combinations of folate deficiency and a diet containing 40% of kcal as Thus, the development of ALD is linked to abnormal methionine metabolism

INTERACTIONS

OF ETHANOL AND

FOLATE

DEFICIENCY

IN

DEVELOPMENT OF

ALCOHOLIC

LIVER

DISEASE

IN

THE

MICROPIG

CHARLES H. HALSTED and (byinvitation)JESUSA. VILLANUEVAand ANGELA M. DEVLIN

DAVIS, CA S. JILL JAMES

JEFFERSON, AR

ABSTRACT

Folate deficiency

is present in most

patients

with

alcoholic

liver

disease

(ALD), whereas folate regulates and alcoholism

perturbs

in-trahepatic

methionine

metabolism, and S-adenosyl-methionine

pre-vents

the

development of experimental

ALD.

Our studies

explored the

hypothesis that abnormal methionine metabolism

is

exacerbated by

folate

deficiency and

promotes

the

development of ALD

in

the setting

of

chronic ethanol

exposure.

Using

the micropig

animal

model, dietary

combinations of folate deficiency and

a

diet containing

40%

of kcal

as

ethanol

were

followed

by

measurements

of

hepatic

methionine

metab-olism and indices

of ALD. Alcoholic liver injury,

expressed

as

steato-hepatitis in terminal 14 week

liver

specimens, was

evident in

mi-cropigs

fed

the

combined

ethanol containing and

folate deficient

diet

but

not

in

micropigs fed each diet

separately.

Perturbations

of

methi-onine

metabolism

included decreased hepatic

S-adenosylmethionine

and

glutathione

with increased

products

of DNA and

lipid

oxidation.

Thus, the development

of

ALD is

linked

to

abnormal methionine

me-tabolism and

is

accelerated

in

the

presence

of folate

deficiency.

INTRODUCTION

Alcohol

is a

frequent

component

of the American diet that

provides

7.1

kcal/g, is essentially devoid of micronutrients,

and, when consumed

in

excess,

influences the availability and hepatic

metabolism of many

From theUniversity ofCalifornia, Davis, California and NationalToxicological Research Center, Jefferson Arkansas.

Addressfor reprints: CharlesH.Halsted, MD, Department ofInternal Medicine, TB156, School ofMedicine, One ShieldsAvenue,University of California Davis, Davis, CA95616.

Phone: 530-752-6778; Fax: 530-752-3470.

nutrients

(1). At least

5%

of the population

consume

alcohol

in excess

and

are at

risk

for

alcoholic liver disease (ALD), the 11th

cause

of

mortality

in

the

United

States

(2). It

has

been increasingly recognized

that one or many

features

of

malnutrition

are present in

virtually all

patients who

develop

ALD.

For

example, varied degrees of protein

calorie malnutrition

that

correlated

with

the

severity

of

ALD were

found universally

in more

than 500 ALD patients

enrolled

in two

multicenter U.S. Veterans Administration studies (3). Among

micro-nutrients,

folate

deficiency

is

the most common

finding

in

chronic

alcoholics,

occurring in up to 80%

of those

at

risk

for

developing ALD

(4-6).

In

clinical studies of alcoholics and

in

animal models of chronic

alcohol

exposure,

folate

deficiency

was

shown

to

result from

poor

diet

and combinations

of decreased folate

absorption, hepatic uptake, and

increased

renal excretion

(7-13).

The

frequent finding

of folate

defi-ciency

in

chronic

alcoholism

suggests a role

for

this vitamin in

the

pathogenesis of ALD.

According

to extensive

studies in animal models, current concepts on

the

pathogenesis

of ALD

involve

ethanol

and

cytokine

induced

produc-tion

of

reactive oxygen species

(ROS), which, together with reduced

antioxidant

defense, results

in

hepatocyte

necrosis,

inflammation, and

eventual

collagen production and

cirrhosis (14-16). The metabolism of

ethanol

in

the liver by the microsomal

enzyme

CYP2E1

generates

oxidative injury through formation of the hydroxyethyl radical (17,18).

At

the

same

time,

gut-derived

lipopolysaccharide (LPS)

enterotoxin

stimulates the

production

in

Kupffer cells of

tumor

necrosis

factor

a

(TNFa),

which,

in turn,

invokes

a

signal

transduction

cascade

in

hepa-tocytes

that involves

production of ceramide

from

membrane

sphingo-myelin and enhances the mitochondrial

generation

of ROS

(19,20).

Oxidative liver

injury is

the

net

result

of

enhanced generation

of ROS

and

depletion

of

antioxidants such

as

glutathione

(GSH). At the

same

time, apoptosis

or

cell death

is

promoted

by stimulation of intracellular

caspases

and enhanced DNA strand

breakage (15,21).

