Deposition

of

fluoride

on

enamel

surfaces

released

from

varnishes

is

limited

to

vicinity

of

fluoridation

site

The

aim

of

the

in-situ

study

was

to

determine

fluoride

uptake

in

non-fluoridated

demineralized

enamel

after

application

of

fluoride

varnishes

on

enamel

samples

located

at

various

distances

from

the

non-fluoridated

samples

All

enamel

samples

used

were

demineralized

with

acidic

hydroxyethylcellulose

before

the

experiment

Intra-oral

appliances

were

worn

by

ten

volunteers

in

three

series

-LRB-

Mirafluorid

F

Duraphat

F

and

unfluoridated

controls

-RRB-

of

days

each

Each

two

enamel

samples

were

prepared

from

bovine

incisors

One

sample

was

used

for

the

determination

of

baseline

fluoride

content

-LRB-

BFC

-RRB-

the

other

was

treated

according

to

the

respective

series

and

fixed

in

the

intra-oral

appliance

for

days

Additionally

from

incisors

each

four

enamel

samples

were

prepared

-LRB-

one

for

BFC

-RRB-

Three

samples

-LRB-

a

c

-RRB-

were

placed

into

each

appliance

at

different

sites

-LRB-

a

-RRB-

directly

neighboured

to

the

fluoridated

specimen

-LRB-

next

-RRB-

-LRB-

b

-RRB-

at

distance

-LRB-

cm

-RRB-

and

-LRB-

c

-RRB-

in

the

opposite

buccal

aspect

of

the

appliance

-LRB-

opposite

-RRB-

At

these

sites

new

unfluoridated

samples

were

placed

at

days

and

which

were

left

in

place

for

day

The

volunteers

brushed

their

teeth

and

the

samples

with

fluoridated

toothpaste

twice

per

day

Both

the

KOH-soluble

and

structurally

bound

fluoride

were

determined

in

all

samples

to

determine

fluoride

uptake

and

were

statistically

analyzed

One

day

after

fluoridation

with

Duraphat

KOH-soluble

fluoride

uptake

in

specimen

a

-LRB-

next

-RRB-

was

significantly

higher

compared

to

the

corresponding

samples

of

both

the

control

and

Mirafluorid

series

which

in

turn

were

not

significantly

different

from

each

other

At

all

other

sites

and

time

points

fluoride

uptake

in

the

enamel

samples

were

not

different

from

controls

for

both

fluoride

varnishes

Within

the

first

day

after

application

intra-oral-fluoride

release

from

the

tested

fluoride

varnish

Duraphat

leads

to

KOH-soluble

fluoride

uptake

only

in

enamel

samples

located

in

close

vicinity

to

the

fluoridation

site

Introduction

It

is

well

established

that

fluoride

products

play

an

important

role

in

the

prevention

and

remineralization

of

carious

lesions

Topical

fluoride

applications

in

the

form

of

varnishes

and

varnishes

may

lead

to

appreciable

acquisition

of

fluoride

on

and

in

both

enamel

and

dentin

samples

which

were

directly

treated

with

the

agent

-LSB-

-RSB-

Especially

the

KOH-soluble

fluoride

deposit

on

the

surfaces

of

dental

hard

tissue

might

be

elevated

depending

on

the

fluoride

concentration

and

composition

of

the

fluoride

varnish

applied

However

the

KOH-soluble

fluoride

deposit

is

continuously

dissolved

in

the

oral

cavity

due

to

the

influence

of

saliva

This

leads

to

a

drastic

reduction

of

the

deposit

within

a

period

of

about

week

after

application

of

the

fluoride

agent

-LSB-

-RSB-

After

the

use

of

fluoride-containing

products

also

salivary

fluoride

levels

are

increased

for

a

certain

period

of

time

depending

on

the

concentration

and

application

form

used

This

effect

is

important

because

fluoride

levels

above

ppm

in

the

surrounding

solution

of

the

dental

hard

tissue

have

been

shown

to

be

related

to

lower

risk

of

caries

progression

in

clinical

studies

-LSB-

-RSB-

The

fluoride

distribution

in

the

oral

cavity

showed

site-specific

variations

which

was

exemplarily

demonstrated

by

Weatherell

at

al.

