In e na ional Jou nal o
En i onmen al Resea ch
and Public Heal h
Re iew
Bioma ke -Based App oaches o Assessing Alcohol
Use Diso de s
Onni Niemelä
Depa men o Labo a o y Medicine and Medical Resea ch Uni , Seinäjoki Cen al Hospi al and Uni e si y o
Tampe e, Seinäjoki 60220, Finland; [email p o ec ed]; Tel.: +358-6-4154719; Fax: +358-6-4154924
Academic Edi o : Ic o Ma emmani
Recei ed: 10 No embe 2015; Accep ed: 20 Janua y 2016; Published: 27 Janua y 2016
Abs ac :
Al hough alcohol use diso de s ank among he leading public heal h p oblems wo ldwide,
haza dous d inking p ac ices and associa ed mo bidi y con inue o emain unde diagnosed. I is
pos ula ed he e ha a mo e sys ema ic use o bioma ke s imp o es he de ec ion o he speci ic ole
o alcohol abuse behind poo heal h. In e en ions should be ini ia ed by ob aining in o ma ion on
he ac ual amoun s o ecen alcohol consump ion h ough ques ionnai es and measu emen s o
e hanol and i s speci ic me aboli es, such as e hyl glucu onide. Ca bohyd a e-de icien ans e in
is a aluable ool o assessing ch onic hea y d inking. Ac i i ies o common li e enzymes can
be used o sc eening e hanol-induced li e dys unc ion and o p o ide in o ma ion on he isk o
co-mo bidi ies including insulin esis ance, me abolic synd ome and ascula diseases. Con en ional
bioma ke s supplemen ed wi h indices o immune ac i a ion and ib ogenesis can help o assess
he se e i y and p ognosis o e hanol-induced issue damage. Many e hanol-sensi i e bioma ke s
espond o he s a us o oxida i e s ess, and hei le els a e modula ed by ac o s o li e s yle,
including weigh gain, physical exe cise o co ee consump ion in an age- and gende -dependen
manne . The e o e, u he a en ion should be paid o de ining sa e limi s o e hanol in ake in a ious
demog aphic ca ego ies and es ablishing common e e ence in e als o bioma ke s o alcohol
use diso de s.
Keywo ds: e hanol; heal h; amino ans e ase; GGT; CDT; ib osis; NASH; obesi y; oxida i e s ess
1. In oduc ion
Alcohol use diso de s, bo h acu e and ch onic, a e signi ican clinical p oblems due o hei
de as a ing heal h impac s and high p e alence h oughou he wo ld [
1
–
5
]. Vi ually all issues in he
body can be a ec ed by excessi e alcohol consump ion and a wide a ie y o alcohol- ela ed diso de s
a e cu en ly known. Fo success ul clinical in e en ions, haza dous d inking should be de ec ed in
an ea ly phase o p e en he a ec ed indi iduals om en e ing a s age o se e e dependence wi h
associa ed issue oxici y.
The occu ence o heal h p oblems in alcohol consume s seems o be p opo ional o he amoun
o alcohol inges ed o e a long pe iod o ime [
1
–
4
]. Ch onic alcohol d inking exceeding 300 g (men) o
200 g (women) pe week is known o sha ply inc ease he isk o damage [
6
,
7
]. In women, ad e se
e ec s may a ise a lowe le els and alcohol- ela ed p oblems conce ning p egnancy could add ano he
dimension o he p oblem o excessi e alcohol consump ion pe se [
3
,
4
,
8
]. In indi iduals wi h isk ac o s
such as obesi y, smoking o hepa i is C in ec ion, heal h p oblems can also be igge ed by ela i ely low
le els o alcohol in ake [
9
–
14
]. Recen Ame ican Associa ion o he S udy o Li e Diseases (AASLD)
guidelines on non-alcoholic a y li e disease (NAFLD) de ined alcohol consump ion exceeding 21
d inks (~250 g) pe week in men and 14 d inks (~170 g) pe week in women as limi s o signi ican
alcohol consump ion [
15
]. Howe e , cu en li e ime isk e alua ions ha e indica ed ha e en le els
In . J. En i on. Res. Public Heal h 2016,13, 166; doi:10.3390/ije ph13020166 www.mdpi.com/jou nal/ije ph
In . J. En i on. Res. Public Heal h 2016,13, 166 2 o 19
o 14 d inks pe week o men o se en d inks pe week o women can inc ease alcohol-a ibu able
mo ali y [16].
Recen de elopmen s in he ea men o pa ien s wi h alcohol use diso de s ha e emphasized
he ole o bioma ke s as an in eg al pa o he assessmen [
17
–
21
]. Bioma ke s a e ma ke s o a
biological p ocess o s a e, which a e use ul o clinicians and pa ien s i hey p o ide in o ma ion
abou he cu en s a us o u u e isk o disease [
22
]. In alcohol use diso de s, bioma ke s should
be used no only o con i m he ae iology bu also o help he in e ac ions be ween physicians and
pa ien s on aising he issue o alcohol use as a possible cause o ad e se heal h ou comes. They can
also imp o e pa ien ollow-up p ocedu es p o iding use ul p ognos ic in o ma ion. Bioma ke -based
e alua ions may also open new insigh s on he p ima y mechanisms o e hanol-induced diseases. The
aim o he p esen con ibu ion is o discuss he cu en ole o bioma ke s in he assessmen o alcohol
consump ion and associa ed heal h p oblems. Fo addi ional in o ma ion, he eade is e e ed o
o he p e ious e iews in his ield [17–21,23].