Over

the past

decade,

it

has

become

apparent

that abnormal

hepatic

methionine metabolism

is

integral

to

ALD

and

perhaps

central to its

pathogenesis.

As

depicted

in

Figure 1,

folate

in

the form

of

5-methyl-tetrahydrofolate (5-MTHF)

and

homocysteine are substrates with

vi-tamin B12

co-factor methionine

synthase

for the

production

of

endog-enous

methionine

that,

in

turn, is

substrate for methionine

adenosyl

transferase

(MAT1)

in

the

production

of 6-8

g/d

of

S-adenosylmethi-onine

(SAM)

in

the

human liver. SAM is the

principal

methyl

donor in

a

multitude of reactions and

regulates

a

number of methionine

cycle

pathways, including

the

generation

of GSH from

homocysteine (16,22).

DNA

DNA MAT M DEMTs

t

PgM

mslylsdDN

ETMI

_{dTMP xDcol4e --PC}

dUMP methionine

Aden

CDPo-wlS\H

transferaMeT(PEMT), (SAMisconverte toS h chinsPA

THF ~~~ ~ ~ ~~~~~~SH 5,0MTHFR homocysteine -/ 5-MTHF ()I[~ -- A cystttlonlne CY(SAH -t l glutathione

FIG. 1. Folate and methionine metabolism in the liver.

5-methyltetrahydrofolate

(5-MTHF), is substrate for themethionine

synthase

(MS)reaction thatgenerates

me-thionine from homocysteine. In an alternate

salvage pathway,

betaine, a

product

of choline

metabolism,

is the substrate for betaine homocysteine

methyltransferase

(BHMT).Methionine is convertedto

S-adenosylmethionine

(SAM)

by

methionine adeno-syltransferase (MAT).

Through

reactions that include DNA methylation andthe syn-thesis of

phosphatidylcholine

(PC) from

phophatidylalcoholamine

(PE)

by

PE

methyl

transferase(PEMT),SAMisconvertedto

S-adenosylhomocysteine

(SAH),whichis also

up-regulated

by

synthesis

from

homocysteine through

the reversible SAH

hydrolase

(SAHh)reaction. SAM

regulates

the

synthesis

of

glutathione

(GSH)by

up-regulation

of

cystathionine dsynthase

and the

homocysteine

transsulfuration

pathway.

SAM

provides

negative regulatory

feedbacktothe

methylenetetrahydrofolate

reductase(MTHFR)

reac-tion

that

converts

5,10

methenyltetrahydrofolate

(5,10-MTHF)

to5-MTHF.

The

ratio

of

SAM

to

its

product

S-adenosylhomocysteine

(SAH)

is

considered

a

comprehensive

measure

of

inhibition of functional SAM

activity,

in

particular

as

related

to

methylation

(23).

Early

clinical

studies in this field

demonstrated

decreased levels of

MATi

activity

and SAM in

liver

biopsies

from ALD

patients

(24,25).

The

administra-tion

of SAM attenuated

the decrease in GSH and the

experimental

development

of ALD in

ethanol

fed

baboons,

and

improved

the

clinical

status

of

patients

with varied

degrees

of

ALD

(26,27).

Recently

it

was

demonstrated in

ethanol fed

rats

that SAM

promotes

the

fluidity

of the

mitochondrial membrane and the

transport

of

intracellular GSH

to

its

mitochondrial site

of

activity

(28).

We

developed

an

animal

model of ALD in the

micropig,

a

species

that

consumes

ethanol

voluntarily

in

the diet.

Initially,

castrated

male

Yucatan

micropigs

were

fed 40%

of kCal

as

ethanol

or

cornstarch

control

and all essential nutrients

including

an excess

of folate.

Among

the ethanol-fed

micropigs

we

observed

typical

features ofalcoholic liver

injury

after

12

months of feeding and cirrhosis

at 21

months, together

with the

accumulation of protein adducts

of

the alcohol metabolite

acetaldehyde

and the lipid oxidant product

malondialdehyde (29,30).

There were no

histological changes in a

subsequent study

of

intact

and

uncastrated

micropigs

fed the

same

diets for

12

mo,

which

we

ascribed

to

changes

in

testosterone

secretion

(31,32).

On the bases of the

high

frequency of folate

deficiency

in

human

ALD, the

essential role of folate in hepatic

methionine metabolism,

and

prior

evidence

for disturbed methionine

metabolism

in

ALD, the

present

study

tests

the

hypothesis that folate

deficiency

promotes

both

abnormal

hepatic methionine metabolism and the development of

ALD.