-LSB-

-RSB-

after

dissolving

a

fluoride

tablet

in

the

oral

vestibulum

With

oral

fluoride-containing

mouthrinses

salivary

fluoride

levels

remain

elevated

for

up

to

h

after

application

-LSB-

-RSB-

The

use

of

fluoride

mouthwashes

twice

daily

leads

to

a

sustained

elevated

fluoride

level

even

between

the

daily

applications

-LSB-

-RSB-

Fluoride

dentifrices

can

cause

increase

in

salivary

fluoride

content

for

a

period

of

about

min

-LSB-

-RSB-

Also

after

application

of

fluoride

varnishes

onto

tooth

surfaces

salivary

fluoride

levels

remain

increased

for

up

to

h

-LSB-

-RSB-

The

above-mentioned

studies

had

made

clear

that

fluoride-treated

tooth

surfaces

and

salivary

fluoride

levels

are

increased

after

administering

local

fluoride

regimes

Besides

these

effects

it

may

be

speculated

that

fluoride

applications

also

lead

to

fluoride

uptake

at

tooth

surfaces

that

were

not

directly

treated

or

covered

with

fluoridation

agents

such

as

varnishes

This

fact

could

especially

be

important

for

demineralized

surfaces

which

were

inaccessible

to

direct

fluoride

application

such

as

inter-proximal

areas

Due

to

toxicological

and

practical

reasons

application

of

fluoride

varnishes

is

mostly

limited

to

some

sites

of

the

dentition

Therefore

the

aim

of

the

present

study

was

to

determine

fluoride

transfer

from

fluoride-varnished

enamel

surfaces

to

demineralized

enamel

surfaces

located

at

various

distances

from

the

fluoridation

site

Materials

and

methods

Enamel

samples

Enamel

cylinders

-LRB-

mm

in

diameter

-RRB-

were

prepared

from

the

buccal

surface

of

bovine

incisors

The

cylinders

were

ground

flat

and

polished

thereby

removing

about

depth

of

the

enamel

as

controlled

with

a

micrometer

Afterwards

all

specimens

were

sterilized

with

gamma

irradiation

-LRB-

kGy

-RRB-

For

rehydration

the

sterilized

samples

were

stored

in

synthetic

saliva

-LSB-

-RSB-

for

a

minimum

of

weeks

Then

all

samples

were

demineralized

with

acidic

hydroxyethylcellulose

-LRB-

pH

days

-RRB-

According

to

this

procedure

each

two

enamel

samples

were

prepared

from

bovine

incisors

and

each

four

samples

were

gained

from

additional

incisors

One

sample

from

each

tooth

was

used

for

the

determination

of

baseline

fluoride

content

of

the

respective

tooth

The

remaining

samples

of

the

bovine

incisors

were

later

treated

according

to

series

The

remaining

samples

of

the

bovine

incisors

were

used

for

the

determination

of

intra-oral

fluoride

transfer

and

acquisition

Set-up

of

the

study

The

study

was

designed

and

conducted

according

to

the

guidelines

of

Good

Clinical

Practice

Ethical

approval

of

the

study

was

granted

by

the

Ethics

Committee

of

the

University

of

Göttingen

-LRB-

-RRB-

Ten

panellists

-LRB-

aged

to

years

-RRB-

participated

in

the

study

They

were

all

residents

of

Göttingen

-LRB-

fluoride

content

in

domestic

water

ppm

F

-RRB-

The

subjects

had

been

instructed

to

avoid

fluoride

uptake

The

participants

had

a

stimulated

salivary

fluoride

rate

within

the

normal

range

of

ml/min

Further

inclusion

criteria

were

that

they

had

at

least

teeth

no

faulty

dental

restorations

no

glass

ionomer

fillings

and

no

current

dental

caries

activity

Intra-oral

lower

jaw

appliances

with

two

buccal

aspects

were

worn

by

ten

volunteers

in

three

series

of

days

each

In

each

series

one

enamel

sample

was

placed

for

days

in

one

buccal

aspect

of

the

appliance

after

treatment

according

to

the

respective

series

-LRB-

central

fluoridated

sample

-RRB-

With

each

five

of

the

participants

the