2. Bioma ke s o Alcohol Consump ion pe se
Bo h he amoun s and pa e ns o e hanol consump ion de e mine he isk o de eloping alcohol
addic ion and associa ed mo bidi y. In o ma ion on he ac ual amoun s o alcohol consump ion can
be collec ed by speci ically designed ques ionnai es such as Alcohol Use Diso de s Iden i ica ion
Tes (AUDIT), CAGE alcohol ques ionnai e (Cu down, Annoyed, Guil y, Eye-opene ), Michigan
Alcoholism Sc eening Tes (MAST) o ime-line ollow-back (TLFB) [
24
,
25
]. While he i s h ee a e
sc eening ools co e ing a ious aspec s o alcohol consump ion, p oblems and dependency, TLFB
calenda assessmen p o ides es ima es o he ac ual amoun s o consump ion. All o hese a e,
howe e , dependen on sel - epo s, which a e memo y-dependen and o en un eliable channels o
in o ma ion. P e ailing a i udes owa ds d inking bo h among pa ien s and heal h ca e pe sonnel
can also in luence he ou come o he ques ionnai es in clinical se ings. The e o e, labo a o y es s a e
o en needed o p o ide addi i e in o ma ion (Table 1).
Measu emen s o e hanol i sel e eal e hanol in oxica ion. They can also be used in he assessmen
o compliance du ing ea men [
17
]. In alcohol-dependen pa ien s, posi i e blood e hanol may be
seen e en a he ime o he clinic isi . Based on blood e hanol indings and clinical obse a ions i is
possible o each conclusions on long- e m d inking habi s. E hanol le els exceeding 1.5
‰
(33 mmol/L)
wi hou any appa en signs o in oxica ion indica e e hanol ole ance, which is a ypical sign among
alcohol-dependen indi iduals. In ac , in heal h ca e se ings, he occu ence o posi i e blood alcohol
le els a any ime should lead o a suspicion o hea y d inking his o y [26].
The sho hal -li e o e hanol o en p e en s physicians om ou inely o de ing hese es s. E hyl
glucu onide (E G), a mino nonoxida i e me aboli e o e hanol, is o med in he li e by enzyma ic
conjuga ion o e hanol wi h glucu onic acid and his me aboli e can be analyzed by immunological
o liquid ch oma og apy-mass spec ome y echniques om di e en ypes o biological luids, hai
o nails [
27
–
32
]. Depending on he sample ype used, E G may emain posi i e o se e al days a e
cessa ion o e hanol in ake and i can he eby p o ide addi ional alue when assessing ecen alcohol
consump ion [
28
]. S udies so a ha e indica ed use ul diagnos ic applica ions o E G in pos -mo em
e alua ions o alcohol d inking [
30
], assessmen o e al alcohol exposu e [
27
,
29
,
33
,
34
] o in pa ien s
scheduled o li e ansplan a ion [
35
]. E hyl sul a e (E S) is ano he conjuga ed me aboli e o e hanol,
which is o med in low amoun s a e alcohol consump ion [
28
,
36
]. Moni o ing bo h E G and E S is,
howe e , usually unnecessa y [
37
]. Phospha idyle hanol (PE h) is a speci ic long hal -li e me aboli e
o e hanol, which is o med in he body only when e hanol is p esen . This phospholipid species
inc eases in a highly sensi i e manne in biological luids as a consequence o alcohol d inking [
38
–
41
].
S abili y o PE h has ecen ly shown o be good in assays om d y blood spo ca ds, which may u he
imp o e he po en ial o PE h o ou ine applica ions [
41
]. Fa y acid e hyl es e s (FAEE) a e o med
by es e i ica ion o e hanol wi h ee a y acids [
42
]. Assays o FAEE by gas ch oma og aphy-mass
spec ome y echniques om hai ha e been sugges ed as possible ools o e ospec i e de ec ion
In . J. En i on. Res. Public Heal h 2016,13, 166 3 o 19
o alcohol abuse du ing p egnancy o in o ensic applica ions [
33
,
34
,
42
]. Ace aldehyde is he i s
me aboli e o e hanol, which, due o i s high eac i i y, is capable o binding o p o eins and cellula
cons i uen s du ing e hanol me abolism [
43
,
44
]. Such binding c ea es dis inc neoan igenic epi opes and
immune esponses, which ha e been sugges ed no only as diagnos ic ools bu also as an impo an
pa hogenic ea u e unde lying alcohol-induced issue oxici y [43–46].
Table 1. Bioma ke s o alcohol consump ion.
Bioma ke Abb e ia ion Biological Sample
Type Ma ke Cha ac e is ics
E hanol E OH Blood
U ine
B ea h
Res ic ed o condi ions whe e e hanol is
s ill p esen in ci cula ion.
E hyl glucu onide/
E hyl sul a e E G/E S
U ine
Se um
Ce eb ospinal luid
Vi eous humou
Hai
Nails
E hanol me aboli e, which emains
posi i e in u ine samples 2–5 days a e
s opping e hanol use. Window o de ec ion
dependen on sample ype.
Phospha idyle hanol PE h Blood
D y blood spo s
E hanol me aboli e, which emains
de ec able 1–2 weeks a e alcohol use.
Measu ed by LC-MS o immunological
echniques.
Fa y acid e hyl es e s FAEE Plasma
Hai
Meconium
E hanol me aboli e de i ed om a
combina ion o a y acid wi h alcohol.