MATERIALS AND METHODS

Twenty-four intact male micropigs each weighing -20 kg were

pur-chased

at 6 mo

age

(Sinclair, Columbia, MO)

and

were

grouped

to

receive

four

different diets for

14

weeks. Each

diet provided

90

kCal/kg

body wt/d

as

polyunsaturated

corn

oil

at 33%

of

kCal,

protein as

vitamin-free

casein at 2

g/lkg

body wt/d, and

cornstarch as

carbohy-drate,

or

the

same

diet

substituting ethanol for cornstarch

at 40%

of

kCal,

equivalent to 5

glkg body wt/d.

Methionine,

choline, and all

essential minerals and

vitamins were

provided with

or

without folic

acid

in

accord

with

the established

requirements

of

growing swine

(33).

The four diets

were

folate-sufficient (FS, or control) with excess folate

at

14.5

ggfkg

body

wt,

folate deficient

containing no

added

folate (FD),

folate

sufficient with ethanol (FSE),

and folate

deficient with ethanol

(FDE).

The animals were weighed and group-paired weekly to ingest

the

same mean

amount as

ingested by the

FDE group.

The U.C.

Davis

Animal

Welfare

Committee

approved the

feeding protocol and all

ex-perimental studies. Animals

were

housed

in

individual kennels

at

the

University of California

Davis

Animal

Resources

Center

facilities,

which

are

approved

by the

National

Institutes of Health and animal

care

followed the

standards and procedures outlined in the National

Academy

of

Sciences

"Guide for the Care and Use of Laboratory

Ani-mals."

Blood samples were obtained for measurements of serum

homocys-teine,

aspartyl aminotransferase (AST),

alanine aminotransferase

(ALT), and malondialdehyde

(MDA) (34,35) as an

index of lipid

per-oxidation. Terminal urine was obtained

by

bladder puncture and was

analyzed for 8-oxo-2'-deoxyguanosine

(Oxo8dG)

as an

index of

DNA

oxidation (36). Terminal liver samples were homogenized and analyzed

for folate

(37), methionine and choline metabolites (38,39), activities of

MS (40) and BHMT (41), and histology.

Data were analyzed by repeated measures or 2-way analysis of

variance, where the independent variables were folate status

(suffl-cient or

deficient), ethanol treatment, and time. When interactions

were

significant, separate sub-group analyses were performed;

other-wise

analyses

were

done with both variable groups pooled.

Correla-tions

between

variables were determined by linear regressions using

all points.

RESULTS

During the 14 weeks of the experiment, micropigs in the FS control

group

gained more than twice the weight of each of the other three

groups. Mean

plasma homocysteine levels were increased in all three

experimental groups from week 6 onward. The greatest

effect occurred

in

the FDE group, where levels were increased 3-fold over those in FS

control and were greater than those each of the other experimental

groups.

Table 1 describes the significant effects of each dietary regimen on

terminal hepatic methionine enzymes and metabolites, urinary

Oxo8dG, plasma MDA, AST,

and ALT. Terminal

hepatic

folate levels

were

decreased

by

one

half in groups FD and FDE,

homocysteine

was

elevated in FSE and

FDE,

and methionine was reduced in

FD,

FSE,

and FDE. Consistent with reduced

methionine,

MS activity was

re-duced in FSE and FDE, whereas the compensatory BHMT

activity

pathway

was

increased

in

FD

and FDE.

Choline

levels were

un-changed,

while

betaine

was

reduced in FD

and

FDE consistent

with its

role as substrate for BHMT. At the same time,

hepatic

PC was reduced

in

all three

experimental

groups

due to an interaction between

ethanol

and folate

deficiency.

Consistent with the effects of

feeding

on

reduc-tions

of liver methionine levels in all three

experimental

groups,

he-patic SAM was reduced in FSE and

FDE,

hepatic

SAH

was

increased

and the SAM to SAH ratio was decreased in all three

experimental

groups due to an interaction between ethanol and folate

deficiency.

Hepatic

GSH, the principal mitochondrial

antioxidant,

was

reduced

in

FSE and FDE,

correlating with levels of SAM. Urinary

Oxo8dG,

a

measure

of DNA

oxidation, was increased

in

FD,

FSE,

and FDE.