fluoridated

samples

were

placed

in

the

right

buccal

aspect

with

the

remaining

subjects

the

fluoridated

samples

were

fixed

in

the

left

aspect

of

the

appliance

A

washout

phase

of

week

elapsed

between

the

three

series

In

series

and

g

of

the

fluoride

varnish

Mirafluorid

-LRB-

-RRB-

or

Duraphat

-LRB-

-RRB-

was

either

applied

on

the

central

enamel

sample

-LRB-

Table

-RRB-

Care

was

taken

to

avoid

contamination

of

the

remaining

enamel

samples

of

the

intra-oral

appliances

with

the

varnish

applied

After

application

of

the

varnish

the

intra-oral

appliances

were

immediately

inserted

into

the

oral

cavities

In

series

the

respective

samples

were

not

fluoridated

with

varnish

and

served

as

controls

Table

and

composition

of

the

fluoride

varnishes

Mirafluorid

and

Duraphat

according

to

manufacturers

Mirafluorid

Duraphat

Fluoride

Fluoride

formSodium

fluorideSodium

fluorideCetylaminehydrofluorideBis

-LRB-

hydroxyethyl

-RRB-

aminpropyl-N-hydroxyethyl-octadecyl-amindihydrofluoridepH4.6

NeutralSolventSolvent-freealcohol

-LRB-

-RRB-

BaseWater-soluble

polymerNatural

resins

-LRB-

colophonium

mastix

shellac

-RRB-

Other

ingredients

Wax

saccharine

flavourManufacturerHager

Werken

Duisburg

Germany

Colgate-Palmolive

Hamburg

Germany

Each

three

unfluoridated

samples

were

placed

into

each

appliance

at

different

sites

-LRB-

a

c

-RRB-

-LRB-

a

-RRB-

directly

neighboured

to

the

central

fluoridated

specimen

-LRB-

next

-RRB-

-LRB-

b

-RRB-

at

distance

-LRB-

cm

-RRB-

and

-LRB-

c

-RRB-

in

the

opposite

buccal

aspect

of

the

appliance

-LRB-

opposite

-RRB-

These

three

samples

were

replaced

after

and

days

by

new

unfluoridated

samples

which

were

removed

after

day

intra-oral

exposition

Thus

each

one

sample

was

worn

at

the

respective

site

for

the

following

periods

days

days

days

and

days

The

central

samples

which

were

fluoridated

on

day

were

not

removed

from

the

appliances

before

completion

of

the

experiment

The

volunteers

brushed

their

teeth

and

the

samples

with

a

pea-size

amount

of

fluoridated

toothpaste

-LRB-

fluoride

as

amine

fluoride

-RRB-

twice

per

day

-LRB-

elmex

GABA

Lörrach

Germany

-RRB-

during

the

experiment

to

simulate

the

common

practice

of

using

a

fluoridated

dentifrice

and

to

follow

the

recommendations

of

the

Ethical

Committee

which

did

not

allow

detaining

a

fluoride

dentifrice

to

the

participants

They

began

to

use

the

dentifrice

days

before

the

start

of

the

trial

After

toothbrushing

the

enamel

samples

in

the

appliances

were

carefully

cleaned

with

ten

brushing

strokes

using

the

toothbrush

-LRB-

impregnated

with

the

remaining

dentifrice

slurry

-RRB-

that

had

been

used

for

brushing

of

the

teeth

before

A

minimum

of

h

elapsed

between

application

of

the

respective

varnish

onto

the

central

specimen

and

first

brushing

of

the

samples

During

the

experiment

the

appliances

were

worn

night

and

day

except

during

meals

during

which

they

were

stored

in

saline

Fluoride

analysis

Immediately

after

removal

from

the

intra-oral

appliances

the

enamel

samples

were

cleaned

with

a

soft

toothbrush

under

running

tap

water

and

analyzed

for

KOH-soluble

and

structurally

bound

fluoride

The

fluoride

was

assayed

by

a

specific

fluoride

electrode

-LRB-

Orion

Research

Cambridge

USA

-RRB-

The

structurally

bound

fluoride

was

determined

in

two

successive

layers

of

μm

each

The

layers

were

removed

by

means

of

a

special

grinding

technique

described

in

detail

previously

-LSB-

-RSB-

The

amount

of

KOH-soluble

fluoride

was

determined

by

the

method

of

Caslavska

et

al.

