Expression, localization and potential physiological significanceof alcohol dehydrogenase in the gastrointestinal tract Julia Vaglenova1,*,‡, Susana E.. Martı´nez1,†,‡, Sergio Porte´1, G
Trang 1Expression, localization and potential physiological significance
of alcohol dehydrogenase in the gastrointestinal tract
Julia Vaglenova1,*,‡, Susana E Martı´nez1,†,‡, Sergio Porte´1, Gregg Duester2, Jaume Farre´s1
and Xavier Pare´s1
1
Department of Biochemistry and Molecular Biology, Universitat Auto`noma de Barcelona, Spain;2OncoDevelopmental Biology Program, The Burnham Institute, La Jolla, CA, USA
ADH1 and ADH4 are the major alcohol dehydrogenases
(ADH) in ethanol and retinol oxidation ADH activity and
protein expression were investigated in rat gastrointestinal
tissue homogenates by enzymatic and Western blot analyses
In addition, sections of adult rat gastrointestinal tract were
examined by in situ hybridization and
immunohistochem-istry ADH1 and ADH4 were detected along the whole tract,
changing their localization and relative content as a function
of the area studied While ADH4 was more abundant in
the upper (esophagus and stomach) and lower (colorectal)
regions, ADH1 was predominant in the intestine but also
present in stomach Both enzymes were detected in mucosa
but, in general, ADH4 was found in outer cell layers, lining
the lumen, while ADH1 was detected in the inner cell layers
Of interest were the sharp discontinuities in the expression found in the pyloric region (ADH1) and the gastroduodenal junction (ADH4), reflecting functional changes The precise localization of ADH in the gut reveals the cell types where active alcohol oxidation occurs during ethanol ingestion, providing a molecular basis for the gastrointestinal alcohol pathology Localization of ADH, acting as retinol dehydro-genase/retinal reductase, also indicates sites of active retinoid metabolism in the gut, essential for mucosa function and vitamin A absorption
Keywords: ethanol; immunohistochemistry; in situ hybridi-zation; retinol; retinoic acid
The major pathway for the elimination of ethanol is
through its oxidation to acetaldehyde that occurs mostly in
liver [1], though ethanol metabolism is also significant in
other tissues [2] Alcohol dehydrogenase (ADH) is the main
enzyme responsible for the first step in ethanol elimination
[3] ADH is expressed in several molecular forms, grouped
in five enzymatic classes [4], and four of them have been well
characterized at the protein level in mammals [5,6] In the
rat, ADH1 has a low Kmfor ethanol and it is responsible for
the hepatic ethanol metabolism [7] ADH2 and ADH3 are
not active at moderate concentrations of ethanol [7,8]
ADH4 has high Kmand kcatvalues for ethanol [9], and it is
found in gastrointestinal mucosa, blood vessels, central
nervous system and many epithelia, but it is absent in
normal liver [2,10,11] Moreover, these ADH forms have
retinol dehydrogenase activity [12–17], and recent genetic studies in knockout mice have demonstrated that ADH1, ADH3, and ADH4 participate in the retinoic acid (RA) synthesis pathway [16,18,19]
Previous studies have shown that the rat ADH system is comprised of single isozyme representatives of each class, making it a simpler system to study, compared to the human ADH [5,6] In spite of several reports on the localization of ADH in rodent [2,7,20–22] and human [23–30] gastrointestinal tissues, these works are only partial This paper presents a complete analysis of the whole gastrointestinal tract in the rat: ADH activity levels were measured by spectrophotometric assays, ADH expression pattern by electrophoretic and Western blot analyses, and the localization of ADH (at mRNA and protein levels) in the distinct cell layers of each gastrointestinal region by
in situ hybridization (ISH) and immunohistochemistry (IHC)
1 Our results demonstrate that ADH1 and ADH4
coexist throughout the gastrointestinal tract and provide new data to understand the physiological role of ADH classes in the gastrointestinal tract and the etiopathogeny related to alcohol abuse
Experimental procedures Animals
Adult Sprague–Dawley rats (n¼ 5; male, 200–250 g) were used Animal protocols were approved by the Ethical Committee of the Universitat Auto`noma de Barcelona After decapitation, gastrointestinal organs were removed and processed rapidly as described below
Correspondence to X Pare´s, Department of Biochemistry and
Molecular Biology, Faculty of Sciences, Universitat Auto`noma de
Barcelona, E-08193 Bellaterra, Barcelona, Spain.
