Aliment Pharmacol Ther 2000; 14: 1511±1518.
Hypochlorhydria induced by a proton pump inhibitor leads
to intragastric microbial production of acetaldehyde from ethanol
È KEVA
È INEN *, J. TILLONEN*, M. SALASPURO*, H. JOUSIMIES-SOMER , H. NUU TINENà
S. VA
È R KKILA
Èà
& M. FA
*Research Unit of Alcohol Diseases, Helsinki University Central Hospital, Helsinki, Finland; Anaerobe Reference Laboratory,
National Public Health Institute, Helsinki, Finland; and àDepartment of Gastroenterology, Helsinki University Central
Hospital, Helsinki, Finland
Accepted for publication 21 July 2000
SUMMARY
Background: Acetaldehyde, produced locally in the
digestive tract, has recently been shown to be carcinogenic in humans.
Aim: To examine the effect of iatrogenic hypochlorhydria on intragastric acetaldehyde production from
ethanol after a moderate dose of alcohol, and to relate
the ®ndings to the changes in gastric ¯ora.
Methods: Eight male volunteers ingested ethanol
0.6 g/kg b.w. The pH, acetaldehyde level and microbial
counts of the gastric juice were then determined. The
experiment was repeated after 7 days of lansoprazole
30 mg b.d.
Results: The mean (‹ S.E.M.) pH of the gastric juice was
1.3 ‹ 0.06 and 6.1 ‹ 0.5 (P < 0.001) before and after
INTRODUCTION
Due to its acidity, the stomach is usually free of
microbes. However, oral microbes may survive in the
stomach with a pH above 4.0 and bacterial proliferation
can be expected when the pH rises over 5.0.1, 2
Therefore, gastric and duodenal microbial overgrowth
is a common ®nding during long-term use of gastric
proton pump inhibitors or H2-receptor antagonists, as
Correspondence: Professor M. Salaspuro, Research Unit of Alcohol Diseases,
Helsinki University Central Hospital, PL 345, Tukholmankatu 8 F, 00029
HUS, Finland.
E-mail: mikko.salaspuro@hus.®
Ó 2000 Blackwell Science Ltd
lansoprazole, respectively. This was associated with a
marked overgrowth of gastric aerobic and anaerobic
bacteria (P < 0.001), by a 2.5-fold (P ˆ 0.003) increase
in gastric juice acetaldehyde level after ethanol ingestion, and with a positive correlation (r ˆ 0.90,
P < 0.001) between gastric juice acetaldehyde concentration and the count of aerobic bacteria.
Conclusions: Treatment with proton pump inhibitors
leads to hypochlorhydria, which associates with intragastric overgrowth of aerobic bacteria and microbiallymediated acetaldehyde production from ethanol. Since
acetaldehyde is a local carcinogen in the concentrations
found in this study, long-term use of gastric acid
secretory inhibitors is a potential risk-factor for gastric
and cardiac cancers.
well as in certain other similar conditions, e.g. achlorhydria caused by atrophic gastritis.3±8
Via alcoholic fermentation mediated by microbial
alcohol dehydrogenases normal gut ¯ora, bacterial
overgrowth and yeast infection can be responsible for
endogenous ethanol production in the digestive tract of
both experimental animals and humans.9±12 Small
amounts of endogenous ethanol have also been found
in the gastric juice of patients receiving cimetidine.13
Under aerobic and microaerobic conditions and in the
presence of excess alcohol the microbial alcohol
dehydrogenase reaction runs in the opposite direction
resulting in marked acetaldehyde production both
in vitro and in vivo.14±16
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È KEVA
È INEN et al.