-LSB-

-RSB-

The

assays

were

prepared

as

described

in

those

studies

resulting

in

a

sample

solution

for

KOH-soluble

fluoride

and

solution

for

structurally

bound

fluoride

determination

respectively

Also

fluoride

content

of

the

fluoridated

samples

was

assessed

after

the

experimental

period

Statistics

According

to

the

manufacturer

sensitivity

of

the

fluoride

electrode

is

given

as

μmol/l

in

a

solution

Bioanalytical

guidelines

recommend

disregarding

the

measurements

that

are

close

to

the

sensitivity

of

an

assessment

method

-LSB-

-RSB-

An

international

cooperation

study

-LSB-

-RSB-

determining

the

lowest

level

of

sensitivity

for

measurements

with

a

fluoride

electrode

showed

that

measurements

below

μmol/l

-LRB-

equivalent

to

KOH-soluble

fluoride

and

structurally

bound

fluoride

-RRB-

were

unstable

not

reproducible

and

showed

high

standard

deviations

and

coefficients

of

variation

-LRB-

-RRB-

Therefore

these

readings

were

regarded

as

to

be

below

detection

limit

Thus

fluoride

uptake

was

not

calculated

by

subtracting

the

baseline

fluoride

content

of

the

respective

tooth

which

was

below

detection

limit

from

the

measured

value

recorded

for

the

specimen

All

groups

were

compared

regarding

their

number

of

readings

above

detection

limit

using

chi-square

tests

For

those

groups

in

which

more

than

of

the

readings

were

above

the

detection

limit

discriminations

were

conducted

using

the

rank-sum

test

according

to

Wilcoxon

and

Mann

Whitney

The

level

of

significance

was

set

at

P

Results

In

Table

the

percentages

of

fluoride

measurements

in

the

respective

groups

which

were

above

the

detection

limits

are

given

It

is

obvious

that

most

of

the

groups

had

more

than

of

their

readings

below

the

detection

limit

Chi-square

tests

proved

that

the

sample

groups

with

more

than

readings

above

detection

limit

were

statistically

significantly

different

from

the

remaining

groups

which

in

turn

did

not

differ

significantly

from

each

other

Table

of

measurements

above

detection

limitsa

for

KOH-soluble

and

structurally

bound

fluoride

in

two

successive

layers

in

enamel

specimens

worn

in

intra-oral

appliances

at

different

sites

and

for

different

periods

-LRB-

days

days

days

days

or

days

-RRB-

after

the

application

of

either

Duraphat

Mirafluorid

or

no

fluoride

varnish

-LRB-

control

-RRB-

on

the

central

specimen

on

day

KOH-soluble

F

Layer

-LRB-

μm

-RRB-

Layer

-LRB-

μm

-RRB-

PeriodSiteMirafluorid

-LRB-

-RRB-

Duraphat

-LRB-

-RRB-

Control

-LRB-

-RRB-

Mirafluorid

-LRB-

-RRB-

Duraphat

-LRB-

-RRB-

Control

-LRB-

-RRB-

Mirafluorid

-LRB-

-RRB-

Duraphat

-LRB-

-RRB-

Control

-LRB-

-RRB-

limits

-LRB-

KOH-soluble

F

structurally

bound

fluoride

-RRB-

In

Table

KOH-soluble

fluoride

and

structurally

bound

fluoride

data

of

the

experimental

groups

are

given

for

which

more

than

of

the

readings

were

above

detection

limit

As

mentioned

above

chi-square

tests

had

proven

that

these

values

were

statistically

significantly

different

from

baseline

content

and

from

the

remaining

groups

For

the

remaining

groups

fluoride

contents

are

not

given

because

statistical

data

such

as

mean

value

or

median

are

not

reasonable

to

calculate

for

values

which

are

not

reliably

measurable

by

definition

Acquisition

of

KOH-soluble

fluoride

was

detectable

in

the

central

fluoridated

samples

of

both

groups

Mirafluorid

and

Duraphat

with

the

latter

once

accumulating

statistically

significantly

more

KOH-soluble

fluoride

-LRB-

P

-RRB-

The

central

fluoridated

Duraphat

samples

also

showed

significant

uptake

of

structurally

bound

fluoride

in

both

enamel

layers

with

significantly

higher

uptake

in

the

first

layer

compared

to

the

second

one

-LRB-

P

-RRB-

One

day

after

fluoridation

the

samples

located

next

to

the

Duraphat-varnished

central

specimens

also

accumulated

measurable

amounts

of

KOH-soluble

fluoride

The

KOH-soluble

precipitate

on

these

samples

was

statistically

significantly

less

pronounced

compared

to

the

central

specimens

-LRB-

P

-RRB-

The

samples

located

next

to

the

Duraphat-treated

showed

measurable

KOH-soluble

fluoride

acquisition

at

days

They

therefore

differed

significantly

from

both

the

control

and

Mirafluorid

series

which

did

not

show

measurable

fluoride

acquisition

at

this

time

point

and

were

in

turn

not

significantly

different

from

each

other

At

all

other

sites

and

time

points

fluoride

uptake

in

the

enamel

samples

was

not

different

from

controls

for

both

fluoride

varnishes

Table

standard

deviation

-LRB-

median

minimum

maximum

-RRB-

of

KOH-soluble

fluoride

and

structurally

bound

fluoride

-LRB-

in

and

layer

-RRB-

given

for

the

experimental

groups

with

more

than

of

the

respective

measurements

above

detection

limit

KOH-soluble

F

-LRB-

-RRB-

Layer

-LRB-

-RRB-

Layer

-LRB-

-RRB-

a

-LRB-

-RRB-

a

c

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

c

-LRB-

-RRB-

Less

than

readings

above

detection

limitSignificantly

different

values

in

the

same

line

-LRB-

aKOH-soluble

F

bstructurally

bound

F

-RRB-

or

ccolumn

are

marked

Discussion

As

stated

in

Introduction

several

investigations

had

demonstrated

that

intra-orally

applied

fluoride

regimes

may

cause

fluoride

release

into

saliva

In

a

previous

study

Dijkman

et

al.