Ace aldehyde adduc s
and associa ed immune
esponses AA-Ab Blood
Tissue specimens
IgA esponse owa ds ace aldehyde
adduc s mos speci ic o alcohol- ela ed
diso de s.
Ca bohyd a e-de icien
ans e in CDT Se um
Ce eb ospinal luid
Speci ic ma ke o ch onic alcohol
consump ion. Lacks sensi i i y o
sc eening pu poses.
Gamma-glu amyl ans e ase
GGT Se um/plasma
Sensi i e ma ke o alcohol use, li e
dys unc ion and oxida i e s ess. Se e al
sou ces o unspeci ici y. No maliza ion
ime 2–3 weeks.
GGT-CDT combina ion GGT-CDT Se um/plasma Imp o es sensi i i y and speci ici y o
de ec ing alcohol abuse. Relies on a
ma hema ical model.
Blood cell coun s Blood
Mean co puscula olume (MCV) o
e y h ocy es ypically ele a ed in
alcoholics. No maliza ion ime 2–4 mon hs.
Mean co puscula haemoglobin (MCH)
and h ombocy es (pla ele coun s) a e also
equen ly al e ed in alcohol abuse s.
Se e al sou ces o unspeci ici y.
T ansaminase enzymes ALT, AST Se um/plasma
Sui able o sc eening o li e dys unc ion
in alcohol use s. Sensi i e o e ec s o
excess body weigh . AST/ALT a io
inc eases in alcoholic li e disease.
Ele a ed le els o se um ca bohyd a e-de icien ans e in (CDT) e eal ch onic alcohol
abuse in a a he speci ic manne (Table 1). Bo h he amoun s o disialo- and asialo-iso o ms o
ans e in inc ease as a esul o hea y alcohol in ake and his abno mal sialyla ion pa e n can
be analyzed by immunological echniques, high pe o mance liquid ch oma og aphy o capilla y
elec opho esis [
18
,
47
,
48
]. In e es ingly, he le els o o al se um sialic acid also inc ease in associa ion
wi h glycop o ein desialyla ion as a esul o hea y alcohol in ake [
49
,
50
]. Unlike many o he
bioma ke s, CDT is mo e sensi i e o changes in e hanol consump ion han o he seconda y e ec s o
li e disease, and i can also help o di e en ia e be ween alcoholic e sus non-alcoholic li e disease.
In . J. En i on. Res. Public Heal h 2016,13, 166 4 o 19
Howe e , i should be no ed ha CDT assays, which a e sensi i e o changes in se um o al ans e in,
also luc ua e in esponse o he s a us o li e disease pe se [
51
]. CDT ele a ions equi e consump ion
o a leas 50–80 g o e hanol pe day o a pe iod o se e al weeks and, hus, i lacks sensi i i y as a
sc eening ool in gene al popula ions. In alcohol-dependen pa ien s, i is, howe e , sensi i e enough
o de ec ing elapses and moni o ing sob ie y [48,52–54].
Gamma-glu amyl ans e ase (GGT) is a memb ane-bound glycop o ein enzyme, which has long
been used as a ma ke o excessi e alcohol in ake (Table 1) [
55
,
56
]. GGT is sensi i e o changes in
alcohol consump ion, bu , due o lack o speci ici y, i is no sui able o sc eening among popula ions
wi h non-alcoholic li e diseases, obesi y o hospi alized pa ien s [
17
,
57
]. In alcoholics, inc eased
ac i i ies usually e u n o no mal wi hin 2–3 weeks upon abs inence, whe eas pe sis en ly abno mal
alues may sugges li e disease.
P e ious wo k has indica ed ha diagnos ic imp o emen in de ec ing alcohol use diso de s could
be achie ed by combining wo o mo e alcohol ma ke s [
17
,
21
]. The con en ional manne o combining
ma ke s is o see whe he ei he is ele a ed [
48
,
58
]. This app oach ob iously gi es imp o ed assay
sensi i i y bu is equen ly associa ed wi h a dec ease in speci ici y. Howe e , combina ion o GGT
and CDT using a ma hema ically o mula ed equa ion GGT-CDT = 0.8
ˆ
ln(GGT) + 1.3
ˆ
ln(CDT) can
imp o e he de ec ion o excessi e alcohol consump ion by inc easing assay sensi i i y wi hou a loss
in speci ici y [
58
]. This ma ke is ele a ed in a highe pe cen age o alcohol abuse s han ei he GGT
o CDT alone and eac s a e egula e hanol consump ion exceeds a h eshold o 40g pe day. The
co ela ions wi h he ac ual amoun s o e hanol consump ion and GGT-CDT a e also highe han hose
o i s pa en componen s [58].
Haza dous d inking p ac ices also c ea e ypical abno mali ies on blood cell coun s and hei
mo phological ea u es, pa icula ly on e y h ocy e and h ombocy e lineages (Table 1) [
59
]. The e
seems o be a dose-dependen esponse be ween e y h ocy e size (mean co puscula olume, MCV) and
e hanol in ake [
60
]. Mean co puscula haemoglobin (MCH) is also ele a ed in hea y d inke s. Upon
abs inence, no maliza ion o ed cell indices may equi e 2–4 mon hs. In hea y d inke s wi hou
co-mo bidi ies, high MCV alues a e ypically seen wi hou anaemia, whe eas in pa ien s wi h
alcoholic li e disease and a concomi an ola e de iciency, megaloblas ic bone ma ow al e a ions
and haemolysis, high MCV and anaemia usually co-exis [
61
]. E y h ocy es om alcoholics a e
p one o damage and sho ened biological hal -li e, which may be associa ed wi h modi ica ions
o p o eins and cell memb ane cons i uen s by ace aldehyde and eac i e aldehydic p oduc s o lipid
pe oxida ion [
44
,
59
]. Blood pla ele coun s a e dec eased in one hi d o he alcoholics [
61
]. Upon
abs inence, he le els e u n o no mal usually wi hin a ew days. A low h ombocy e coun associa ed
wi h inc eased li e ansaminase (aspa a e amino ans e ase, AST, and alanine amino ans e ase,
ALT) enzymes—and possibly inc eased AST/ALT a io—can be conside ed an ea ly wa ning sign o
de eloping alcoholic li e disease.
