot hybridization
Detection; estimates relative abundance
Detection; estimates relative abundance
Monitoring of community shifts; rapid
(From Zoetendal et al. (116).)
Table 2. Composition of fecal microbiota of adults (n=11) and elderly people (n=15)
determined by FISH or DAPI staining 1
Eubacterium low G+C2
E. cylindroides group
1.0 ± 0.5 27.7
0.8 ± 0.4 22.7
0.4 ± 0.3 11.9
0.4 ± 0.3 10.8
0.4 ± 0.5 10.3
0.2 ± 0.1 4.8
0.04 ± 0.07 1.4
0.02 ± 0.03 0.6
0.006 ± 0.01 0.2
0.002 ± 0.004 0.08
0.0004 ± 0.0009 0.01
0.5 ± 0.9 10.3
0.2 ± 0.2 3.8
0.4 ± 0.5 7.9
0.6 ± 0.4 12.5
0.2 ± 0.2 4.9
0.3 ± 0.6 6.0
0.1 ± 0.2 3.2
0.02 ± 0.05 0.5
0.2 ± 0.6 2.2
0.02 ± 0.03 0.4
0.001± 0.001 0.0
0.01± 0.02 0.3
0.006 ± 0.001 0.1
1 Values are means ± SD or %.
2 per g feces, wet weight
3Percentage of Bacteria (Eub338).
-: No data were reported
(From Harmsen et al. (123) and He et al. (124))
3.2.2 In vivo
Studies on metabolic activities of the colonic microbiota are carried out in
experimental animals and human volunteers. Laboratory animals with conventional
gut microbiota, particularly rodents (131) and pigs (132,133), have been used for
studies of the gut microbiota. However, differences exist in the composition of the
Adults (20-55 yr)
Cells (1010)2 % bacteria3 Cells (1010) 2 bacteria3
Bacterial groups Stain or probes
Bacteria Eub338 3.5 ± 1.6 100
5.1 ± 2.6 100
colonic microbita between animals and man. To circumvent this problem, human
microbiota-associated rodents were explored (134-136).
Studies with human subjects are rare, considering difficulties in quantitative
delivery of substrates to the colon and sampling in situ in the colon. Techniques
using stable isotopes provide alternative approaches to study colonic metabolism in
vivo (137-139). For studying colonic fermentation of a certain substrate in vivo
using stable isotopes, a labeled tracer is infused at a constant and known rate until a
new steady-state is reached. Then the substrate of interest is administered, orally or
delivered with a certain device. Blood samples are collected at regular intervals.
From the isotopic enrichment in blood at a steady-state, the production or
elimination rates of metabolites of the substrate of interest can be calculated based
on a principle of isotope-dilution. Furthermore, colon-delivery catheters (104) and
capsules (140) can be considered for quantitative delivery of substrates of interests
to the colon.
3.3 Proteomics and metagenomics in studying metabolism and structure of
the colonic microbiota
Proteins are important for cell structure and function. Whereas their basic
biological functions are encoded by genes, the structure and function of proteins
are also regulated by post-translational modifications. Proteomics is the large-scale
study of proteins, usually by biochemical methods. Proteomic techniques have
been applied to explore bacterial proteomes. New methodologies are being
developed (141,142). In proteomic studies of microorganisms, two main
approaches can be envisaged. The first one is to establish a systematic cartography
of a bacterium in a given state. The second one is a differential approach,
consisting of comparing protein patterns of a given strain, submitted to different
environmental conditions (143). A few proteomic studies have been devoted to
intestinal bacteria. Recently, proteomic profiles of Bifidobacterium infantis (144)
and B. longum (145) were described. Bifidobacterium is one of the predominant
bacterial groups in the human colon and generally regarded as health-promoting
Recently, metagenomics has emerged as a powerful tool to study the intestinal
microbiota. Metagenomics has been defined as the science of biological diversity.
It combines the use of molecular biology and genetics to identify and characterize
genetic material from complex microbial environments. A full metagenomic
approach is a comprehensive study of nucleotide sequence, structure, regulation,
and function, providing a picture of the dynamics of complex microbial
communities. The combination of metagenomics and subsequent quantification of
each identified species using molecular techniques allows the relatively rapid
analysis of a whole bacterial population, including uncultured microorganisms
(147,148). With metagenomic analysis, Gill et al. defined the gene content and
encoded functional attributes of the gut microbiome in healthy humans (149).
Manichanh et al. observed reduced diversity of fecal microbiota in patients with
Crohn’s disease with a metagenomic approach (150).