S. VA
Many recent epidemiological studies have shown that
the risk of ethanol-associated digestive tract cancers is
greatly increased in Asian subjects with a genetically
de®cient ability to metabolize acetaldehyde.17±23 After
adjustment for age, daily alcohol consumption, and
amount of cigarette smoking, signi®cantly increased
risks (odds ratios) in the presence of mutant aldehyde
dehydrogenase-2 have been found for oropharyngolaryngeal (11.1), oesophageal (12.5), stomach (3.5),
colon (3.4), lung (8.2) and oesophageal cancer concomitant with oropharyngolaryngeal and/or stomach
cancer (54.2).20 Very recently, we found that individuals with this gene de®ciency have two to three times
higher in vivo salivary acetaldehyde levels after a
moderate dose of alcohol than those with normal
aldehyde dehydrogenase-2.24 Accordingly, aldehyde
dehydrogenase-2-de®cient heavy drinkers are exposed
for years or decades to markedly higher local acetaldehyde levels than those with the normal aldehyde
dehydrogenase-2 genotype. When this information is
combined with the above mentioned epidemiological
data, our ®nding provides strong evidence for the local
carcinogenic action of acetaldehyde in humans. Therefore, all conditions possibly associating with enhanced
local production of acetaldehyde can be considered as
potential risk-factors for upper digestive tract cancers.
The aim of this study was to examine in humans the
effect of 1 week of treatment with a proton pump
inhibitor on gastric ¯ora and its capacity to produce
acetaldehyde from ethanol in vivo after ingestion of a
moderate dose of alcohol.
MATERIALS AND METHODS
Hospital, and an informed consent to participate in the
study was obtained from the subjects. A paired study
design in which each subject served as his own control
was used. Two study days were separated by a 1-week
interval. The volunteers fasted for at least for 6 hours
before the study. The participants were admitted to the
Department of Gastroenterology of Helsinki University
Central Hospital, and all studies started between 13:30
and 14:00 hours. Ethanol (0.6 g/kg body weight) was
diluted in water at 15% v/v concentration. The alcohol
had to be consumed within 20 min, and afterwards the
volunteers stayed on their left sides for 40 min to avoid
total gastric emptying before the gastroscopy was
performed. Just before the endoscopy 2 mL of paraf®n
stimulated saliva was collected from each volunteer.
Gastroscope (Olympus, GIF-Q140) was lubricated with
Lidocain gel (Orion, Medipolar, Oulu, Finland), containing no ethanol, and gastroscopy was performed. Gastric
juice was aspirated into collectors immediately after the
endoscope entered the stomach. Thereafter, gastric juice
was transferred into gas chromatograph vials as
described below, and to cryovials that were frozen to
± 80 °C for later microbial analysis. The pH of the
obtained gastric juice was determined using a glass
electrode and a digital pH meter (WTW pH-521,
Weilheim, Germany). During the 7 days between the
experiments, the volunteers received 30 mg lansoprazole (Lanzo, Wyeth Lederle Nordiska Ab, Solna, Sweden)
orally b.d. The drug intake was started on the evening of
the ®rst study day and the last tablet was taken on the
morning of the second study day. Experiments were
exactly the same on both study days, with the exception
that routine gastric mucosal biopsies were only taken
during the ®rst endoscopy.
Subjects
Eight healthy men, with an age range of 21±25 years
volunteered for the study. Their mean body weight was
73 ‹ 3 kg and body mass index 22.7 ‹ 0.7 kg/m2.
None of the subjects had received any antibiotics for
4 weeks preceding the study or was using any other
drugs during the study days. One of the volunteers was a
light smoker and all were normal social drinkers, with an
average consumption of 90 g or less of ethanol per week.
Study design
The study was approved by the Ethical Committee of the
Department of Medicine, Helsinki University Central
In vivo intragastric and salivary acetaldehyde production
In order to measure acetaldehyde production capacity,
450 lL of gastric juice or saliva was immediately
transferred into a vial, containing 50 lL of 6 M
perchloric acid to stop the acetaldehyde production.
Acetaldehyde and ethanol were analysed by using headspace gas chromatography as described previously.25
Six parallel samples were used for measurements, and
the mean value was calculated. To control for nonenzymatic artefactual acetaldehyde formation from
ethanol during protein precipitation, perchloric acid
was added simultaneously with ethanol into additional
incubation vials. Acetaldehyde concentration of these
Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 1511±1518
INTRAGASTRIC PRODUCTION OF ACETALDEHYDE
1513
control samples were subtracted from in vivo acetaldehyde values.