Plasma

MDA, a measure of lipid peroxidation, was increased in FSE

and FDE. Levels

of

plasma AST and

ALT,

each a

measure

of liver

TABLE1

Effectsof Experimental Diets on HepaticMethionine Enzymes andMetabolites,DNAand LipidPeroxidation, and LiverInjury Enzymes

Variable FD FSE FDE

Folate (-) * Homocysteine (+) t t Methionine (-) * t t MS(-) t t BHMT(±) * * Choline Betaine(-) * * PC (-) * t SAM (-) t SAH(+) * .t *t SAM/SAH (-) * t GSH(-) t t Oxo'dG(urine) (+) * t t MDA(plasma) (+) t t AST (plasma) (+) t tt ALT (plasma) (+) t t

(+)Positiveeffect, (-) negative effect, * significant effect of folate deficiency, t significant effect of ethanolfeeding, tsignificant interaction offolatedeficiencyand ethanolfeeding.A significant positive correlation was found by linear regressions between individual values of hepatic SAM and GSH. Significant negative correlations were found between values of hepatic GSHorthe SAMtoSAH ratio and levels ofurineOxo'dGandplasma MDA, and between the SAMtoSAH ratio and values of plasma ALT.

injury,

were

increased

in

FSE

and

FDE. Levels

of urinary

Oxo8dG

and

plasma

MDA were

each correlated negatively

to

the SAM

to

SAH

ratio

and

to

levels

of

hepatic GSH. The level of plasma

ALT was

correlated

negatively

to

the SAM

to

SAH

ratio.

While there

were no

changes

in

liver

histology

in

FS

control,

FD

and

FSE

micropigs,

livers from

5

of

the

6

animals in group FDE

demon-strated

lesions

characteristic

of alcoholic liver

injury,

specifically

dif-fuse intralobular

hepatocytes

steatosis

and

necrosis

with

infiltration

of

inflammatory

cells.

DISCUSSION

The

significance of the present

findings

relate to

the potential

im-portance of

perturbations

of

methionine

metabolism in chronic

alco-holism

during

development

of

ALD. In addition to rodent ethanol

feeding

studies

describing

reduction in MS

activity

and SAM

produc-tion

with

compensatory

increase in BHMT

(42-44),

a

clinical

study

demonstrated

decreased transcripts of

MATl1A,

BHMT,

C,3S

and MS in

liver biopsies from ALD patients

(45).

Using the

intragastric

ethanol-fed

rat

model,

others recently demonstrated decreased

hepatic levels

of

methionine and SAM, together

with decreased MAT1 but

increased

MAT2 activity

and increased DNA

strand

breaks

in

the

ethanol

group

(46).

In a

previous study of uncastrated male micropigs fed control or

ethanol

containing

diets identical

to

the present FS and FSE groups for

12

months, we

found

that ethanol feeding

significantly

reduced

MS

and the

SAM

to

SAH

ratio,

while

causing

DNA

nucleotide

imbalance

and

increasing

hepatocellular

apoptosis (31).

In

the present

studies,

we

tested

the hypothesis that the

develop-ment

of ALD involves

perturbations

in

hepatic

folate-regulated

methi-onine

metabolism that result from

chronic

ethanol

ingestion and are

enhanced in

the

presence

of folate

deficiency

in

the

micropig. The

experimental design permitted analysis of the

separate,

additive,

and

synergistic

effects of folate deficiency

and chronic

ethanol

exposure on

methionine and

choline metabolism. By deleting folate

from the

diet,

we

achieved

-50%

lower

liver

folate

levels in

animals fed folate

defi-cient diets

with

or

without ethanol

(Table 1).

Ethanol

feeding acted

alone in

elevating

liver

homocysteine and plasma

MDA and ALT

and

in

reducing MS activity,

SAM,

and GSH. Folate deficiency acted alone

in

increasing BHMT activity

and reducing liver

folate

and

betaine.

Ethanol

feeding and folate deficiency

were

additive

in

elevating

serum

homocysteine

levels

and liver SAH and

Oxo8dG

levels.

On

the other

hand, ethanol

feeding

and

folate deficiency

were

interactive

and

syn-ergistic

in

reducing the

liver

SAM

to

SAH

ratio

and PC

levels,

in

elevating

serum

AST

levels,

and

in

production

of

the

histopathological

features

of

steatonecrosis.

The

finding of

a

positive

correlation

of SAM

to

GSH

is

explained by

the known

regulatory role

of

SAM

in

the

transsulfuration

pathway

(47),

and links

methionine metabolism

to

the

anti-oxidative

GSH

re-sponse.

The finding of

a

negative

correlation

of the SAM to SAH ratio

and of GSH to

plasma

MDA

levels

links

increased lipid

peroxidation

to

SAM

through

its

regulation of GSH production. Similarly, the finding

of a

negative

correlation

of

the

SAM to SAH ratio

and

of GSH

to

urinary

Oxo8dG

levels links

increased

DNA oxidation

to

SAM

through

its

regulation

of the

anti-oxidant GSH.

At the same time,

the negative

correlation of the

hepatic

SAM

to

SAH ratio

to

the

plasma

ALT

level

links

hepatic liver injury to

aberrations in the methionine

cycle.