Fax: + 34 93 5811264, Tel.: + 34 93 5813026,
E-mail: xavier.pares@uab.es
Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde
dehydrogenase; IHC, immunohistochemistry; ISH, in situ
hybridization; RA, retinoic acid.
Note: These authors made equal contributions to this study.
*Present address: Department of Pharmacal Sciences, 401 Pharmacy
Bldg., Auburn University, Auburn, AL 36849, USA.
Present address: Biology Department, Boston College, 321 Higgins
Hall, 140 Commonwealth Ave., Chesnut Hill, MA 02467, USA.
(Received 27 February 2003, revised 21 April 2003,
accepted 28 April 2003)
Eur J Biochem 270, 2652–2662 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03642.x
Trang 2ADH activity assay and starch gel electrophoresis
Tissues from gastrointestinal tract were dissected carefully
and subsequently washed in ice-cold homogenization
buffer (50 mM sodium phosphate, pH 7.6, 0.5 mM
dithio-threitol) The specimens were cut into small fractions
and homogenized at 4C Crude homogenates were
centrifuged (24 000 g, 4C, 30 min) and supernatants
were used for activity assay or analysis by starch gel
electrophoresis [2] After electrophoresis, gels were stained
for ADH activity using 100 mM 2-buten-1-ol as a
substrate Also, ADH activity of homogenates was
monitored at 340 nm in a UV-VIS spectrophotometer
(Cary 400Bio; Varian), in 0.1MNaCl/Pi, pH 7.5, 2.4 mM
NAD+, at 25C, using 10 mM ethanol or 1M ethanol
as a substrate At 10 mM ethanol, we determined the
contribution of ADH1 (Km¼ 1.4 mM, kcat¼ 40 min)1)
[7] At 1Methanol, the observed activity was mainly due
to ADH4 (Km¼ 2.4M, kcat¼ 2600 min)1) [9] At this
ethanol concentration, ADH1 shows substrate inhibition
[31] and the contribution of ADH3 is still negligible
because of its extremely low activity at pH 7.5 [7] One
activity unit corresponds to the reduction of 1 lmol
NAD per min Protein concentrations were estimated by
the method of Bradford [32] using bovine serum albumin
as standard
In situ hybridization analysis (ISH)
Generation of ADH1 and ADH4 specific sense and
antisense riboprobes was performed as reported previously
[11] The gastrointestinal tract was removed and divided
into regions corresponding to the various tissues After
dissection, digestive samples were immediately rinsed in
NaCl/Pi(0.1M sodium phosphate buffer, pH 7.4, 0.15M
NaCl) and immersed in 4% (w/v) paraformaldehyde
in NaCl/Pifor 12 h The paraffin-embedded tissues were
sliced into serial 8-lm sections using a Leica microtome
and attached to coated microscope slides I n situ
hybrid-ization and subsequent immunochemical chromogenic
detection of digoxigenin-labeled hybrids was performed
as previously described [11] The hybridization signal
corresponding to each probe appeared highly specific, as
demonstrated by the negative controls performed with the
sense RNA probes
Protein immunoblotting and Western blot analysis
Homogenates were prepared from fresh adult rat tissue
as reported previously [11], except that 1 mM
phenyl-methanesulfonyl fluoride, 1 lgÆmL)1 leupeptin, and
1 lgÆmL)1 pepstatin were added as protease inhibitors
Protein blots were incubated with affinity-purified rabbit
antiserum raised against mouse ADH4 (1 : 500) [21]
Immunodetection was carried out using goat anti-(rabbit
IgG)-alkaline phosphatase conjugate (Bio-Rad) for 1 h at
room temperature Alkaline phosphatase activity was
then visualized by incubation with 0.1M Tris/HCl,
pH 9.5, containing 5-bromo-4-chloro-3-indolylphosphate
and nitroblue tetrazolium as substrates according to the
instructions of the Alkaline Phosphatase Conjugate
Substrate kit (Bio-Rad)
Immunohistochemistry Rat gastrointestinal tissues were fixed, processed routinely, and embedded in paraffin as described for ISH Localiza-tion of ADH4 was investigated using affinity-purified antibodies specific for ADH4 [21] diluted to 1 : 500 on serial 5-lm tissue sections Slides were treated with xylene and hydrated through a graded series of decreasing ethanol concentrations Endogenous peroxidase activity was blocked with 1% (v/v) hydrogen peroxide for 15 min After rinsing in Tris/HCl-buffered saline, slides were blocked with 2% (v/v) of normal serum, and the primary antibody was applied for 1 h Biotinylated goat anti-(rabbit IgG) Ig (Dakopatts) was used as a secondary antibody and was visualized by avidin–biotin complex (Strept–ABCom-plex–HRP; Dakopatts; dilution 1 : 400 in blocking solu-tion) with peroxidase detection using the Vectastain Universal Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA) 