Microbial analysis
The gastric juice samples were thawed, and diluted in
peptone yeast extract broth. An aliquot of 100 lL of the
undiluted sample and its 100-fold dilutions were
inoculated and spread on several selective and nonselective agar media for enumeration and isolation of
total counts and main groups of aerobic and anaerobic
bacteria and yeasts. The aerobic plates were incubated
at 35 °C in an atmosphere containing 5% CO2 for up to
5 days; anaerobic plates were incubated in anaerobic
jars ®lled with the evacuation replacement method with
mixed gas (90% N2, 5% CO2, 5% H2) for 7 days for the
®rst inspection and up to 14 days for the ®nal
inspection. The bacteria were enumerated and identi®ed
by established methods.26,27
Figure 1. The effect of lansoprazole treatment (30 mg b.d.) for
7 days on the pH of the gastric juice in eight volunteers after
ethanol (0.6 g/kg b.w.) ingestion.
Statistical analysis
The statistical signi®cance of the differences before and
during lansoprazole intake was analysed by paired
t-test. Logarithmic transformation was performed when
appropriate. The possible correlations were tested by
using Spearman Rank Order Corralation. A P-value less
than 0.05 was considered to be signi®cant. The results
are expressed as mean ‹ S.E.M.
RESULTS
Lansoprazole treatment raised the mean (‹ S.E.M.) pH
level of the gastric juice from 1.3 ‹ 0.06 to 6.1 ‹ 0.5,
P < 0.001 (Figure 1). This was associated with a
signi®cant increase in the mean intragastric acetaldehyde level from 22.1 ‹ 2.3 lM to 55.4 ‹ 8.0 lM,
P ˆ 0.003. The intragastric acetaldehyde levels
increased in all volunteers during the medication; the
highest measured level was 100.5 lM (Figure 2). The
mean ethanol concentration of the gastric juice was
1.7% (range 0.7±3.4%) and 2.6% (range 1.2±4.1%),
P ˆ 0.054, non-signi®cant, before and during the
treatment, respectively. Lansoprazole did not change
the mean salivary acetaldehyde level (44.7 ‹ 6.8 lM
before the medication and 36.1 ‹ 6.4 lM during it,
P ˆ 0.21, non-signi®cant).
Before the medication, minor growth of aerobic bacteria was detected in the gastric juice of two volunteers,
Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 1511±1518
Figure 2. The effect of lansoprazole treatment (30 mg b.d.) for
7 days on the acetaldehyde level of the gastric juice in eight volunteers after ethanol (0.6 g/kg b.w.) ingestion.
1 ´ 101 and 2 ´ 101 colony forming units(cfu)/mL, the
other aerobic cultures were negative. The anaerobic
bacterial cultures of the gastric juices before the
treatment were all negative. During lansoprazole, the
total aerobic counts were 1.3 ‹ 0.7 ´ 106 cfu/mL and
the total anaerobic counts were 1.5 ‹ 0.8 ´ 106 cfu/mL.
The increase in both total counts was highly signi®cant
(P < 0.001). The cultures of yeasts were negative both
before and during lansoprazole treatment. A vast
selection of oral bacterial species were present; Table 1
summarizes the bacteriological results. There was also a
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È KEVA
È INEN et al.
S. VA
Aerobes
Total counts
Stomatococci spp.
Viridans group Streptococci
Neisseria spp.
Corynebacterium spp.
Staphylococcus aureus
Coagulase-negative Staphylococci
and Micrococci spp.
Other**
Other gpb***
Anaerobes
Total counts
Pigmented Prevotella spp.
Actinomyces spp.
Fusobacterium spp.
Bacteroides ureolyticus-like group
Lactobacillus spp.
Nonpigmented Prevotella spp.
Anaerobic cocci
Capnocytophaga spp.
Before
lansoprazole
During
lansoprazole
negative*
negative
negative
negative
negative
negative
negative
1.3
1.4
1.0
1.3
4.8
6.8
1.0
negative
negative
6.3 ´ 103
3.8 ´ 103
negative
negative
negative
negative
negative
negative
negative
negative
negative
1.5
1.3
5.5
1.0
5.1
1.4
1.4
2.8
3.4
´
´
´
´
´
´
´
´
´
´
´
´
´
´
´
´
106
105
106
104
103
101
103
106
105
104
104
103
105
104
105
101
Prevalence
8/8
7/8
7/8
4/8
4/8
1/8
Table 1. The effect of lansoprazole on the
mean gastric bacterial counts (cfu/mL) in
eight volunteers
(100%)
(87.5%)
(87.5%)
(50%)
(50%)
(12.5%)
1/8 (12.5%)
1/8 (12.5%)
6/8
6/8
5/8
4/8
3/8
2/8
2/8
1/8
(75%)
(75%)
(62.5%)
(50%)
(37.5%)
(25%)
(25%)
(12.5%)
*Detection threshold 101 cfu/mL.