A wide a ie y o o he labo a o y ma ke s a e also al e ed in esponse o excessi e alcohol
use, al hough wi hou su icien speci ici y o se e as bioma ke s o alcohol abuse. Hea y alcohol
consump ion inc eases se um u ic acid, a compound wi h ee adical sca enging p ope ies, which
may indica e an inc eased need o an ioxidan capaci y unde such condi ions [
62
–
64
]. U ic acid also
co ela es wi h he ac i i ies o li e enzymes in alcohol consume s [
62
]. In lipid p o iles om hea y
d inke s, inc eased high densi y lipop o ein-choles e ol (HDL) is obse ed e en ollowing egula
alcohol in ake o less han i e d inks pe day. Excess d inking also equen ly leads o dys egula ed
a me abolism, as e lec ed in inc eased le els o se um iglyse ides and ee a y acid e hyl es e s.
Such indings also associa e wi h inc eased hepa ic a con en , glucose dys egula ion, and low-g ade
in lamma ion [65].
3. Li e Enzymes as Indica o s o Hepa ic and Ex ahepa ic E ec s o Alcohol
The li e is a majo a ge o e hanol oxici y due o i s p ima y ole in e hanol me abolism [
2
–
4
].
The e o e, unexpec ed abno mali ies in li e enzyme ac i i ies, GGT o ALT, a e equen ly he i s
In . J. En i on. Res. Public Heal h 2016,13, 166 5 o 19
clinical signs o excessi e alcohol consump ion. Measu emen s o hese enzymes a e also widely
used as sc eening ools o abno mal li e unc ion and in decisions o selec pa ien s needing he
closes moni o ing.
Fa y li e disease associa ed wi h obesi y (NAFLD) is he mos common non-alcoholic cause
o inc eased GGT and ALT ac i i ies [
14
,
15
,
66
–
69
]. Alcohol use and obesi y o en co-exis and c ea e
oxici y in a syne gis ic manne [
9
,
69
–
72
]. Alcoholic li e disease (ALD) and NAFLD can also be
o e lapping phenomena and he h eshold le els o ha m ul alcohol consump ion in indi iduals wi h
a ying body weigh s ha e no ye been es ablished. In obese pe sons, inc eased GGT and issue
mo phology simila o alcohol excess is common e en in hose d inking an a e age o wo d inks pe
day [
9
]. This may be explained by induc ion o common pa hways o oxida i e s ess since GGT plays
a key ole in he me abolism o glu a hione (GSH) and in he egula ion o oxida i e s ess [
9
,
13
,
73
–
78
].
GGT could also be in e p e ed as a bioma ke o oxida i e s ess indica ing an inc eased need o
main ain in acellula GSH le els [73,79,80].
In e es ingly, in cu en popula ions, he e seems o be a end e en owa ds pe manen
GGT inc eases [
79
]. S udies ha e u he shown an associa ion be ween GGT le els and a
a ie y o ex ahepa ic ch onic diseases, which a e associa ed wi h oxida i e s ess, including
ca dio ascula diseases, diabe es, me abolic synd ome, cance , neu odegene a i e diseases and
heuma oid a h i is [
81
–
87
]. While he speci ic ole o alcohol as a possible igge o such mo bidi y
has emained unknown, i should be no ed ha ecen s udies ha e indica ed ha e en ligh o
mode a e alcohol d inking can lead o an ele a ed isk o cance [
88
] and an inc ease in all-cause
mo ali y [
16
,
89
]. Ele a ed GGT is associa ed wi h inc eased ca dio ascula isk especially in men
wi h simul aneous e idence o hepa ic s ea osis [
90
–
93
]. Fu he mo e, ecen s udies ha e linked he
de elopmen o a y li e and ea ly a he oscle osis wi h he abili y o GGT o igge i on-dependen
oxida ion o low densi y lipop o ein (LDL) in co ona y plaques [
94
]. S udies ha e also no ed signi ican
co ela ions be ween LDL-choles e ol and GGT le els, especially in men [
95
]. Howe e , GGT le els a e
also associa ed wi h mo ali y ou comes independen ly o a y li e [87].
Alcohol abuse is also a common cause o inc eased se um amino ans e ase (ALT,AST) ac i i ies.
ALT o igina es p ima ily om he hepa ocy es, whe eas AST is also abundan in hea , skele al
muscle issue, kidneys, and he b ain. Thus, se um ALT has been conside ed a mo e speci ic
ma ke o li e a ec ion, whe eas AST o en shows inc eased ac i i ies due o ex ahepa ic easons,
including muscle diseases o s enuous exe cise [
96
]. Cu en es ima es ha e indica ed ha o e
hal o he amino ans e ase abno mali ies in Wes e n coun ies esul om obesi y and ela ed
como bidi ies [
14
,
97
,
98
]. The occu ence o alcohol consump ion and adiposi y oge he also inc eases
he isk o abno mal ansaminase ac i i ies and while GGT enzyme seems o be ela i ely mo e
sensi i e o e hanol in ake, ALT may be he p edominan esponde owa ds inc easing BMI [
9
,
71
,
99
].