3,3¢-Diaminobenzidine tetra-hydrochloride (DAB; Sigma-Aldrich) was used as a chromogen (50 mg DAB in 100 mL 0.05MTBS, pH 7.4, with 33.3 lL H2O2, prepared prior to use) Tissues were then rinsed in Tris/HCl, dehydrated and mounted using a xylene-based medium (ENTELLAN neu; Merck) Adja-cent slides were stained with Harris hematoxylin (Vecta-stain), dehydrated through a graded series of increasing ethanol concentrations, followed by two xylene washes, and cover-slipped with ENTELLAN neu (Merck) Both the omission of anti-ADH4 IgG and the preadsorption of anti-ADH4 IgG with excess of purified recombinant ADH4 abolished the positive reaction in the control sections, demonstrating the specificity of the staining Control experiments had showed that anti-ADH4 IgG immunoreacted with recombinant purified rat ADH4 but did not cross-react with any other ADH classes
Image analysis Following ISH and IHC techniques, digestive tract sections were examined under a Leica DMRD fluorescense micro-scope with a Hamamatsu C5310 CCD or a Leica DC200 camera Image acquisition was carried out with IMAGE PROPLUS software and imported into Adobe PHOTOSHOP v5.5 (Adobe) Color images were transformed into black and white images using a grey-scale function, and brightness and contrast were adjusted All sections were examined concurrently and compared to published pictures and schemes [33]
Results ADH expression in rat gastrointestinal homogenates Homogenates from gastrointestinal tissues (tongue, eso-phagus, stomach, duodenum, jejunum, ileum, caecum, colon and rectum) were analyzed for the presence of ADH at activity and protein levels by using starch gel electrophoresis, spectrophotometric measurements, and immunoblotting (Fig 1) Both ADH1 and ADH4 were detected throughout the entire gastrointestinal tract but with a differential tissue distribution ADH1 was detected mainly in duodenum and the colorectal region, while ADH4
Trang 3was highly expressed in the upper (mainly esophagus and
stomach) and colorectal regions ADH3 was detected in all
tissues examined No large differences were found in activity
or in the tissue distribution of the ADH forms between
different animals (Fig 1)
Localization of ADH in tongue and esophagus
Immunohistochemistry (IHC) of rat tongue showed ADH4
in the mucosa The signal was detected in the papillae,
specifically in the stratified squamous epithelium (Fig 2B,
C,E) ADH4 was also detected in the endothelium of
microvessels (Fig 2C) I n situ hybridization (ISH) analysis
of esophagus revealed that ADH1 mRNA was only
localized in the base line of the stratified squamous
epithelium (data not shown) In contrast, ADH4 mRNA
was present at high level in all cell layers of stratified
squamous epithelium (Fig 2G) No specific signal was
detected in the lamina propia and muscularis mucosae In
good agreement, ADH4 immunostaining was detected in the stratified squamous epithelium (Fig 2I) Interestingly, a strong ADH4 protein signal was observed in the keratinized layer of epithelium, where the ADH4 mRNA was not detected
Localization of ADH in stomach and the gastroduodenal junction
ADH1 and ADH4 mRNAs were both expressed in the gastric mucosa from cardiac to pyloric stomach but each form was confined to distinct layers and cell types In the stomach body, ADH1 was localized in the medium and basal layers of the mucosa, and muscularis mucosae but not
in mucus-secreting cells (Fig 3A) However, towards the pyloric region, ADH1 gradually appeared in the mucus-secreting epithelium as well (cf Fig 3B,C,D) In contrast, ADH4mRNA was detected in the mucus-secreting cells,
in some of the inner cell layers, and in muscularis mucosae
Fig 1 Detection of ADH1 and ADH4 in tis-sue homogenates of the gastrointestinal tract (A) Starch gel electrophoresis stained for activity using 100 m M 2-buten-1-ol as a sub-strate (B) Graphic representation of ADH1 (black bars) and ADH4 (grey bars) activity levels Activity assays were performed with 0.1 M sodium phosphate, pH 7.5, at 25 C, with 10 m M (ADH1) or 1 M (ADH4) ethanol
as a substrate and 2.4 m M NAD + as a coen-zyme Values are expressed as the arithmetical mean ± SD of measures from four different animals, each determination run in duplicate (C) Immunoblot analysis of tissue extracts (30 lg) using affinity-purified rabbit anti-(mouse ADH4) IgG Lanes: T, tongue;
E, esophagus, S, stomach; D, duodenum;
J, jejunum; I, ileum; C, caecum; Cl, colon,
R, rectum Liver (L) was used as a control.