**Haemophilus parain¯uenzae.
***Bacillus spp.
highly signi®cant positive correlation (r ˆ 0.90,
P < 0.001) between the individual total aerobic bacterial counts and the individual gastric juice acetaldehyde
levels during the medication (Figure 3A). The correlation between individual acetaldehyde levels and anaerobic bacterial counts was also positive (r ˆ 0.76,
P ˆ 0.021) (Figure 3B).
Histologically gastric mucosal biopsies were within
normal limits in all of the volunteers, and all of them
were Helicobacter pylori-negative.
DISCUSSION
It has been shown that acetaldehyde can be formed
from ethanol in vitro by incubating human bronchopulmonary washings, and in vivo in mouthwashes of
ethanol-rinsing volunteers, both thought to be of
bacterial origin.28, 29 Moreover, we have shown the
production of marked amounts of acetaldehyde in the
saliva after moderate alcohol ingestion.14 This acetaldehyde production can be signi®cantly reduced by the
use of antiseptic chlorhexidine mouthwash, indicating a
microbial background for acetaldehyde production from
ethanol by oral microbes.14 In this study salivary
acetaldehyde levels after alcohol ingestion were comparable to those reported earlier.14
Diminished gastric acid secretion is associated with an
increased number of microbes in the gastric juice.8,30
This was con®rmed in this study. The baseline aerobic
and anaerobic cultures of bacteria and yeasts from the
acidic gastric juices were nearly negative, which also
con®rms earlier ®ndings.8, 30±31 During the induction of
hypochlorhydria with the proton pump inhibitor, both
aerobic and anaerobic bacteria were found more
frequently and in higher loads from the gastric juices.
Stomatococcus, Streptococcus and Neisseria species were
the most prevalent and numerous aerobic species. Since
these bacteria are common species colonizing the
oropharynx, the microbes that are able to survive in
the neutral gastric milieu originate from the oral cavity.
This is in accordance with earlier ®ndings.31
The characteristics of microbially mediated acetaldehyde production from ethanol both in vitro and in vivo
have been established in several studies.14±16, 25,32±35
This is the ®rst study to show the in vivo acetaldehyde
production from ethanol by the microbial overgrowth in
Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 1511±1518
INTRAGASTRIC PRODUCTION OF ACETALDEHYDE
Figure 3. (A, B) Correlation between individual total aerobic and
anaerobic bacterial counts and individual gastric juice acetaldehyde
levels in eight volunteers after ethanol (0.6 g/kg b.w.) ingestion and
during lansoprazole (30 mg b.d.) treatment.
the hypochlorhydric stomach. Acetaldehyde production
was enhanced in all our volunteers after lansoprazole
treatment and this paralleled with the increased number of microbes in the gastric juice. In fact, we were able
to show a highly signi®cant positive correlation between
the individual total aerobic bacterial counts and the
individual acetaldehyde levels during the medication.