3.4 Modulating the colonic microbiota with pre-, pro-, and synbiotics to
alleviate lactose intolerance
The human gut microbiota influences health and well-being through its
involvement in nutrition, pathogenesis and immunology of the host (112). The
targeted use of dietary supplementation of e. g. pre-, pro- and synbiotics, may
modulate the composition and some metabolic activities of the colonic microbiota
such that certain health-benefits or remedial effects can be achieved (151,152).
Probiotics are defined as “a preparation of or a product containing viable, defined
microorganisms in sufficient numbers, which alter the microflora (by implantation
or colonization) in a compartment of the host and by that exert beneficial health
effects in this host” (153). In several reviews (15,154,155), probiotics are regarded
to be able to improve lactose digestion and eliminate symptoms of intolerance. The
mechanisms by which probiotics exert their effects are not clear, but may involve
modifying gut pH, providing bacterial ß-galactosidase, positive effects on intestinal
functions and colonic microbiota. However, in a systematic review by Levri et al.
(156), it is concluded that probiotic supplementation in general did not alleviate
the symptoms and signs of lactose intolerance in adults.
Prebiotics are non-digestible food ingredients that beneficially affect the host by
selectively stimulating the growth and/or activity of one or a limited number of
bacterial species already resident in the colon, and thus attempt to improve host
health (112). The traditional targets for prebiotics are Bifidobacterium spp. and
Lactobacillus spp. Recently, Palframan et al. (157) devised a prebiotic index for in
vitro comparison of the prebiotic effect of different oligosaccharides. The prebiotic
index takes into account the levels of bifidobacteria, lactobacilli, clostridia and
bacteroides. Most prebiotic research has been done with ?(2–1) fructans, but
prebiotic potential has also been shown for galacto-oligosaccharides, xylo-
oligosaccharides, soyabean oligosaccharides, polyols and polydextrose (158-160).
The term synbiotic is used when a product contains both probiotics and prebiotics.
Because the word alludes to synergism, this term should be reserved for products in
which the prebiotic compound selectively favors the probiotic compound (153).
This combination could improve the survival of the probiotic organism, because its
specific substrate is readily available for its fermentation, and result in
advantagesto the host that the live microorganism and prebiotic offer (151).
Scope of the thesis
Colonic metabolism of lactose, in addition to lactase activity and transit in the
small intestine, might be involved in the pathophysiology of lactose intolerance.
However, few studies have been conducted to investigate this topic. This
dissertation is mainly devoted to describing the possible role of composition and
metabolic activities of the colonic microbiota in lactose intolerance and the
possibility to modulate the colonic microbiota with targeted use of dietary
supplementations. Results of these studies may not only aid to design strategies for
dietary management of lactose intolerance, but will also lend more understanding
to colonic metabolism of other undigestible carbohydrates, such as prebiotics, and
the health relevance of this process. For studying colonic metabolism of lactose,
proteomic techniques can be promising tools for interpreting the metabolic
pathways. Furthermore, studies on the small intestinal transit and digestion of
lactose would help to clarify possible involvement of the colon in lactose
The aims of this dissertation were:
(i) to study transit and digestion of lactose in the small intestine for clarifying
possible involvement of the colon in lactose intolerance;
(ii) to investigate whether the composition and metabolic activities of the
colonic microbiota play a role in lactose intolerance;
(iii) to investigate effects of dietary supplementation of pre- and probiotics on
the colonic microbiota and lactose-induced symptoms;
(iv) to explore proteomic techniques for studying metabolism of lactose by the
colonic microbiota and for monitoring the alteration in metabolic activities
of the colonic microbiota during dietary supplementation.
To address the above questions, the following studies have been carried out:
Scope of the thesis
1. Transit and digestion of lactose in the small intestine in lactose
digesters and maldigesters
To investigate the effect of lactose on OCTT and the possible mechanisms
underlying the effects, degree of lactose digestion in the small intestine (indicated
with lactose digestion index, LDI) and OCTT of lactose and glucose were
compared between lactose digesters (n=13) and maldigesters (n=20). The two
parameters were also compared between well-classified lactose tolerant (n=7) and
intolerant maldigesters (n=5) to investigate whether the difference in onset of
symptoms could result from differences in digestion and transit of lactose in the
small intestine (Appendix 1).
2. Do composition and metabolic activities of the colon microbiota play
a role in lactose intolerance?