In obesi y, ALT ac i i ies co ela e wi h ec opic a deposi ion, and he alues decline wi h weigh
loss [
100
,
101
]. Inc eased ALT le els a e also linked wi h ex ahepa ic heal h isks, such as ype 2
diabe es, me abolic synd ome, and insulin esis ance [
72
,
83
,
102
–
104
]. They also p edic ascula
mo bidi y [72,92,102–108].
When in e p e ed oge he , amino ans e ases can p o ide in o ma ion on he na u e o li e
dys unc ion. The ele a ion o he AST/ALT a io o e one has been conside ed sugges i e o alcoholic
ae iology [
96
,
109
–
111
]. Such indings may be explained by deple ion o py idoxine (B6) i amin o
ALT biosyn hesis, mo e p onounced hepa ic mi ochond ial damage o skele al o ca diac muscle inju y
(alcoholic myopa hy), which elease AST in o ci cula ion [
109
,
112
]. Ele a ed AST/ALT a ios ha e,
howe e , also been epo ed om non-alcoholic s ea ohepa i is (NASH) pa ien s wi h a high ib osis
isk [55,113,114].
4. Impac s o Gende , Age and Li e S yle
Many e hanol-induced
biochemical changes ake place in a gende -dependen manne [3,9,62,95,115]
.
The indi idual suscep ibili y o diso de s such as li e ci hosis, b ain damage, hea disease o

In . J. En i on. Res. Public Heal h 2016,13, 166 6 o 19
alcohol-induced cance is ma kedly highe in women despi e he ac ha women gene ally d ink less
alcohol o e hei li e ime [
3
,
88
,
115
]. Lowe limi s o sa e d inking le els a e also ecommended o
women [
16
]. Women ha e less wa e in hei body and he e o e i is belie ed ha women a e exposed
o highe concen a ions o alcohol and i s oxic me aboli es du ing pe iods o alcohol d inking and
e hanol me abolism. In women, GGT le els a e also ele a ed a e inges ion o lowe le els o alcohol
han in men (Figu e 1).
In .J.En i on.Res.PublicHeal h2016,13,166
6

Figu e1.Th esholdle elso alcoholconsump ion(s anda dd inkuni s/week) o ini ia ingGGT
ac i a ioninindi idualsbelowandabo e40yea so age.Alcoholconsump ionwas eco ded om
hepas oneyea p io  osampling[116].Thele elsleading oGGTinc easesa ema kedlylowe 
han hecu en limi so hea yd inkinginmanyWes e ncoun ies(men:24d inks,women:16d inks).
Recen s udiesha ealsoemphasizedinc easingageasanimpo an de e minan o 
alcohol‐ ela ed oxici y.Inindi idualso e 40yea so ageonlyeigh s anda dd inks o menand
ou d inks o womenasle elso  egula e hanolconsump ionpe weeklead o i s signso GGT
ac i a ion(Figu e1).Al houghin hosebelow40yea sold heco esponding h esholddosesa e
highe ,i shouldbeno ed ha bo hle elso consump iona eclea lylowe  han hecu en lyused
limi so hea yd inkinginmanycoun ies.Since ecen popula ions udiesha eemphasizedhigh
mo ali y a esamongolde indi idualsconsumingalcohol[117], heconcep o sa elimi s o 
e hanolin akeshouldob iouslybe e isi edno onlybe weengende sbu alsoamongdi e en age
ca ego ies.Inaddi ion,inexpe imen alanimals,aginghasbeenshown op omo e hede elopmen 
o die ‐induceds ea ohepa i isandinduc iono li e enzymele els[118].
Thecomposi iono  hedie and hep esenceo absenceo obesi ya eimpo an co‐ ac o sin
de e miningbody esponses oalcoholconsump ion[9,11,71,119].Induc iono li e enzyme
ac i i ies oge he wi hele a edbloodlipidle elsmaybeseene enamongyoungindi idualswi h
o e consump iono  heWes e ndie [95,118].Inexpe imen alanimals,ad e see ec so e hanol
a eagg a a edbyhigh‐ a ‐die s[120]o die sde icien in ola e[121].Excessdie a yi onalso
exace ba ese hanol oxici y[120].Gene ic a ia ioninadiponu in(PNPLA3)o inalcohol‐me abolizing
enzymesalsoseem oplaya oleincon e ingsuscep ibili y o issuedamage[14].
Recen s udiesha e u he indica edasyne gis ic oxice ec o smokingone hanol‐induced
li e pa hologyandac i a iono GGTenzyme[10,122].On hecon a y,inhea yd inke swi h
egula co eeconsump ion,GGTle elsseem obe ela i elylowe  haninhea yd inke swi hou 
anyco eeconsump ion,indica ingapossiblep o ec i ee ec o co ee owa dsalcohol‐induced
li e damageandassocia edoxida i es ess[123–125].Co eeconsump ionseems omodula e he
e ec o e hanolinadose‐andgende ‐dependen manne , hemos s ikinge ec sbeing ound
amongmenwhod inko e  ou cupso co eepe day[123].Regula ae obicexe cise educes
hepa iclipidse enin heabsenceo bodyweigh  educ ionandcouldalsop o idep o ec ion
owa dsoxida i es ess[126,127].