This correlation was more obvious than the one
between acetaldehyde levels and the number of total
anaerobic bacteria. This is in line with our earlier
®ndings, which show that, by and large, only aerobes
representing normal human gut ¯ora possess alcohol
dehydrogenase activity.15,34
The subjects, who had the lowest gastric juice pH
levels and therefore low bacterial counts after the
Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 1511±1518
1515
medication, had only slight increases in their gastric
juice acetaldehyde levels. These ®ndings suggest that
acetaldehyde in the stomach is produced by oral
aerobic microbes which are able to grow in the
neutral gastric milieu. However, small amounts of
acetaldehyde were also detected from the acidic gastric
juices. Part of the acetaldehyde that is formed from
ethanol in the oral cavity is evidently distributed with
saliva via the oesophagus to the stomach. Since no
microbes were able to grow in the acidic gastric juice,
it can be assumed that acetaldehyde in the acidic
stomach is most probably originated from the oral
cavity. This concept is strongly supported by our
earlier ®ndings indicating that microbial acetaldehyde
production is almost totally abolished at pH 4.0 or
lower.25
Alcohol consumption is a well-known risk-factor for
upper digestive tract cancers.36±39 However, the
tumour-promoting effect of alcohol has so far been
obscure, since ethanol itself is not carcinogenic. In
contrast, acetaldehyde, the ®rst metabolite of ethanol,
has been shown to have many mutagenic and carcinogenic effects in cell cultures and animal models.40±43
As a highly reactive compound it can, for example, form
adducts with macromolecules, proteins and DNA.44±46
Acetaldehyde adducts can interfere with normal cellular
functions and lead to cellular destruction and promote
carcinogenesis. Moreover, acetaldehyde can cleave
folate, which has been shown to lead to local folate
de®ciency and, thereby, to the hypomethylation of DNA
and the promotion of carcinogenesis.47 Acetaldehyde is
also known to be one of the major components of
tobacco smoke.48
Stronger evidence for acetaldehyde as the major factor
responsible for ethanol-associated carcinogenesis is
derived from recent studies linking the genotypes of
ethanol-metabolizing enzymes to markedly enhanced
tumour risk.17±23, 49,50 In Asian `¯ushers', single point
mutation in the gene that codes low Km aldehyde
dehydrogenase-2 leads to nearly total inactivation of
this enzyme. Thereby, aldehyde dehydrogenase-2-de®cient individuals are exposed after alcohol ingestion to
markedly higher local acetaldehyde levels than those
with the normal aldehyde dehydrogenase-2 enzyme.24
Alcoholics with cancer of oropharynx, larynx, oesophagus, stomach, colon or lungs had markedly higher
frequencies of this mutant aldehyde dehydrogenase2*1/*2 genotype than cancer-free alcoholics.20 Interestingly, microbial acetaldehyde production from
1516
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È INEN et al.
S. VA
ethanol has been described in all these organs.14,28,29,33
This human `knockout model' for de®cient acetaldehyde removal provides strong evidence for the local
carcinogenic action of acetaldehyde in humans.
It should be noted that the gastric juice acetaldehyde
levels found in this study were equal to the salivary
acetaldehyde levels found in aldehyde dehydrogenase-2de®cient subjects after a moderate dose of alcohol.24
Furthermore, many of the in vitro and animal studies
dealing with the carcinogenic action of acetaldehyde
have been carried out with acetaldehyde concentrations
less than 500 lM.43,51,52 Accordingly, acetaldehyde
levels of the gastric juice found in this study can be
considered to be comparable to those suggested to be
carcinogenic in other studies.
It has also been shown that Caucasians with rapid
metabolizing alcohol dehydrogenase (alcohol dehydrogenase-3*1) allele, which leads to higher and quicker
production of acetaldehyde, have greater risk for upper
digestive tract cancers than those with slow metabolizing allele if high amounts of alcohol are consumed.49,50
These studies also provide convincing evidence for the
carcinogenicity of acetaldehyde in humans. Aldehyde
dehydrogenase-2-de®cient genotype does not exist
amongst Finns. The possible role of alcohol dehydrogenase-3*1 allelle on our results remains to be established in future studies.
In conclusion, the use of gastric proton pump inhibitors leads to hypochlorhydria and overgrowth of
bacteria in the gastric juice. This associates with
enhanced intragastric production of acetaldehyde via
alcohol dehydrogenase mediated ethanol oxidation
carried out by the aerobic bacteria representing normal
oral micro¯ora. Since acetaldehyde is a local carcinogen, long-term use of the inhibitors of gastric acid
secretion may increase the risk for upper digestive tract
cancers, especially amongst frequently drinking subjects with aldehyde dehydrogenase-2-de®cient or
alcohol dehydrogenase-3*1 genotype.
ACKNOWLEDGEMENTS
The technical assistance of Mikko Blom for the
microbial analysis is gratefully acknowledged. This
study was supported by grants from the YrjoÈ Jahnsson
Foundation, the Mary and Georg C Ehrnrooth Foundation, the Finnish Foundation for Alcohol Studies,
and the Helsinki University Central Hospital Research
Funds.
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