2.1 Bacteria with ß-galactosidase activity in feces from lactase non-persistent
ß-galactosidase is the bacterial enzyme that catalyzes the first step of lactose
fermentation in the colon and is often measured as an indication of the capacity of
the colonic microbiota to ferment lactose. The percentage and composition of
bacteria in feces with ß-galactosidase activity were determined in 28 lactase non-
persistent subjects and compared between lactose tolerant (n=7) and intolerant
subjects (n=5). The method used to determine those bacteria combines a colony-lift
filter assay with X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) as
substrate for differentiation and the fluorescent in situ hybridization technique for
identification (Appendix 2).
2.2 Comparison of the colonic capacity to ferment lactose in vitro between
lactose tolerant and intolerant subjects
Feces from well-classified lactose tolerant (n=7) and intolerant subjects (n=5) was
incubated with lactose in vitro for 5 h. During the incubation, medium was
Scope of the thesis
collected for measurement of lactose, glucose, galactose, SCFA and D- and L-
lactate. Fecal bacterial composition was determined by FISH (Appendix 3).
3. Effects of yogurt and bifidobacteria supplementation on the colonic
microbiota and lactose-induced symptoms in lactose intolerant
After lactose intolerant subjects (n=11) had ingested yogurt and bifidobacteria for 2
w, ß-galactosidase activity in their feces was measured and fecal bacterial
composition was determined by FISH. PCR and DGGE were used to study
dynamics of the bifidobacterial population in feces. Furthermore, the effects of the
supplementation on the endogenous lactase activity in the small intestine and
lactose-induced symptoms were studied as well (Appendix 4).
4. Exploring proteomic techniques for studying lactose metabolism by
the colonic microbiota
Bifidobacterium was chosen as a model bacterium for studying fermentation of
lactose by the colonic microbiota. Differences in cytoplasmic protein expression of
Bifidobacterium animalis, breve and longum grown on different carbohydrates
(lactose, glucose, glactose) were analyzed with the SELDI-TOF MS Proteinchip
technology and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE). After fractionation by SDS-PAGE, differentially-expressed proteins
among different carbohydrates were identified with LC-MS/MS (Appendix 5).
Results and discussion
1. Fermentation of lactose by the colonic microbiota may play a role in
The lactose tolerant and intolerant subjects did not differ in LDI or OCTT of
lactose, which suggests the involvement of other pathologic mechanisms in lactose
intolerance. We hypothesize that colonic metabolism of lactose is one of these
mechanisms (Appendix 1). During the in vitro incubation of feces with lactose,
the lactose intolerant group produced D- and L-lactate, acetate, propionate and
butyrate significantly faster than the tolerant group. In the intolerant group, the
amount of acetate, propionate, butyrate and L- lactate produced was higher than
that in the tolerant group. The results indicate that a faster and higher production of
microbial intermediate and end metabolites during colonic fermentation of lactose,
may be related to the development of lactose-induced symptoms (Appendix 3).
However, the degree and rate of lactose hydrolysis in the colon does not play a role.
During colonic fermentation, lactose is first hydrolyzed to glucose and galactose,
which is catalyzed by ß-galactosidase. We found that bacterial ß-galactosidase
activity is abundant in the colon as 80.6% (mean, SD: 12.1, range: 47.8%-100%) of
the cultured fecal bacteria possess this activity. The lactose tolerant and intolerant
subjects did not differ in the percentage or composition of the bacteria with ß-
galactosidase activity or ß-galactosidase activity in feces (Appendix 2). We
assume that lactose itself will not present a large osmotic burden to the colon as it
should be quickly degraded by the colonic microbiota. This assumption is
supported by observations that the tolerant and intolerant groups did not differ in
the rate or degree of hydrolysis of lactose or production of glucose and galactose
(Appendix 3). We propose that after lactose is hydrolyzed, the subsequent
fermentation of glucose and galactose may play a role in the pathophysiology of
Whether colonic fermentation of lactose would influence lactose intolerance,
either aggravates or alleviates it, depends on the balance between the ability of the
colonic microbiota to ferment lactose and the ability of the colon to remove the
fermentation metabolites. We assume that the absorption rate of the colon is not
sufficient to remove all the SCFA and other metabolites produced from rapid
Results and discussion
fermentation of lactose. This leads to temporary accumulation of SCFA and other
metabolites in the colon, which plays a role in the onset of lactose-induced
symptoms possibly through their osmotic load, altering intestinal motility or
causing colonic hypersensitivity.
The in vitro results suggest that colonic fermentation of lactose may play a role
in lactose intolerance. This provides the basis for the following studies: (i) to
investigate whether the colonic microbiota could be modulated by dietary
supplementation for the purpose of alleviating symptoms; (ii) to explore
proteomics techniques to study metabolic pathways of lactose fermentation by the
colonic microbiota; (iii) to design an in vivo study to verify the observations of the
in vitro study.
