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  • Название: New approaches for detecting alcohol abuse
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JOHANNA HIETALA

Novel Use of Biomarkers and Their Combinations
for Detecting Excessive Drinking

ACADEMIC DISSERTATION
To be presented, with the permission of
the Faculty of Medicine of the University of Tampere,
for public discussion in the auditorium of
Mediwest Health Technology Center, Koskenalantie 16, Seinäjoki,
on February 23rd, 2007, at 12 o’clock.
(Simultaneous video conference connection
in the small auditorium of Building K,
Medical School of the University of Tampere,
Teiskontie 35, Tampere)

U N I V E R S I T Y O F TA M P E R E

ACADEMIC DISSERTATION
University of Tampere, Medical School
Seinäjoki Central Hospital, Department of Laboratory Medicine and Medical Research Unit
Finland

Supervised by
Professor Onni Niemelä
University of Tampere

Reviewed by
Docent Kari Pulkki
University of Turku
Professor Kaija Seppä
University of Tampere

Distribution
Bookshop TAJU
P.O. Box 617
33014 University of Tampere
Finland

Tel. +358 3 3551 6055
Fax +358 3 3551 7685
taju@uta.fi
www.uta.fi/taju
http://granum.uta.fi

Cover design by
Juha Siro

Printed dissertation
Acta Universitatis Tamperensis 1210
ISBN 978-951-44-6856-8 (print)
ISSN 1455-1616

Tampereen Yliopistopaino Oy – Juvenes Print
Tampere 2007

Electronic dissertation
Acta Electronica Universitatis Tamperensis 594
ISBN 978-951-44-6857-5 (pdf )
ISSN 1456-954X
http://acta.uta.fi

To my family

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Abstract

Excessive alcohol consumption and consequent medical disorders create a major burden for modern
health care. In addition to medical and social problems, excessive drinking causes considerable
strains on the national economy. In order to improve the diagnosis and treatment of patients suffering from ethanol-related health problems, reliable and accurate methods for recognizing excessive
alcohol consumption in its early phase need to be developed. Currently, excessive drinkers tend to
escape detection, which may lead to delays in intervention.
The present study was set out to develop new approaches for detecting excessive drinking based
on conventional and new laboratory tests and their combinations and to address the relationships
between such markers and alcoholic liver disease. Conventional laboratory markers of excessive
drinking (GGT, CDT, MCV, AST, ALT), a mathematically formulated combination of GGT and
CDT, and autoimmune responses to proteins modified with acetaldehyde, the first metabolite of
ethanol, were measured in alcoholics with or without liver disease, moderate drinkers and abstainers. Cytokine profiles of subjects were also studied in order to clarify the associations between alcohol intake and pathogenesis of alcoholic liver disease.
The results show that even moderate drinking may increase levels of gamma-glutamyl transferase (GGT). When GGT was combined logarithmically with carbohydrate-deficient transferrin
(CDT), the diagnostic performance of the combination GGT-CDT was found to markedly exceed
that of the traditional markers, reaching a sensitivity of 90 % whereas the sensitivities of its parent
components remained at 63 % (CDT) and 58 % (GGT).
Alcohol consumption was also found to induce alterations in the immune system. An association
between cytokine levels and alcohol use was observed, with most evident alterations in alcoholics
with liver disease. Pro-inflammatory cytokines (IL-2, IL-6, IL-8, TNF-α) were found to increase in
alcoholic liver disease whereas the levels of anti-inflammatory cytokines, particularly TGF-β1
showed a slight decrease.
Acetaldehyde adducts are formed when acetaldehyde reacts with proteins and cellular constituents. Antibodies directed against acetaldehyde-modified proteins were found in the circulation of
alcoholic patients. The highest anti-adduct IgA and IgG titres occurred in patients with alcoholic
liver disease, while specific class IgM antibodies were most abundant in alcoholics without liver
disease. The possible usefulness of anti-adduct IgA as a marker of excessive drinking was subsequently studied in alcoholics without liver disease, and the mean anti-adduct IgA levels were significantly higher than those in the moderate drinkers or abstainers (p < 0.001), while the difference
between the moderate drinkers and abstainers was also significant (p < 0.05). Mean daily ethanol
consumption during the previous month was found to correlate significantly with anti-adduct IgA
levels. Based on these findings, anti-adduct IgA antibodies could serve as markers of alcohol con-

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sumption. A combination of the anti-adduct IgA and CDT results showed improved diagnostic performance for this marker (IgA-CDT) as compared to traditional ones.
In the light of these results, it may be suggested that GGT, CDT and IgAs against acetaldehydederived epitopes either alone or combined by means of mathematical equations can serve as useful
markers of alcohol consumption. The elevation of GGT in moderate drinkers as compared with abstainers may affect its reference ranges, since they are usually calculated from general population in
which the proportion of abstainers is decreasing. The results also indicate that the antibody responses and alterations in cytokine balance may contribute to the pathogenesis and progression of
alcoholic liver disease in humans.