-LSB-

-RSB-

had

demonstrated

that

no

measurable

fluoride

enrichment

could

be

detected

in

untreated

sound

enamel

controls

located

in

a

distance

of

mm

from

fluoride

varnish-treated

samples

after

a

period

of

week

The

present

study

made

clear

that

within

the

first

day

after

application

the

highly

concentrated

fluoride

varnish

Duraphat

KOH-soluble

fluoride

deposition

in

close

vicinity

to

the

fluoridation

occurs

However

in

consistence

with

the

investigation

by

Dijkman

et

al.

-LSB-

-RSB-

no

measurable

fluoride

transfer

could

be

revealed

after

the

first

day

Although

when

interpreting

the

results

of

the

present

study

it

should

be

noted

that

a

more

sensitive

method

for

fluoride

determination

might

have

shown

slightly

different

results

In

the

present

study

no

fluoride

acquisition

could

be

detected

in

samples

located

at

a

distance

of

cm

from

the

fluoridated

site

or

fixed

in

the

opposite

vestibulum

of

the

participants

This

means

that

fluoride

transfer

via

saliva

was

strictly

limited

to

the

close

neighbourhood

of

the

fluoridation

site

This

finding

corresponds

to

a

previous

study

which

demonstrated

that

significant

elevation

of

salivary

fluoride

concentrations

is

more

or

less

restricted

to

the

fluoridation

site

-LSB-

-RSB-

With

exception

for

the

samples

directly

treated

with

Duraphat

no

measurable

acquisition

of

structurally

bound

fluoride

was

observed

in

any

other

sample

group

In

a

previous

study

application

of

Mirafluorid

did

not

also

result

in

uptake

of

structurally

bound

fluoride

-LSB-

-RSB-

However

also

the

samples

which

were

located

next

to

the

specimens

treated

with

Duraphat

for

day

and

which

showed

a

KOH-soluble

fluoride

deposit

did

not

show

increase

of

structurally

bound

fluoride

This

finding

could

be

explained

by

the

fact

that

formation

of

structurally

bound

fluoride

is

a

time-dependent

process

during

which

some

of

the

KOH-soluble

deposit

is

dissolved

allowing

fluoride

to

diffuse

into

the

underlying

enamel

and

to

be

structurally

incorporated

into

the

enamel

crystallites

-LSB-

-RSB-

However

in

these

samples

structurally

bound

fluoride

was

assessed

already

day

after

insertion

into

the

appliance

It

might

be

speculated

that

with

longer

exposition

in

the

oral

cavity

structurally

bound

fluoride

might

have

been

formed

by

dissolution

from

the

KOH-soluble

fluoride

deposit

In

the

present

study

demineralized

enamel

specimens

were

used

for

assessing

fluoride

acquisition

Recent

studies

had

proven

that

fluoridation

measures

lead

to

significantly

higher

uptake

of

fluoride

in

demineralized

samples

as

compared

to

sound

enamel

-LSB-

-RSB-

It

could

therefore

be

assumed

that

fluoride

acquisition

of

sound

enamel

surfaces

due

to

fluoride

transfer

in

the

oral

cavity

might

be

actually

more

limited

as

compared

to

the

present

results

Under

clinical

conditions

a

varnish

is

often

applied

onto

more

sites

which

might

elevate

salivary

fluoride

to

a

presumably

higher

extent

as

in

the

present

study

where

the

varnishes

were

applied

to

a

single

site

only

Due

to

the

results

of

the

present

study

it

could

be

concluded

that

within

the

first

day

after

application

intra-oral

fluoride

release

from

the

tested

fluoride

varnish

Duraphat

leads

to

KOH-soluble

fluoride

uptake

only

in

demineralized

enamel

samples

which

are

located

in

close

vicinity

to

the

fluoridation

site

Owing

to

the

negligible

transfer

of

fluoride

from

the

varnish

to

more

remote

sites

this

finding

means

for

the

clinical

situation

that

all

sites

in

the

oral

cavity

requiring

fluoridation

have

to

be

directly

treated

with

the

varnish

O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O B O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O B O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B B O O B O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O B O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O B O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O B O O O B O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O B O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Embolization with the Amplatzer Vascular Plug in TIPS Patients Vessel embolization can be a valuable adjunct procedure in transjugular intrahepatic portosystemic shunt -LRB- TIPS -RRB- . During the creation of a TIPS , embolization of portal vein collaterals supplying esophageal varices may lower the risk of secondary rebleeding . And after creation of a TIPS , closure of the TIPS itself may be indicated if the resulting hepatic encephalopathy severely impairs mental functioning . The Amplatzer Vascular Plug -LRB- AVP ; AGA Medical , Golden Valley , MN -RRB- is well suited for embolization of large-diameter vessels and has been employed in a variety of vascular lesions including congenital arteriovenous shunts . Here we describe the use of the AVP in the context of TIPS to embolize portal vein collaterals -LRB- n = 8 -RRB- or to occlude the TIPS -LRB- n = 2 -RRB- . Portal vein collaterals supplying esophageal varices may give high risk for secondary rebleeding . Those collaterals might require embolization to decrease this risk , despite transjugular intrahepatic portosystemic shunt -LRB- TIPS -RRB- placement . Embolization of the collaterals may be particularly difficult when these vessels are large . Moreover , after creation of a TIPS , closure of