5.Di e en ialDiagnosiso Alcoholic e susNon‐AlcoholicCauseso TissueToxici y
Clinicalsymp omso alcohol oxici ya eo enunspeci icandmaya ise om i uallyany
issue[3,6,18,128].Alcohol‐consumingpa ien s,howe e , end oescapespeci ic ea men because
hecliniciansabili y ode ec alcoholabuseiso encons ainedby hedi icul iesinob aining
eliable epo sonalcoholin ake[19].Inaddi ion oob ainingin o ma iononcu en d inking
habi sbyspeci icques ionnai esandbioma ke s,awideselec iono bioma ke sisalsoa ailable o
0
4
8
12
16
Men Women
E hanol doses pe week
< 40 yea s ≥ 40 yea s
Figu e 1.
Th eshold le els o alcohol consump ion (s anda d d ink uni s/week) o ini ia ing GGT
ac i a ion in indi iduals below and abo e 40 yea s o age. Alcohol consump ion was eco ded om
he pas one yea p io o sampling [
116
]. The le els leading o GGT inc eases a e ma kedly lowe han
he cu en limi s o hea y d inking in many Wes e n coun ies (men: 24 d inks, women: 16 d inks).
Recen s udies ha e also emphasized inc easing age as an impo an de e minan o
alcohol- ela ed oxici y. In indi iduals o e 40 yea s o age only eigh s anda d d inks o men
and ou d inks o women as le els o egula e hanol consump ion pe week lead o i s signs
o GGT ac i a ion (Figu e 1). Al hough in hose below 40 yea s old he co esponding h eshold doses
a e highe , i should be no ed ha bo h le els o consump ion a e clea ly lowe han he cu en ly
used limi s o hea y d inking in many coun ies. Since ecen popula ion s udies ha e emphasized
high mo ali y a es among olde indi iduals consuming alcohol [
117
], he concep o sa e limi s o
e hanol in ake should ob iously be e isi ed no only be ween gende s bu also among di e en age
ca ego ies. In addi ion, in expe imen al animals, aging has been shown o p omo e he de elopmen o
die -induced s ea ohepa i is and induc ion o li e enzyme le els [118].
The composi ion o he die and he p esence o absence o obesi y a e impo an co- ac o s
in de e mining body esponses o alcohol consump ion [
9
,
11
,
71
,
119
]. Induc ion o li e enzyme
ac i i ies oge he wi h ele a ed blood lipid le els may be seen e en among young indi iduals wi h
o e consump ion o he Wes e n die [
95
,
118
]. In expe imen al animals, ad e se e ec s o e hanol a e
agg a a ed by high- a -die s [
120
] o die s de icien in ola e [
121
]. Excess die a y i on also exace ba es
e hanol oxici y [
120
]. Gene ic a ia ion in adiponu in (PNPLA3) o in alcohol-me abolizing enzymes
also seem o play a ole in con e ing suscep ibili y o issue damage [14].
Recen s udies ha e u he indica ed a syne gis ic oxic e ec o smoking on e hanol-induced
li e pa hology and ac i a ion o GGT enzyme [
10
,
122
]. On he con a y, in hea y d inke s wi h egula
co ee consump ion, GGT le els seem o be ela i ely lowe han in hea y d inke s wi hou any co ee
consump ion, indica ing a possible p o ec i e e ec o co ee owa ds alcohol-induced li e damage
and associa ed oxida i e s ess [
123
–
125
]. Co ee consump ion seems o modula e he e ec o e hanol
in a dose- and gende -dependen manne , he mos s iking e ec s being ound among men who d ink
o e ou cups o co ee pe day [
123
]. Regula ae obic exe cise educes hepa ic lipids e en in he
absence o body weigh educ ion and could also p o ide p o ec ion owa ds oxida i e s ess [
126
,
127
].
In . J. En i on. Res. Public Heal h 2016,13, 166 7 o 19
5. Di e en ial Diagnosis o Alcoholic e sus Non-Alcoholic Causes o Tissue Toxici y
Clinical symp oms o alcohol oxici y a e o en unspeci ic and may a ise om i ually any
issue [
3
,
6
,
18
,
128
]. Alcohol-consuming pa ien s, howe e , end o escape speci ic ea men because
he clinicians abili y o de ec alcohol abuse is o en cons ained by he di icul ies in ob aining
eliable epo s on alcohol in ake [
19
]. In addi ion o ob aining in o ma ion on cu en d inking
habi s by speci ic ques ionnai es and bioma ke s, a wide selec ion o bioma ke s is also a ailable
o ule ou possible non-alcoholic e iologies (Table 2). Fo example, in pa ien s wi h suspec ed
li e a ec ion, NAFLD is known o be he mos common non-alcoholic e iology and e alua ion
o me abolic co-mo bidi ies wi h measu emen s o body mass index, wais ci cum e ence, and o al
glucose ole ance a e help ul [
14
,
18
]. Many compe ing and co-exis ing causes o abno mal li e unc ion
can be excluded by app op ia e se ological and gene ic es s (Table 2). In a simila manne , combined
use o issue-speci ic labo a o y ma ke s wi h ma ke s o e hanol consump ion, such as CDT, can be
used o de ec he possible alcoholic o igin in panc ea ic diso de s [129].
Table 2. Bioma ke -based di e en ial diagnosis o abno mal li e unc ion.