2654 J Vaglenova et al (Eur J Biochem 270) FEBS 2003
Trang 4throughout the cardiac, fundic, and pyloric regions
(Fig 3E,I,J) A strongly positive and specific signal was
found in the epithelial cells lining the surface of gastric pits
of the gastric body (Fig 3F) Detection by IHC confirmed
expression of ADH4 in the mucus-secreting cells of pylorus
(Fig 3G) Therefore, in the surface epithelium, ADH1 and ADH4 only overlapped in the gastric region close to the pylorus The endothelium lining small blood vessels within the gastric mucosa and submucosa also showed ADH4 expression (data not shown)
Fig 2 Localization of ADH4 in tongue and esophagus byISH and IHC analyses Hematoxylin-stained section of filiform (A) and fungiform (D) tongue papillae Immunodetection of ADH4 protein in stratified epithelium of tongue mucosa (B, C and E) Control section incubated with anti-ADH4 IgG preadsorbed with 15 lg recombinant mouse anti-ADH4 (F) Detection of anti-ADH4 mRNA in stratified squamous epithelium in sections of esophagus hybridized with antisense riboprobe (G) Control section of esophagus hybridized with ADH4 sense riboprobe (H) Immunodetection of ADH4 protein in queratinized stratified epithelium of esophageal mucosa (I) and control section of rat esophagus incubated with anti-ADH4 IgG preadsorbed with 15 lg recombinant mouse ADH4 (J) C, circumvallate papilla; E, squamous stratified epithelium; FI, filiform papilla; LP, lamina propia; MM, muscularis mucosae Calibration bars: A–F (shown in F), G–H (shown in H) and I–J (shown in J), 50 lm.
Trang 52656 J Vaglenova et al (Eur J Biochem 270) FEBS 2003
Trang 6Localization of ADH in small intestine
In duodenum, ISH showed abundance of ADH1 mRNA
in the absorptive mucosa and muscularis mucosae layer
(data not shown), following the same pattern observed in
the lower pyloric region (Fig 3D) While ADH4 was
abundant in stomach mucosa outer cell layers (Fig 3E), it
was absent in the duodenum external mucosa after a sharp
transition at the gastroduodenal junction (Fig 3I,J)
ADH4 was only detected in muscularis mucosae In the
jejunum and ileum, both ADH1 and ADH4 mRNAs were
detected throughout the epithelium in intestinal villi and
crypts of Lieberku¨hn (Fig 4A,B) By IHC, ADH4 was
prominent in the epithelial cells lining intestinal villi, in contrast to crypts of Lieberku¨hn, which stained weakly (Fig 4C) Connective tissue, lamina propia, and muscularis mucosae were not stained
Localization of ADH in the colorectal region Analysis of colorectal sections showed that ADH1 mRNA was localized primarily in the cells of the lower part of the crypts of Lieberku¨hn (Fig 5A,B) In contrast, ADH4, at mRNA and protein levels, was detected uniformly along the crypts of Lieberku¨hn and in the surface brush-border epithelium (Fig 5D,E,G,H) ADH4 immunostaining was
Fig 4 Localization of ADH1 and ADH4 in jejunum and ileum ADH1 (A) and ADH4 (B) mRNA detection in jejunal mucosa Immunodetection of ADH4 protein in ileal mucosa (C) Omission of anti-ADH4 IgG in an adjacent control section of ileum (D) CL, crypt of Lieberku¨hn; LP, lamina propia; MM, muscularis mucosae; SM, submucosa; V, villi; v, vessel Calibration bars (shown in D): A,B, 200 lm; C,D, 50 lm.