the TIPS itself may be indicated when hepatic encephalopathy severely impairs mental functioning . The Amplatzer Vascular Plug -LRB- AVP ; AGA Medical , Golden Valley , MN -RRB- is a device well suited to embolize large vessels and has been employed in a variety of vascular lesions such as pulmonary arteriovenous malformations and congenital shunts -LSB- 1 , 2 -RSB- . This report describes the use of the AVP in the context of TIPS to embolize portal vein collaterals -LRB- n = 8 -RRB- or to occlude the TIPS -LRB- n = 2 -RRB- . Description of Procedures Embolization of Portal Vein Collaterals Eight patients were referred for TIPS , seven because of multiple recurrent bleeding episodes from esophageal varices -LRB- n = 5 -RRB- , gastric fundus varices -LRB- n = 1 -RRB- , or both -LRB- n = 1 -RRB- that could not be managed by endoscopic therapy . One other patient -LRB- n = 1 -RRB- was in hypovolemic shock because of recurrent massive bleeding from rectal hemorrhoids . Three TIPS procedures were elective and five were done as emergency procedures for uncontrolled bleeding after initial hemodynamic stabilization had been achieved using blood transfusions , endoscopic treatment , intravenous octreotide infusion , and/or placement of a Sengstaken double balloon tube , alone or in combination . TIPS procedures were carried out under general anesthesia . As an adjunct procedure , we obliterated the portal vein collaterals supplying the bleeding varices using AVP embolization -LRB- Figs. 1 and 2 -RRB- . This was done either before or after creation of the TIPS based on operator preference and clinical circumstances . Embolization early in the procedure with early control of the potential bleeding site was considered relevant in emergency procedures in hemodynamically unstable patients . Also , at least theoretically , AVP embolization may be done more easily prior to TIPS whenever the collateral target vein is still widened because of portal hypertension and easily opacified because of preferential flow . After the creation of a TIPS both these phenomena may have been altered . Fig. 1Portal venous angiogram in a 53-year-old patient referred for TIPS creation because of recurrent variceal bleeding not controlled by medication plus multiple treatments with endoscopic band ligation and sclerotherapy . The varices are supplied by a single coronary veinFig . 2The same patient as in Fig. 1 immediately following TIPS creation and AVP embolization of the coronary vein -LRB- a ; thin arrow -RRB- . The unsubtracted image -LRB- b -RRB- shows the radiopaque sclerosing agent from prior endoscopic sclerotherapy -LRB- fat arrow -RRB- in the esophageal varices Whenever AVP embolization preceded TIPS placement , the guiding catheter for AVP delivery was introduced transhepatically into the right portal vein . At our institution the portal vein is routinely catheterized prior to TIPS with a 3 - or 4-Fr catheter to visualize the portal vein as a fluoroscopic target for transjugular transhepatic puncture . For transhepatic AVP placement this catheter was exchanged for a 6 - to 8-Fr hepatic introducer sheath . After AVP embolization and initial creation of the hepatic-to-portal vein shunt tract for TIPS , the hepatic introducer sheath was withdrawn in multiple small successive steps over the course of the remainder of the procedure , so that a solid sealing clot could form in the hepatic parenchymal tract . No additional embolization of the tract was performed . Whenever vein embolization was performed after the creation of a TIPS , AVP placement was done via the transjugular route . In all patients , the large-diameter coronary/gastric vein supplying extensive esophageal/gastric varices was embolized using a single AVP plug 10 , 12 , or 14 mm in diameter . For placement , a 6 - to 8-Fr guiding catheter was first negotiated into the proximal part of the coronary vein . Then the AVP device was advanced through the guiding catheter and initially deployed by retracting the guiding catheter but without detaching the device . A control injection was made , and if needed , the AVP was retrieved into the guiding catheter and repositioned . When in a satisfactory position , the AVP was detached by anticlockwise rotation of the AVP guide wire to unscrew it from the AVP device proper . Within minutes of deployment , the collateral flow through the coronary vein had completely stopped . TIPS creation was done by placement of e-PTFE-covered stent-grafts -LRB- Viatorr ; Gore , Flagstaff , AZ -RRB- 8 -- 10 mm in diameter and 6 ± 2 cm long , resulting in a postprocedural portosystemic pressure difference of 5 -- 10 mm Hg . All patients were hemodynamically stable immediately after the procedure . During follow-up there were no recorded episodes of rebleeding with a follow-up of 1 year maximum . The patient who was treated for bleeding rectal hemorrhoids died after 3 days because of combined liver and respiratory failure . Another patient who received emergency TIPS while in hypovolemic shock due to massive unstoppable bleeding from esophageal varices died 46 days after the procedure because of liver and kidney failure complicated by ileus . TIPS Occlusion A 47-year-old male was referred for closure of a TIPS that had been created 3 years earlier for alcoholic Child A liver cirrhosis with recurrent episodes of esophagus variceal bleeding . He now presented with problems of amnesia , difficulties concentrating , and a general , overwhelming feeling of tiredness . Spectral EEG findings were consistent with grade 2 hepatic encephalopathy . The symptoms had not improved using conservative treatment . At angiography , a patent TIPS was seen , with a portosystemic pressure difference of 10 mm Hg . Closure of the TIPS with a 14-mm AVP