By

demonstrating the presence of steatonecrosis

only

in

the FDE

group

and its absence in the FSE group, we

determined

that folate

deficiency

promotes

while folate

sufficiency

attenuates

the

develop-ment

of ALD. Further underscoring the

significance

of folate

deficiency

histo-pathology

in

the

FDE group

micropigs after

14

weeks of

feeding

con-trasts

with absence of

overt

histopathology in

our

previous 12

mo

study

of micropigs fed the

identical folate

sufficient control

or

ethanol

con-taining

diets (31).

How

might the observed perturbations of methionine

metabolism

triggered

by ethanol

and

accentuated by

folate

deficiency

be

related

to

the

pathogenesis

of ALD?

A prior

study showed induction of

apoptosis

of

hepatocytes

in rats

fed

diets

deficient

in

folate, methionine, and

choline,

in

association

with nucleotide imbalance,

as

described

by

elevation

in

dUMP and reduction of

dTMP

levels (48). Since

we

achieved the

same

findings

in

ethanol fed but folate

replete micropigs

(31),

we can presume

that this apoptotic mechanism is

magnified

in

pigs

fed

a

combination

of folate deficient and ethanol containing diets.

Our

present

findings linking decreased SAM

to

GSH are relevant in

view

of the

known effects of chronic alcoholism and ALD on reduction

of liver

GSH and

its correction

by SAM (26,27),

and

are

consistent

with

concepts on

the

production of ethanol induced oxidative

liver injury

and the

important

role of GSH antioxidant defense (15,16). Lastly,

a

prior

finding

of SAM attenuation of plasma TNFa and

LPS-stimulated

steatonecrosis in

choline

deficient

rats

suggests

that SAM

deficiency

accentuates

TNFa

mediated liver

injury

(49).

The

present

study has potential

implications

for

the prevention

and

treatment

of ALD. As demonstrated previously

in

the baboon

model

(26),

the

provision

of SAM would be expected

to correct

the

observed

decrease

in

SAM

levels

and

increase

levels

of

the

antioxidant

GSH in

the ethanol

fed

groups,

thereby

countering

the

process

of oxidative

liver

injury.

Through

its

positive

effect in maintaining 5,10-MTHF for

the TS reaction,

SAM would

also

predictably

maintain

DNA nucleotide

balance and

prevent

DNA strand breakage

and

hepatocellular

apopto-sis.

Although mechanisms

were not

provided,

a

multicenter

clinical

trial

demonstrated

the

efficacy

of

SAM

treatment

of

patients with

established alcoholic hepatitis,

an

intermediate

form of

ALD

(27).

Further, provision of PC has been shown

to prevent

the development of

ALD in the

baboon model by

uncertain

mechanism

(50). Based on our

finding

of

diminished PC

in

all experimental

groups

and choline

path-ways, the salutary effect of correction of PC deficiency could be

ex-plained by

enhancement

of

betaine as

substrate for BHMT and

methi-onine and SAM

replenishment,

or

by enhancing

sphingomyelin

synthesis

and

thereby decreasing

the

availability

of

ceramide

for

the induction of oxidative

hepatocellular

necrosis

(15,51).

Finally,

the

present

finding

that steatonecrosis was limited to

animals

fed the

combined folate deficient and ethanol FDE diet suggests that

mainte-nance of normal folate stores

through provision of supplemental folic

acid or 5-MTHF may

delay the onset or mitigate the severity of ALD.

The present model of ALD in

micropigs fed ethanol and folate deficient

diets lends itself to further

exploration of these potential mechanisms

of liver injury and their potential correction by various modalities

targeted at methionine

metabolism

in

the liver.

ACKNOWLEDGMENTS

This work was supported byUSPHS Grants DK 45301 and DK 35747 to CHH. The authors areindebtedtothefollowing individuals for expert technical assistance: Tim Garrow, Universityof Illinois, Urbana, IL; Steven Zeisel, University of North Carolina, Chapel Hill, NC; and Lynn Wallock and Mark Shigenaga, Children's Hospital of Oak-land Research Institute, OakOak-land,CA.

REFERENCES

1. Lieber CS. Medical disorders ofalcoholism. N Engl J Med 1995;333:1058-1065. 2. National InstituteonAlcohol Abuse and Alcoholism. Tenthspecial reporttothe US

Congress on alcohol and health. Rockville, MD: US Department of Health and HumanServices, 2000.

3. Mendenhall C, Roselle GA, Gartside P, Moritz T. Relationship of protein calorie malnutrition toalcoholic liver disease: a reexamination of data fromtwoVeterans Administration CooperativeStudies. Alcohol Clin Exp Res 1995;19:635-641. 4. LeevyCM, Cardi L, Frank 0, Gellene R, Baker H. Incidence and significance of

hypovitaminemiainarandomly selected municipal hospitalpopulation. Am J Clin Nutr 1965;17:259-271.