Trang 7absent in the submucosa, lamina propia and muscularis
mucosae
Discussion
Although several works had provided information on the
ADH distribution in rodent digestive organs [2,7,20,21,34],
the present report represents the most thorough study on the localization of the ethanol-metabolizing ADHs in the digestive tract tissues of adult rat In previous reports, ADH1 was, in general, undetected in upper digestive organs, including stomach while ADH4 was not found in several intestinal regions [21,34] Notably, here we demon-strate that ADH1 and ADH4 are expressed throughout the
Fig 5 Localization of ADH1 and ADH4 in the colorectal region ADH1 (A,B) and ADH4 (D,E) mRNA detection in the longitudinal (A,D) and transversal (B,E) section of crypts of Lieberku¨hn Control sections hybridized with ADH1 (C) and ADH4 (F) sense riboprobe Immunodetection of ADH4 protein in longitudinal (G) and transversal (H) section of the Lieberku¨hn glands of colorectal mucosa Control section incubated with anti-ADH4 IgG preadsorbed with 15 lg recombinant mouse anti-ADH4 protein (I) CL, crypt of Lieberku¨hn; G, goblet cell; LP, lamina propia; MM, muscularis mucosae; SB, striated border of enterocytes Calibration bars: A–F (shown in F) and G–I (shown in I), 50 lm.
2658 J Vaglenova et al (Eur J Biochem 270) FEBS 2003
Trang 8rat gastrointestinal tract Each ADH form, however, is
confined to specific regions and cell populations Thus,
ADH1 is localized predominantly in the intestinal area
whereas ADH4 is prominent in the most external parts
(esophagus, stomach and colorectum) of the digestive
system In each tissue, except for the duodenum, ADH1 is
confined to the inner cell layers of the mucosa, while ADH4
is localized in the outer cell layers exposed to the lumen
Interestingly, duodenum is the only region where ADH4 is
absent from the external cell layers of the mucosa The
precise colocalization of mRNA, protein and activity
demonstrates that these enzymes are present in the same
regions where their mRNA is found However, the
restric-tion of the ADH1 and ADH4 expression to a relatively
small number of cell types in specific regions could explain
the previous difficulty of demonstrating their presence in
various digestive organs [2,7,20,24,35] Also, it should be
considered that there may exist some rat/mouse species
differences in ADH localization along the gastrointestinal
tract that account for the slightly different ADH localization
reported here for rat as compared to that previously
reported for mouse [21]
Although ADH1 and ADH4 are found in all digestive
tube organs, discontinuity exists regarding the cellular layers
where the enzymes are expressed Thus, while ADH1 is not
expressed in the gastric pits of most of the stomach mucosa,
it is of interest the progressive increase in expression in this
external area as the mucosa reaches the pyloric region
(Fig 3B,C,D) Even more impressive is the sharp
disap-pearance of ADH4 expression from the mucosa outer cell
layers in the gastroduodenal junction (Fig 3J) The sudden
change in functional requirements in the transition between
stomach and duodenum is therefore also reflected by
marked differences in the expression levels of the ADH
enzymes
Comparison of the present data from rat with the partial
information available from human [24–25,29,30, and
S Porte´, S E Martı´nez , J Farre´s and X Pare´s, unpublished
results
2 ], indicates that the general pattern of ADH
distribu-tion in the gastrointestinal tract is similar in the two species
The present results along with previous in vitro studies
on the substrate specificity of ADH1 and ADH4
[2,7,9,12–14] provide the basis to hypothesize some
physiological functions for these enzymes in the
gastro-intestinal tract However, precaution should be taken
when extrapolating conclusions to human because of
different ADH4 Km values for ethanol between rat and
human (2.4Mvs 37 mM, respectively) [9] and differences
in diet, intestinal flora, etc
Role of gastrointestinal ADH in retinoid metabolism
The expression of ADH1 and ADH4 in certain cell layers of
gastrointestinal tissues, and its colocalization with the
biochemical apparatus associated with RA responsiveness
and metabolism [36–42], support the contribution of ADH1
and ADH4 (both exhibiting retinol dehydrogenase activity
[12–15,17]) to RA generation in adult gastrointestinal tract
ADH1 and ADH4 displayed some nonoverlapping
locali-zation which might reflect distinct roles, as has been
suggested by studies with knockout animals [43] ADH4,
located in the most external tissues and cell layers with a
high epithelial cell turnover, is well suited to fulfill a function