device -LRB- Fig. 3 -RRB- resulted in a dramatic improvement in cognitive functioning and normalization of the spectral EEG . Two years after closure of the TIPS , the patient is still free of variceal bleeding . Recently , however , he developed esophageal varices grades I -- II , which are currently treated with band ligation . This will be repeated every 2 weeks until complete obliteration . Fig. 3AVP device for TIPS occlusion in a patient with life-incapacitating hepatic encephalopathy . The 14-mm-diameter AVP is positioned through a guiding catheter in the central portion of the 10-mm-diameter e-PTFE stent-graft -LRB- a -RRB- . By retraction of the guide wire , the unconstrained AVP sets in the intended place -LRB- b -RRB- . At this point , the AVP can still be retracted into the guiding catheter if in an unsatisfactory position . The AVP is fully deployed by unscrewing the guide wire from the AVP device proper -LRB- c -RRB- Another patient , a 65-year-old male , had been referred for TIPS placement for refractory ascites following liver transplantation , which required weekly paracentesis . In the days following placement of a 10-mm-diameter e-PTFE-covered stent-graft TIPS , liver failure developed , necessitating its closure . Closure of the TIPS was technically successfully achieved using a 16-mm-diameter AVP but did not substantially improve the clinical status . Because of liver failure and refractory ascites , the patient underwent a second liver transplantation 2 days after TIPS occlusion . Discussion In this report we have described the use of AVP embolization in the context of TIPS . For closure of collateral veins supplying bleeding varices , we consider the AVP a useful device in a defined subset of patients . The AVP 's relative advantages are its efficiency to occlude large-diameter vessels in a single-step procedure and its reliability of placement . Unlike standard embolization coils , the AVP can be positioned precisely , its position can be checked with contrast injections before actual detachment , and it can be repositioned if required . Migration of the AVP during or after deployment is unlikely . Disadvantages of the AVP include the higher cost of the device itself and of the additionally required guiding catheter . Furthermore , the 6 - to 8-Fr guiding catheter markedly increases the bulk of the introducer system relative to the 4 - to 5-Fr catheter alternatively used for coil embolization . In our practice we are inclined to use the AVP whenever there is a single , large-diameter collateral vein which would otherwise require the insertions of multiple embolization coils and/or whenever accurate placement of the embolic device close to the portal vein may be critical . Embolization of portal vein collaterals to esophageal varices may be indicated even after successful portal venous decompression by creation of a TIPS . Especially in the life-threatening emergency situation of acute esophageal bleeding in hemodynamically unstable patients , we feel more confident providing additional control of the bleeding site by hemodynamically separating the weak-walled varices from the high-flow and potentially high-pressure portal venous bed . TIPS has proven efficient in the secondary prevention of variceal bleeding but this protection is not absolute . The rebleeding rate of TIPS alone -LRB- without embolization -RRB- has been reported as between 9 % and 40.6 % , with a median of 18.4 % -LSB- 3 -RSB- . These values apply to bare-stented TIPS and initial experience suggests that the rebleeding rate is lower with e-PTFE-covered stented TIPS -LSB- 4 -RSB- . Whether or not performing embolization of the portal vein collaterals as an adjunct procedure provides additional protection against rebleeding is unknown . Under the assumption that it does , we routinely combine TIPS with embolization of all venous collaterals supplying the esophageal varices when creating a TIPS in the context of variceal bleeding . Another potential use for the AVP device is for closure of the TIPS itself , in circumstances where hepatic encephalopathy and/or liver failure because of the shunt may prove to be life-incapacitating . Here , the effectiveness of the AVP in occluding large-diameter high-flow vessels , its resistance to migration , and its ease of placement are particularly helpful . A potential concern when occluding a TIPS is the acute increase in portal venous pressure , which can lead to recurrence of variceal bleeding and ascites and , also , to hemodynamic changes with increased cardiac loading -LSB- 5 -RSB- . As mentioned , we routinely place a transhepatic catheter into the portal vein as a target for puncturing the portal vein during TIPS creation . In our experience , this increases the control over the exact entrance point into the portal vein and also reduces the number of puncture attempts while creating the TIPS tract . We have found that the reduced procedure time for the TIPS procedure proper more than makes up for the extra time it takes to insert this initial transhepatic catheter into the portal vein . We have encountered no complications because of this approach in more than 100 TIPS procedures . To our knowledge , this is the second reported use of the AVP in the portal venous system . In a recent case report , Kessler and Trerotola described a patient with gastric varices and TIPS in whom they used the AVP device for transhepatic obliteration of the portal vein collateral supplying the varices -LSB- 6 -RSB- , a patient similar to one of our patients with flow-mediated gastric varices . We would like to emphasize that the AVP may be considered also for embolization of the more common collateral veins to esophageal varices and , if need be , the TIPS stent itself .