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Contents

ABSTRACT .................................................................................................................5
CONTENTS .................................................................................................................7
ABBREVIATIONS....................................................................................................10
LIST OF ORIGINAL PUBLICATIONS ...................................................................13
1. INTRODUCTION..................................................................................................15
2. REVIEW OF THE LITERATURE........................................................................16
2.1. Ethanol: past and present................................................................................................. 16
2.2. Main features of ethanol metabolism .............................................................................. 16
2.3. Effects of ethanol on health ............................................................................................. 17
2.3.1. Ethanol and the liver ........................................................................................... 18
2.3.2. Ethanol and extrahepatic tissues ......................................................................... 19
2.3.3. Ethanol and the immune system ......................................................................... 20
2.3.4. Ethanol, psychiatric disorders and accidents ...................................................... 20
2.3.5. Suggested positive effects of ethanol.................................................................. 21
2.4. Assessing alcohol consumption....................................................................................... 22
2.4.1. Amount and pattern of drinking.......................................................................... 22
2.4.2. Self-reported alcohol consumption ..................................................................... 22
2.4.3. Laboratory markers of alcohol consumption ...................................................... 23
2.4.4. Laboratory markers of ethanol-induced tissue injury ......................................... 25
2.4.5. Gender issues ...................................................................................................... 25
2.5. Routinely used biomarkers of ethanol consumption ....................................................... 25
2.5.1. Ethanol concentration in blood, breath or urine.................................................. 25
2.5.2. Gamma-glutamyl transferase .............................................................................. 25
2.5.3. Carbohydrate-deficient transferrin ...................................................................... 26
2.5.4. Mean corpuscular volume ................................................................................... 27
2.5.5. Aminotransferases............................................................................................... 27

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2.6. New biomarkers of ethanol consumption........................................................................ 27
2.6.1. Acetaldehyde adducts and anti-adduct antibodies .............................................. 27
2.6.2. 5-Hydroxytryptophol .......................................................................................... 28
2.6.3. Ethyl glucuronide ................................................................................................ 28
2.6.4. Fatty acid ethyl esters.......................................................................................... 28
2.6.5. Phosphatidylethanol ............................................................................................ 29
2.6.6. Sialic acid ............................................................................................................ 29
2.6.7. Other postulated markers .................................................................................... 29
2.7. Marker combinations....................................................................................................... 30
2.8. Laboratory markers for the follow-up of alcoholics........................................................ 31
2.9. Immune responses related to alcohol consumption......................................................... 31
2.9.1. Immune responses to ethanol metabolites........................................................... 31
2.9.2. Alcohol-induced cytokine responses................................................................... 33

3. AIMS OF THE PRESENT RESEARCH...............................................................34
4. MATERIALS AND METHODS ...........................................................................35
4.1. Subjects............................................................................................................................ 35
4.2. Measurements of laboratory markers (I-II, IV) ............................................................... 36
4.3. Determination of marker combinations (II, IV) .............................................................. 37
4.3.1. GGT-CDT (II)..................................................................................................... 37
4.3.2. IgA-CDT ............................................................................................................. 37
4.4. Measurement of antibodies against acetaldehyde adducts (III-IV) ................................. 37
4.5. Serum cytokines .............................................................................................................. 38
4.6. Statistical methods........................................................................................................... 38

5. RESULTS...............................................................................................................39
5.1. Influence of drinking levels on markers (I, II, IV) .......................................................... 39
5.2. Clinical characteristics of marker combinations (II, IV)................................................. 39
5.2.1. GGT-CDT (II)..................................................................................................... 39
5.2.2. IgA-CDT (IV) ..................................................................................................... 40
5.3. Antibody and cytokine responses in alcoholics (III–IV)................................................. 41
5.3.1. Alcohol-related changes in cytokine and antibody production (III-IV).............. 41
5.3.2. Anti-adduct IgAs as a marker of alcohol consumption (IV)............................... 41

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5.4. Markers in the follow-up of alcoholics (II-IV)................................................................ 42

6. DISCUSSION.........................................................................................................43
6.1. Influence of moderate drinking on marker levels (I-IV) ................................................. 43
6.2. Marker combinations (II, IV) .......................................................................................... 44
6.3. Antibody and cytokine responses in alcohol abusers (III-IV)......................................... 45
6.3.1. Changes in antibody and cytokine production attributable to alcohol and liver
disease (III-IV) .............................................................................................................. 45
6.3.2. Anti-adduct IgAs as a marker of alcohol consumption....................................... 47
6.4. Follow-up studies ............................................................................................................ 47
6.5. Possible limitations of this study..................................................................................... 48
6.6. Future considerations....................................................................................................... 48