in RA synthesis In this sense, esophageal, gastric and colorectal mucosa show NAD+-dependent RA formation from all-trans-retinol, that is disturbed by inhibitors of ADH and aldehyde dehydrogenase (ALDH) [44,45] On the other hand, b-carotene absorbed by intestinal enterocytes is converted to retinal which is subsequently reduced to retinol for transport and storage [46] Thus, ADH1 and ADH4 (kcat/Km for retinal¼ 500 mM )1Æmin)1 [13] and
1750 mM )1Æmin)1[14], respectively) could be also involved
in the step to generate retinol that would be immediately esterified in vivo This could shift the reaction equilibrium towards retinal reduction, even in the absence of a favorable NAD/NADH ratio Interestingly, we have shown that ADH4 is not present in duodenal enterocytes, where most b-carotene cleavage occurs [46] Therefore, ADH1 would be the main ADH for the physiological retinal reduction in duodenum, although microsomal retinal reductases may also contribute to this function [47,48] ADH4, specialized
in retinal generation from retinol in specific tissues [21], could not be necessary in duodenal enterocytes where retinal
is directly formed from b-carotene
Role of gastrointestinal ADH in alcohol metabolism and pathology
Substrate specificity predicts that both ADH1 and ADH4 participate in the elimination of ingested alcohols and aldehydes, ethanol generated by intestinal microbial flora, and products of lipid peroxidation [12,13] ADH4, located in the upper part of the gastrointestinal tract and the luminal part of the mucosa, would be in contact with the highest concentrations of ingested alcohols and aldehydes, and in areas subjected to high levels of oxidative stress Therefore, ADH4 could act as a first metabolic barrier Likewise, ADH1, that is positioned more internally along the tract and within the mucosa, could act as a second metabolic barrier The localization of ADH4 suggests its contribution to the first-pass metabolism [49–53], mostly at high ethanol concentration (Km¼ 2.4M) [9] In addition, we have demonstrated here that ADH1 is also present in the upper digestive tract and therefore it may have a role as well in the first-pass metabolism, mostly at low ethanol concentrations (Km¼ 1.4 mM) [7] In the lower gastrointestinal tract, colonic flora is the major source of endogenous ethanol in mammals that is produced constantly [54–56] The main function of the high amount of ADH1 in colon might be the elimination of this endogenous ethanol
The presence of ADH throughout the gut can be related
to alcohol pathology Thus, ethanol and acetaldehyde have been associated with epithelial hyperegeneration of the mucosa and cancer [57–59] On the other hand, disturbance
of RA metabolism may be related to carcinogenesis [58, 60–62], and ethanol is a competitive inhibitor of retinol oxidation by ADH [12,51,63–66] The esophagus and the colorectal region are especially vulnerable to alcohol injury [58,59], and these are tissues with the highest ADH activity (Fig 1) where acetaldehyde-metabolizing ALDH2 is virtu-ally absent or scarce [25] Thus, 50 lM acetaldehyde hampers RA formation [45], suggesting that acetaldehyde produced by ADH could also disturb RA generation catalyzed by retinal-active ALDH1 which has been also
Trang 9detected in these gastrointestinal areas [25,30,41,42,67,68].
The impairment of RA formation by ethanol and
acetal-dehyde could be an explanation for mucosal damage,
increased cell proliferation and the high incidence of
esophageal and colorectal neoplasia in alcohol abusers
In conclusion, we have detected ADH1 and ADH4 in
distinct cell types of specific regions throughout the
gastrointestinal tract, which evidences a local level of
ethanol metabolism Active ethanol oxidation in specific
gastrointestinal regions can be related to some deleterious
effects of ethanol The involvement of ADH1 and ADH4 in
retinol oxidation makes these enzymes relevant to
gastro-intestinal functions that require RA The impairment of
retinol oxidation by inhibition of ADH during ethanol
consumption may be an additional mechanism of
gastro-intestinal alcohol pathology
Acknowledgements
Supported by grants from the Direccio´n General de Investigacio´n
Cientı´fica (BMC2002-02659 and BMC2000-0132) and the
Commis-sion of the European Union (BIO4-CT97-2123) to X P and J F.,
and by the National Institutes of Health grant AA09731 to G D We
are grateful to Dr Salvador Bartolome´ (Laboratori d’Ana`lisi i
Fotodocumentacio´ d’Electroforesis, Autoradiografies i
Luminesce`n-cia, Universitat Auto`noma de Barcelona) for his help in image
analysis.
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