5. EichnerER, Hillman RS. The evolution of anemiainalcoholic patients.AmJMed 1971;50:218-232.

6. WuA, Chanarin I, SlavinG, Levi AJ. Folate deficiencyinthe alcoholic-its rela-tionshiptoclinical and haematological abnormalities, liver disease and folatestores. BrJ Haematol 1975;29:469-478.

7. Herbert V,Zalusky R, Davidson CS. Correlation of folate deficiency with alcoholism and associated macrocytosis, anemia, and liver disease. Ann Intern Med 1963;58:977-988.

8. HalstedCH,RoblesEA, Mezey E. Decreased jejunal uptake of labeled folic acid (3 H-PGA) in alcoholic patients: roles of alcohol and nutrition. N Engl J Med 1971;285:701-706.

9. Romero JJ, Tamura T, Halsted CH. Intestinal absorption of [3H]folic acid inthe chronic alcoholicmonkey.Gastroenterology 1981;80:99-102.

10. TamuraT,RomeroJJ,WatsonJE,Gong EJ,Halsted CH.Hepatic folate metabolism inthe chronicalcoholic monkey.JLab Clin Med 1981;97:654-661.

11. TamuraT, Halsted CH. Folateturnoverinchronically alcoholic monkeys.JLab Clin Med 1983;101:623-628.

12. McMartin KE, Collins TD, Eisenga BH, Fortney T, Bates WR, Bairnsfather L. Effects of chronic ethanol and diettreatment onurinaryfolateexcretionand devel-opment of folatedeficiencyintherat.J Nutr1989;119:1490-1497.

pro-longed turnover time of folic acid during ethanol ingestion. Am J Clin Nutr 1983;38:64-70.

14. Lieber CS. Ethanol metabolism, cirrhosis and alcoholism. Clin Chim Acta 1997;257:59-84.

15. Tilg H, Diehl AM. Mechanisms of Disease:Cytokines inAlcoholic and Nonalcoholic Steatohepatitis. N Engl J Med 2000;343:1467-1476.

16. Tsukamoto H, Lu SC. Current conceptsinthe pathogenesis of alcoholic liver injury. Faseb J 2001;15:1335-1349.

17. Albano E, Clot P, Morimoto M, Tomasi A,Ingelman-Sundberg M, French SW. Role ofcytochrome P4502E1-dependent formation of hydroxyethyl free radical in the development of liver damageinratsintragastrically fed with ethanol. Hepatology 1996;23:155-163.

18. Roman J, Colell A, Blasco C, et al. Differential role of ethanol and acetaldehydein theinduction of oxidativestress inHEP G2 cells:effectontranscription factors AP-1 andNF-kappaB. Hepatology 1999;30:1473-1480.

19. Garcia-Ruiz C, Colell A, Mari M, Morales A, Fernandez-Checa JC. Direct effect of ceramideonthemitochondrial electron transport chain leadstogenerationof reac-tive oxygenspecies. Roleof mitochondrial glutathione. Journal of Biological Chem-istry 1997;272:11369-11377.

20. Hannun YA. Functions of ceramide in coordinating cellular responses to stress. Science 1996;274:1855-1859.

21. Deaciuc IV, Nikolova-Karakashian M,Fortunato F, Lee EY, Hill DB, McClain CJ. Apoptosis and dysregulated ceramide metabolism in a murine model of alcohol-enhancedlipopolysaccharide hepatotoxicity [In Process Citation]. Alcohol Clin Exp Res 2000;24:1557-1565.

22. LuSC. Methionine adenosyltransferase and liver disease: it's all about SAM [edi-torial; comment]. Gastroenterology 1998;114:403-407.

23. Mato J MAL, Corrales FJ, Pajares MA. S-adenosylmethionine and the liver. In: Arias IM BJ, Fausto N, Jakoby WB, Schachter DA, Shafritz DA, ed. The liver: biology and pathobiology. 3rd ed. New York: Raven, 1994:461-470.

24. DuceAM, Ortiz P, Cabrero C, Mato JM. S-adenosyl-L-methionine synthetase and phospholipid methyltransferase are inhibited in human cirrhosis. Hepatology 1988;8:65-68.

25. Cabrero C, Duce AM, Ortiz P, Alemany S, Mato JM. Specific loss of the high-molecular-weight form of S-adenosyl-L-methionine synthetase in human liver cir-rhosis. Hepatology 1988;8:1530-1534.