2. Yogurt and bifidobacteria supplementation modifies the colonic
microbiota and alleviates lactose-induced symptoms in lactose
Our results suggest that colonic fermentation of lactose may play a role in lactose
intolerance (Appendix 3). This raises the question whether we can modulate the
composition and metabolic activities of the colonic microbiota in such a way that
lactose intolerance could be attenuated. In this study (Appendix 4), 2-w
supplementation of probiotic bacteria Bifidobacterium longum and a yogurt
enriched with Bifidobacterium animalis increased the numbers of total cells, total
bacteria and Eubacterium rectale/Clostridium coccoides group and ß-galactosidase
activity in feces of lactose intolerant subjects. The supplementation did not increase
the endogenous lactase activity in the small intestine. Symptoms of lactose
intolerance decreased after the supplementation. The increase in bacterial numbers
could be attributed to the lactose contained in the yogurt which can be considered
as a prebiotic for people with lactose maldigestion (162,163). Reduction in
symptoms could be caused by adaptation to lactose consumption (80) and changes
in the composition and metabolism of the colonic microbiota. In conclusion,
supplementation of yogurt and a probiotic strain modified the amount and probably
the metabolic activities of the colonic microbiota of lactose intolerance subjects.
The changes in the colonic microbiota may play a role in alleviation of intolerant
3. Exploring proteomic techniques for studying lactose metabolism by
the colonic microbiota
In the above studies (Appendix 2-4), the composition of the colonic microbiota
was determined by FISH, DGGE and an X-gal assay, and the metabolic activities
of the colonic microbiota were investigated by in vitro incubation and by
determination of enzyme activities. Development of new techniques will facilitate
the studies on the role of the colonic microbiota in health and disease. Proteomic
techniques might aid to interpret the metabolic pathways of lactose metabolism by
the colonic microbiota. In this study (Appendix 5), the SELDI-TOF MS
Proteinchip technology and sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) could discern differences in protein expression of
bifidobacteria grown on lactose, glucose and galactose. With LC-MS/MS, proteins
related to Bifidobacterium were identified, which included enzymes for metabolism
of lactose, glucose and galactose. The applied approaches are promising in
studying metabolism of lactose and other substrates in a complex bacterial
ecosystem such as the colonic microbiota, but need further development. For
instance, when fractionation by SDS-PAGE and identification by LC-MS/MS is
combined with stable isotope labeling, it will facilitate identification of proteins of
which expression is induced by specific substrates.
4. Lactose accelerates the oro-cecal transit in lactose maldigesters
Studies on digestion and transit of lactose in the small intestine would help to
clarify possible involvement of the colon in lactose intolerance. In the study
(Appendix 1), we observed that ompared to glucose, lactose triggers a faster transit
in the small intestine in lactose maldigesters, but not in digesters. The accelerated
transit is not the result of intestinal distention caused by osmotic load from
malabsorbed lactose as suggested earlier (31,97). Based on LDI, we estimated the
Results and discussion
amount of fluid the maldigested lactose would attract to the intestine in lactose
digesters and maldigesters. The amounts are unlikely to cause intestinal distention
and the difference in the amount between digesters and maldigesters is unlikely to
cause the difference in OCTT. We hypothesize that presence of maldigested lactose
in the intestinal lumen plays a role in the alteration of intestinal transit by affecting
the intrinsic factors that regulate intestinal motility. Postprandial motility of the
gastrointestinal tract is controlled by nerves, hormones and paracrine mediators
(164). It might be possible that undigested lactose alters the motility of the intestine
by stimulating the secretion of certain gastrointestinal hormones, or by stimulating
the neural activities of certain chemosensitive receptors or osmoreceptors.
We speculate that this hypothesis can be extended to explain the accelerated
intestinal transit in malabsorption of other sugars and some food components, for
instance, fructose and sorbitol (165-170). Malabsorption of lactose, fructose and
sorbitol can be related, as fructose and sorbitol malabsorption are common when
lactose malabsorption is present (168).
The lactose tolerant and intolerant subjects did not differ in degree of lactose
digestion (LDI) or OCTT of lactose. This suggests the involvement of other
pathologic mechanisms in lactose intolerance, e.g. the colonic metabolism of
Comparison of colonic metabolism of lactose in vivo between lactose tolerant
and intolerant subjects using stable isotopes
Our in vitro study indicates that a faster and higher production of microbial
intermediate and end metabolites during colonic fermentation of lactose, may play
a role in lactose-induced symptoms (Appendix 3). However, the in vitro system
may not be a perfect reflection of the in vivo situation. The culturing conditions are
different from those in the colon. Colonic factors, e.g. removal of the metabolites,
gut secretions and immunology and interaction with mucosal cells, are not studied.