Condi ion Suppo ing Labo a o y Da a O he Diagnos ic Tools
Fa y li e
Alcoholic Alcohol, E G, GT, CDT, ALT, AST,
MCV Ques ionnai es: AUDIT, TLFB,
CAGE, MAST
Non-alcoholic (obesi y) ALT, AST, glucose, OGT, iglyce ides,
PNPLA3 geno yping BMI, wais ci cum e ence,
abdominal ul asonog aphy
Vi al hepa i is
A: an i-HAV IgM; B: HBsAg, PCR,
an i-HBc IgM; C: an i-HCV, PCR;
D: an i-HDV; E: an i-HEV; G:
an i-HGV
Li e ci hosis Albumin, bili ubin, p o h ombin ime,
immunoglobulins, ma ke s o immune
ac i a ion and ib ogenesis
Li e biopsy, xenobio ic me abolism
and exc e ion es s, li e imaging:
ul asound, MRI, Fib oscan, measu es
o hepa ic unc ion: Child-Pugh,
CCLI, CMI
D ug oxici y T ansaminases, he apeu ic d ug
moni o ing, blood eosinophils Case his o y
Hemoch oma osis I on s a us, ans e in i on sa u a ion,
e i in, HFE-geno yping
(C282Y mu a ion) Li e biopsy (hepa ic i on index)
Au oimmune diseases
Au oimmune hepa i is Immunoglobulins, an inuclea
an ibodies, an ismoo h muscle an igen
P ima y bilia y ci hosis AP, IgM, an imi ochond ial an ibodies
P ima y scle osing cholangi is ANCA, AP ERCP
α1-an i ypsin de iciency α-1-an i ypsin pheno yping
Wilson’s disease Ce uloplasmin, u ine and
hepa ic coppe
Celiac disease Tissue ansglu aminase an ibodies
S enuous exe cise AST, ALT, myoglobin,
c ea inine kinase
Malignan condi ion AFP Ul asound
Idiopa hic Absence o ma ke s Li e biopsy
ALT: alanine amino ans e ase; ANCA: an i-neu ophil cy oplasmic an ibody; AP: alkaline phospha ase;
AST: aspa a e amino ans e ase; AUDIT: alcohol use diso de s iden i ica ion es ; BMI: body mass
index; CAGE: alcohol ques ionnai e; CDT: ca bohyd a e-de icien ans e in; ERCP, endoscopic e og ade
cholangiopanc ea og aphy; E G: e hyl glucu onide; GGT: gamma-glu amyl ans e ase; MAST; Michigan
alcoholism sc eening es ; MCV, mean co puscula olume o e y h ocy es; OGT: o al glucose ole ance; PCR,
polyme ase chain eac ion; PNPLA3: pa a in like phospholipase-3; TLFB: ime line ollow-back.
In . J. En i on. Res. Public Heal h 2016,13, 166 8 o 19
6. Ma ke s o Disease P ognosis
Sco ing sys ems based on selec ed combina ions o bioma ke s ha e been de eloped o assessing
se e i y o alcohol-induced issue damage. In pa ien s wi h li e disease, algo i hms such as he
Child–Tu co e–Pugh sco e, Model o End-S age Li e Disease, and Combined Clinical and Labo a o y
Index e lec o e all li e unc ion, li e expec ancy and su gical mo ali y [
19
,
130
] (Table 3). These
pa ame e s co ela e wi h disease p ognosis and help o s a i y expec ed disease ou come and o
iden i y high- isk pa ien s o he apy. The labo a o y indices selec ed in hese models also show
signi ican co ela ions wi h impo an mo phological indices o disease se e i y, such as combined
mo phological index (CMI) [130].
Table 3. Bioma ke -based sco ing sys ems o he se e i y o alcoholic li e disease.
Sco e Full Name Clinical and His ological
Componen s Labo a o y Componen s
CPT Child-Pugh-Tu co e Asci es, encephalopa hy Albumin, bili ubin, p o h ombin ime
MELD Model o end-s age
li e disease Bili ubin, c ea inine, INR
MDF Madd ey disc iminan unc ion Bili ubin, p o h ombin ime
GAH Glascow alcoholic
hepa i is sco e Age Whi e blood cell coun , u ea, p o h ombin
ime, bili ubin
CCLI Combined clinical and
labo a o y index Asci es, encephalopa hy,
colla e al ci cula ion, edema
Hemoglobin, albumin, bili ubin, alkaline
phospha ase, p o h ombin ime
CMI Combined mo phological index Nec osis, in lamma ion,
cMallo y bodies Co ela es wi h labo a o y indices o
p ognos ic signi icance
Among he mos high-impac bioma ke s o assessing he se e i y o alcoholic li e disease
(ALD) a e se um bili ubin and li e -de i ed p o eins. Bili ubin is an insoluble b eakdown p oduc o
heme, which is conjuga ed o glucu onic acid in he li e [
55
]. S ongly (5–10 old) ele a ed bili ubin
le els ha e been shown o be a highly signi ican p ognos ic de e minan and is included in mos
algo i hms (Table 3) [
130
]. Concen a ions o se um albumin, e i in, and blood clo ing ac o s also
show cha ac e is ic changes in esponse o li e disease s age [
55
]. The hal -li e o albumin is abou
20 days, whe eas ha o clo ing ac o s is only abou one day. Se um albumin, which also plays
a unc ional ole as a ci cula ing an ioxidan , is o en sligh ly ele a ed in hea y d inke s de oid o
li e disease [
131
,
132
]. In pa ien s wi h ad anced li e disease p o ein syn hesis a es a e ma kedly
dec eased and le els below 25 g/L associa e wi h poo p ognosis [
55
,
133
]. In alcohol consume s
wi hou appa en li e disease, se um e i in syn hesis a es a e also inc eased, which can be associa ed
wi h dis u bances in cellula i on homeos asis and he isk o seconda y i on o e load [
133
,
134
]. I on
and alcohol can also ac in a syne gis ic manne o enhance lipid pe oxida ion, oxida i e s ess and
associa ed li e inju y [
12
,
120
,
135
,
136
]. On he o he hand, se um e i in can seques e ca aly ically
ac i e ee i on, which has been conside ed a possible de ense mechanism owa ds e hanol-induced
oxida i e s ess [137].