26. LieberCS, Casini A, DeCarli LM, et al. S-adenosyl-L-methionineattenuates alcohol-induced liver injuryinthe baboon.Hepatology 1990;11:165-172.

27. Mato JM, Camara J,Fernandez de Paz J, et al.S-adenosylmethionineinalcoholic liver cirrhosis: arandomized, placebo-controlled, double-blind, multicenterclinical trial. JHepatol 1999;30:1081-1089.

28. ColellA, Garcia-Ruiz C, Morales A,etal.Transport ofreducedglutathioneinhepatic mitochondriaandmitoplastsfrom ethanol-treated rats: effect of membranephysical properties andS-adenosyl-L-methionine. Hepatology 1997;26:699-708.

29. Halsted CH, Villanueva J, ChandlerCJ, et al. Centrilobular distributionof

acetal-dehydeandcollageninthe ethanol-fedmicropig.Hepatology 1993;18:954-960. 30. Niemela 0, Parkkila S, Yla-Herttuala S, VillanuevaJ, Ruebner B, Halsted CH.

Sequential acetaldehyde production, lipidperoxidation,andfibrogenesisinmicropig

model of alcohol-induced liver disease.Hepatology 1995;22:1208-1214.

methionine metabolism and increases hepatocellular apoptosis and proliferation. Hepatology 1996;23:497-505.

32. Niemela0, Parkkila S, Pasanen M, Viitala K, Villanueva JA, Halsted CH. Induction of cytochrome P450 enzymes and generation of protein-aldehyde adducts are asso-ciatedwith sex-dependent sensitivity to alcohol-induced liver disease in micropigs. Hepatology 1999;30:1011-1017.

33. Agriculture SoSNBo. Nutrient Requirements of Swine. 10th ed. Washington DC: National Research Council, 1998.

34. Yeo HC, Liu J, Helbock HJ, Ames BN. Assay of malondialdehyde andother alkanals inbiological fluids by gas chromatography-mass spectrometry. Methods Enzymol 1999;300:70-78.

35. BasnakianAG, James SJ. Quantification of 3'OH DNA breaks by random oligonu-cleotide-primed synthesis (ROPS) assay. DNA Cell Biol 1996;15:255-262.

36. HelbockHJ, Beckman KB, Shigenaga MK, et al. DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc NatlAcad Sci U S A 1998;95:288-293.

37. Tamura T. Microbiological assay of folates. In: Picciano M, Stokstad ELR, Gregory JF III,ed. Folic Acid Metabolism in Health and Disease. NewYork, NY: Wiley-Liss,

1990:121-138.

38. Melnyk S, Pogribna M, Pogribny IP, Yi P, James SJ. Measurement of plasma and intracellular S-adenosylmethionine and S-adenosylhomocysteine utilizing coulomet-ricelectrochemical detection: alterations withplasma homocysteine and pyridoxal 5'-phosphate concentrations. Clin Chem 2000;46:265-272.

39. Pomfret EA, daCosta KA, Schurman LL, Zeisel SH. Measurement of choline and choline metabolite concentrations usinghigh-pressure liquid chromatography and gaschromatography-mass spectrometry. Anal Biochem 1989;180:85-90.

40. KondoH, Osborne ML, Kolhouse JF, et al. Nitrous oxide has multipledeleterious effectsoncobalamin metabolism andcausesdecreases in activities ofboth mamma-liancobalamin-dependent enzymes in rats. J Clin Invest 1981;67:1270-1283. 41. Garrow TA. Purification, kinetic properties, and cDNA cloningof mammalian

be-taine-homocysteine methyltransferase. J Biol Chem 1996;271:22831-22838. 42. BarakAJ,Beckenhauer HC, Tuma DJ, Badakhsh S. Effects of prolonged ethanol

feedingonmethionine metabolism inratliver. Biochem Cell Biol 1987;65:230-233. 43. Finkelstein JD, Cello JP, Kyle WE. Ethanol-induced changes in methionine

metab-olism inratliver.BiochemBiophysResCommun 1974;61:525-531.

44. TrimbleKC, Molloy AM, Scott JM, Weir DG. The effect of ethanolonone-carbon metabolism:increased methionine catabolism and lipotrope methyl-group wastage. Hepatology 1993;18:984-989.

45. Avila MA, Berasain C, Torres L, et al. Reduced mRNA abundance of the main enzymesinvolved in methionine metabolism in human liver cirrhosis and

hepato-cellular carcinoma. [InProcessCitation]. J Hepatol 2000;33:907-914.

46. LuSC, Huang ZZ, Yang H, Mato JM, Avila MA, Tsukamoto H.Changes in methi-onine adenosyltransferaseand S-adenosylmethionine homeostasisinalcoholic rat liver.Am JPhysiol Gastrointest Liver Physiol 2000;279:G178-G185.