Therefore, our in vitro observations need to be verified by in vivo studies.
In vivo studies on colonic metabolism of certain substrates with humans are
scant as they are hampered by difficulties in sampling in situ and quantitative
delivery of substrates to the colon. To circumvent these difficulties, several
approaches using stable isotopes can be considered.
1. Oral administration of 13C-lactose-ureide
Background: The human gut tissue possesses no allantoicase-like activity to split
the bond between glycosyl and ureide (171). Therefore, glycosyl ureides cannot be
absorbed in the small intestine. In the colon, glycosyl ureides can be degraded by
Clostridium innocuum strains which belong to the normal intestinal microbiota of
infants and adults (172). After the glycosyl-ureide bond is split, the glycosyl and
ureide will be further metabolized by colonic bacteria. Glycosyl ureides are used as
a marker for measurement of the OCTT (173).
Approaches: Tracer amount of 13C-lactose-ureide and 25 g of lactose dissolved in
water will be administered orally in lactose maldigesters. Peripheral blood samples
will be collected for measurement of 13C-acetate. Breath samples will be collected
for determination of OCTT.
intestine and will enter the colon. After the lactose ureide bond is split, 13C-lactose
13C-lactose-ureide is not absorbed in the small
Results and discussion
will be fermented by the colonic bacteria together with the maldigested unlabeled
lactose. The kinetics of 13C-acetate in the peripheral blood will reflect the kinetics
of colonic fermentation of maldigested lactose.
Disadvantages: The hydrolysis of the lactose-ureide bond by bacterial enzymes is
the rate-limiting step in bacterial degradation of lactose-ureide (174). However,
there is no detailed information available on how long this step takes or whether
the time varies among individuals. If there is a large inter-individual variation, the
fermentation of 13C-lactose-ureide cannot represent the fermentation of lactose.
2. Isotope-dilution technique
Background: The principle of the isotope-dilution technique is as following: in a
closed volume at steady-state (production and elimination rates are at equilibrium),
the labeled tracer is infused at a constant and known rate until a new steady-state is
reached. Blood samples are collected at regular intervals. From the isotopic
enrichment in blood at steady-state the production or elimination rates of the
metabolite of interest can be calculated (139). The isotope-dilution technique has
been used to quantitatively estimate colonic fermentation of non-digestible
Approaches: Lactose maldigesters will receive a primed, constant and intravenous
infusion of [l-13C]acetate for 7 h. 25 g of lactose will be ingested 1 h after the tracer
infusion starts. Arterialized venous blood samples will be collected for
determination of the total production and the production rate of [l-13C] acetate.
Disadvantages: As absorption of lactose in the small intestine varies among lactose
maldigesters, the amount of lactose entering the colon varies. The amount of
lactose in the colon may influence the rate of bacterial fermentation. Therefore, the
possible differences in total production and the production rate of [l-13C] acetate
measured in blood can be derived from the difference in amount of lactose entering
Results and discussion
3. Colon-delivery capsules
Various colon-specific drug delivery systems have been developed (176). The
release triggering mechanisms of these capsules can be pH- or time-dependent,
microbiota-activated, pressure-dependent, etc. However, the delivery capacity of
these systems may not be sufficient to deliver the amount of lactose averagely
maldigested after ingestion of 25 g of lactose in maldigester (~20 g).
4. Colon-infusion catheters
Different types of catheters have been implanted into the colon for various
purposes (104,177). Although the procedure can be experience-dependent and
somewhat invasive, colon-infusion catheters allow quantitative delivery of large
amounts of substrates. Therefore, for our purpose of studying colonic fermentation
of lactose in vivo, colon-infusion catheters can be an appropriate approach. An in
vivo study on colonic fermentation of lactose using stable isotopes delivered via a
colon-infusion catheter is proposed (Appendix 6).
The results presented in this dissertation suggest that colonic metabolism of lactose,
in addition to the small-intestinal lactase activity and transit, may play a role in the
pathophysiology of lactose intolerance. During fermentation of lactose by the
colonic mictobiota, the rate and magnitude of production of metabolites could be
among the factors contributing to the onset of lactose-induced symptoms. This
observation implies that in studies on colonic fermentation of carbohydrates, i.e.
prebiotics, the rate and magnitude of fermentation can be of health relevance.
Dietary supplementation of pre-, pro- or synbiotics provides a promising
approach for dietary management of lactose intolerance.
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