7. CONCLUSIONS ....................................................................................................49
ACKNOWLEDGEMENTS .......................................................................................50
REFERENCES ...........................................................................................................51

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Abbreviations

ADH
ALDH
ALT
Apo J
AST
AUC
AUDIT
BMI
CAGE
CCLI
CDT
%CDT
CHD
CMI
CYP2E1
DSM-IV
EOA
EtG
EtS
FAE
FAEEs
FAS
GGT
HA
HDL
HIAA
HTOL
IFN
IgA, IgG, IgM
IL
LOA
LPS
MAST
MCV
MEOS
NPV
PBS
PEth

Alcohol dehydrogenase
Aldehyde dehydrogenase
Alanine aminotransferase
Apolipoprotein J
Aspartate aminotransferase
Area under the curve
Alcohol Use Disorders Identification Test
Body mass index
Cut down, Annoyed, Guilty, Eye-opener (acronym)
Combined clinical and laboratory index
Carbohydrate-deficient transferrin
CDT as a percentage of total transferrin
Coronary heart disease
Combined morphological index
Cytochrome P450 IIE1
Diagnostic and Statistical Manual of Mental Disorders. Fourth Edition.
Early onset alcoholics
Ethyl glucuronide
Ethyl sulphate
Fetal alcohol effects
Fatty acid ethyl esters
Fetal alcohol syndrome
Gamma-glutamyl transferase
Hyaluronic acid
High density lipoprotein -cholesterol
Hydroxyindole-acetic acid
Hydroxytryptophol
Interferon
Immunoglobulin A, G, M
Interleukin
Late onset alcoholics
Lipopolysaccharide
Michigan Alcoholism Screening Test
Mean corpuscular volume
Microsomal ethanol oxidizing system
Negative predictive value
Phosphate-buffered saline
Phosphatidylethanol

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PINP
PIIINP
PPV
ROC
SA
SD
SIJ
TGF-β1
TLFB
TNF-α

Aminoterminal propeptide of type I collagen
Aminoterminal propeptide of type III collagen
Positive predictive value
Receiver-operating characteristic
Sialic acid
Standard deviation
Sialic acid index of apolipoprotein J
Transforming growth factor-β1
Timeline follow-back -method
Tumor necrosis factor-α

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List of original publications

I

Hietala J, Puukka K, Koivisto H, Anttila P, Niemelä O (2005) Serum gamma-glutamyl
transferase in alcoholics, moderate drinkers and abstainers: effect on GT reference intervals at population level. Alcohol Alcohol 40: 511–514.

II

Hietala J, Koivisto H, Anttila P, Niemelä O (2006) Comparison of the combined
marker GGT-CDT and the conventional laboratory markers of alcohol abuse in heavy
drinkers, moderate drinkers and abstainers. Alcohol Alcohol 41: 528–533.

III

Latvala J, Hietala J, Koivisto H, Järvi K, Anttila P, Niemelä O (2005) Immune responses to ethanol metabolites and cytokine profiles differentiate alcoholics with or
without liver disease. Am J Gastroenterol 100: 1303–1310.

IV

Hietala J, Koivisto H, Latvala J, Anttila P, Niemelä O (2006) IgAs against acetaldehyde-modified red cell protein as a marker of ethanol consumption in male alcoholic
subjects, moderate drinkers, and abstainers. Alcohol Clin Exp Res 30: 1693–1698.

The original articles are referred to in the text with the above Roman numerals.

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1. Introduction

Excessive alcohol consumption and consequent medical disorders are considerable problems in
many Western countries (Österberg 2006, Blocker, Jr. 2006). In addition to medical and social
problems at the individual level, excessive drinking causes significant problems for the national
economy. Resources should therefore be focused on reducing the prevalence of alcoholism through
more effective diagnosis and early intervention (Anderson 1993, Sharpe 2001, Fleming et al. 2002,
Latt and Saunders 2002, Niemelä 2002, Rehm et al. 2006). It has been estimated that only 20–50 %
of patients with alcoholism are actually identified in health care and thus more reliable and accurate
methods of recognizing excessive alcohol consumption in clinical work are urgently needed (Reid
et al. 1986, Moore et al. 1989, Sharpe 2001).
The diagnosis of excessive alcohol consumption is often based on patients' own reports and answers to questionnaires. This approach suffers from a lack of reliability, because patients are usually
unwilling to admit excessive drinking (Rosman and Lieber 1994, Sharpe 2001, Niemelä 2002).
Laboratory markers have been integrated into the diagnostics and may lead to a considerable improvement in detecting excessive drinking (Lieber 1995, Niemelä 2002). The main advantage of
laboratory markers is their objective nature. The results of laboratory tests revealing excessive alcohol consumption and possible tissue injury could motivate patients more than a verbal report by a
clinician (Allen et al. 1992, Rosman and Lieber 1994). It has also been suggested that a better understanding of the physiological effects