47. Taoka S, Widjaja L,

Banerjee

R. Assignment of enzymatic functions to specific regionsof thePLP-dependent heme protein cystathioninebeta-synthase. Biochem-istry 1999;38:13155-13161.

48. James SJ,MillerBJ, Basnakian AG, PogribnyIP, Pogribna M,Muskhelishvili L. Apoptosis andproliferationunderconditions ofdeoxynucleotide pool imbalancein liveroffolate/methyl deficientrats.Carcinogenesis 1997;18:287-293.

S-adenosyl-methioninedeficiency and TNF-alpha inlipopolysaccharide-induced hepatic injury. Am JPhysiol1998;275:G125-G129.

50. LieberCS, Robins SJ, Li J, et al. Phosphatidylcholine protects againstfibrosis and cirrhosis in the baboon [see comments]. Gastroenterology1994;106:152-159. 51. Yen CL, Mar MH, Zeisel SH. Cholinedeficiency-induced apoptosisinPC12 cellsis

associated with diminished membranephosphatidylcholine andsphingomyelin, ac-cumulation of ceramide and diacylglycerol, and activation of a caspase. Faseb J 1999;13:135-142.

DISCUSSION

Oates, Nashville:Iwonder if you couldcomment onthe cardiactoxicity of ethanol and how it's altered with these interventions?

Halsted, Davis: We did not do cardiac studies on our micropigs. Ingeneral, thereare twodichotomous views about alcohol and the heart. Oneisthat alcoholic consumption promoteselevated circulating HDLcholesterol levels and therefore alcoholicsare pro-tected against coronary artery disease. On the other hand, the entity of alcoholic cardiomyopathy, considered a direct toxic effect of alcohol, is probably much more commonthanwethink, occursinheavydrinkers, and resultsincongestiveheart failure. Thiamindeficiency, also commoninheavily drinking alcoholics is a nutritionalcauseof high output cardiac failure, or "wet beri-beri."

Fallon, Charleston: It has been some time sinceIdelved intothis field, and I'm glad you are continuingindoing so well.Ihad a couple of questions.IthinkIheard youthat the folate deficient animals alone hadnoabnormalitiesinliverhistology. Is that correct?

Halsted: That's correct.

Fallon: And that the development of the alcoholic lesion also required the addition of folate deficiency.

Halsted: That is correct in this model of early changes after 14 weeks'feeding. Fallon:Well the problem that has always been in this field, and I know that you are well awareof this is that not allhumans who develop alcoholic liver diseasearefolate deficient. Itdependsalittlebitonsocio-economic class andwhether therearebankers who tippleorwhether they are down and out and malnourished. SoIwonderhow you put this together back into the framework of the original problem which we've all struggled with is; why do20%ofalcoholic humansdevelop alcoholic liver disease?

Halsted:The question why some people who drink a lot get liver disease and some do not isstillunanswered. As you know, an old study of well-nourished German executives foundadirect correlation between the amount of ethanol consumed and the likelihood of developing cirrhosis accordingtotheir liverbiopsies. Ican't tell you whethersomeof them had folate deficiency, but suspect that our model is more reflective of derelict malnourished alcoholics who are typically folate-deficient. At the same time, our mi-cropig model suggests thatpeople whoarefolate-sufficient,i.e.,well-nourished, maybe atlessrisk, ormaytakelongertodevelop alcoholic liver disease than folate-deficient derelictalcoholics.

Billings, Baton Rouge: You may have a slide that you are about to show us,which presents the data forthe group thatweretreated withfolate to a more than sufficient level.We here attheHomestead, are, of course, goingtohavemorethanenough folate and, maybemore thanenough alcohol.Whenwe return home should wegoonadditional folatetoreduceourpotential alcohol inducedhepatoxicity?

Halsted: I knew someone wasgoingto askthat. First of all we are allgetting an adequateamountof folateinourdietsthanks tothe US national food folatefortification

program. Whether that's going to make a difference in the incidence of alcoholic liver disease inthis country is an interesting question. It is noteworthy that many physicians don't determine whether their patients drink too much and are at risk for liver disease. Sincealcohol consumption is conducive of folate deficiency for a variety of reasons, I would recommend folic acidsupplementation for any man consuming more than 2-3 drinksdaily, and since women are more susceptible to alcoholic liver

disease,

Iwould recommend folic acidsupplementation for any woman consuming more than 1 drink daily. There areretrospective epidemiologicalstudiessuggesting that 3-5 drinks per day may be thecutoffpoint before an individual is at risk for alcoholic liver disease. I doubt thatmany of us at thismeeting have reached that point.