7. Bioma ke s o Fib ogenesis
Fib osis in alcoholics is a esponse o inju y, cell dea h and in lamma ion, cons i u ing a majo
de e minan o pa ien ou come [
20
,
138
,
139
]. Al hough p og ession o ib osis o i e e sible ci hosis
is la gely dependen on he amoun s o alcohol consumed o e a long pe iod o ime, i may also
occu in an unp edic able manne in suscep ible indi iduals. The gold s anda d o diagnos ics is he
mo phological examina ion o biopsy specimens, which is, howe e , a cos ly and in asi e app oach
wi h a possible isk o complica ions. The e o e, bioma ke s o ollowing he ac i i y o excess
connec i e issue deposi ion a e also equi ed. O e he pas decades, se e al non-in asi e ools ha e
been in oduced o allow epea ed examina ions du ing pa ien ollow-up (Table 4). In addi ion o
In . J. En i on. Res. Public Heal h 2016,13, 166 9 o 19
speci ically designed imaging echniques (Fib oscan), bioma ke s based on collagen ype-speci ic
pep ides and a ious labo a o y algo i hms ha e become a ailable [18,20,140].
Type I and ype III collagens a e he main ypes o collagen accumula ing in hepa ic issue in
esponse o alcoholic inju y. The la e is mo e pliable and he e o e ype III p ocollagen de i ed
agmen s ha e been p e e ed as bioma ke s [
18
,
141
]. The amino e minal p opep ide o ype III
p ocollagen (PIIINP), is ele a ed in ALD and he measu emen s help o iden i y pa ien s wi h
p og essi e collagen deposi ion [
141
]. Hyalu onic acid (HA), a mucopolysaccha ide syn hesized
by ib oblas s and hepa ic s ella e cells, also inc ease in ALD co ela ing wi h he p og ession o
pe isinusoidal ib osis and ci hosis [138].
The inabili y o collagen deg ada ion o keep pace wi h inc eased biosyn hesis is a ypical
ea u e o p og essi e ib osis. The deg ada ion o ex acellula ma ix is egula ed by issue
inhibi o s o me allop o einases (TIMPs), which a e usually ele a ed in alcoholics wi h p eci ho ic
s a es [
138
,
139
]. In se e e s ages o ALD, he e seems o be p ominen ele a ions in se um PIIINP and
p oin lamma o y cy okines (IL-2, IL-6, IL-8, TNF-
α
), which coincides wi h low le els o ma ke s o
ib olysis and an i-in lamma o y cy okines (IL-10, TGF-
β
) [
142
,
143
]. Assays e lec ing he dis u bed
balance be ween collagen syn hesis and deg ada ion ha e been p oposed o p o ide mo e accu a e
es ima es o he collagen deposi ion a es han analyses o any single connec i e- issue de i ed
pep ide [
20
,
138
–
140
,
144
,
145
] (Table 4). A his ime, Fib o es is he mos widely used such algo i hm
in Eu ope [
140
]. ELF (Enhanced Li e Fib osis), a es combining se um PIIINP, hyalu onic acid
and TIMP, has also shown signi ican co ela ions wi h his ological indings in he ollow-up o
ib ogenesis [
146
]. O he ma ke s include combina ions o connec i e issue componen s wi h blood
pla ele le els [147] (Table 4).
Table 4. Bioma ke s o ib ogenesis.
Ma ke Abb e ia ion Componen s in Combina ion
Connec i e issue de i ed pep ides
Aminop opep ide o p ocollagen ype III PIIINP
Aminop opep ide o p ocollagen ype I PINP
Ca boxyp opep ide o p ocollagen ype I PICP
Ca boxy e minal elopep ide o ype I collagen ICTP
Hyalu onic acid HA
β-C osslaps β-CTX
Tissue inhibi o o ma ix me allop o einase TIMP
Combina ion ma ke s
Fib o es GGT, ALT, α-2-mac oglobulin,
hap oglobin, apo A1, bili ubin
Enhanced li e ib osis ELF PIIINP, hyalu onic acid, TIMP
AST/pla ele a io APRI AST, pla ele coun
T a ic ligh es TLT
PIIINP, hyalu onic acid, h ombocy es
8. Ma ke s o Immune Ac i a ion in Alcohol Use Diso de s
Table 5summa izes use ul con en ional and no el bioma ke s o immune ac i a ion in alcoholic
pa ien s. The p esence o absence o in lamma ion is a key de e minan o pa ien ou come in
he pa hogenesis o alcohol use diso de s. In alcoholic li e disease, an al e ed balance be ween
p o- and an i-in lamma o y s a us is ela ed wi h p og ession o ib ogenesis. P o eins exp essed
by immunologically ac i e cells, such as soluble u okinase plasminogen ac i a o ecep o (suPAR)
is inc eased as a esul o hea y alcohol consump ion and u he wi h he de elopmen o li e
In . J. En i on. Res. Public Heal h 2016,13, 166 16 o 19
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