B O O B I I O O O O B O O O O O O O B I I I O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O B I I I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O


Polymorphisms

in

the

Low-Density

Lipoprotein

Receptor-Related

Protein

-LRB-

-RRB-

Gene

Are

Associated

with

Peak

Bone

Mass

in

Non-sedentary

Men

Results

from

the

Odense

Androgen

Study

Purpose

To

investigate

the

impact

of

the

-LRB-

exon

-RRB-

and

-LRB-

exon

-RRB-

polymorphisms

of

the

low-density

lipoprotein

receptor-related

protein

-LRB-

-RRB-

gene

on

peak

bone

mass

in

young

men

Family

and

twin

studies

have

demonstrated

that

genetic

factors

account

for

of

the

inter-individual

variation

in

bone

mineral

density

-LRB-

BMD

-RRB-

in

both

women

and

men

-LSB-

-RSB-

Polymorphisms

in

a

number

of

genes

affect

the

bone

phenotype

regarding

BMD

bone

size

or

fracture

risk

-LSB-

-RSB-

Only

a

small

part

of

the

overall

variation

in

BMD

however

has

been

explained

by

the

polymorphisms

identified

so

far

The

transmembrane

proteins

low-density

lipoprotein

receptor-related

protein

-LRB-

-RRB-

and

are

essential

in

Wnt

signaling

They

are

expressed

in

many

tissues

including

osteoblasts

and

have

been

implicated

in

the

adaptive

response

of

bone

to

mechanical

load

-LSB-

-RSB-

Located

on

chromosome

activating

mutations

in

the

gene

are

responsible

for

rare

conditions

with

a

high

bone

mass

phenotype

-LSB-

-RSB-

as

well

as

autosomal

dominant

osteopetrosis

type

-LSB-

-RSB-

and

transgenic

mice

carrying

the

mutation

have

increased

bone

mass

and

bone

biomechanical

properties

-LSB-

-RSB-

Conversely

inactivating

mutations

are

responsible

for

the

osteoporosis

pseudoglioma

syndrome

-LSB-

-RSB-

and

this

syndrome

is

recapitulated

by

inactivation

in

mice

-LSB-

-RSB-

In

addition

to

these

mutations

a

number

of

polymorphisms

have

been

described

in

the

gene

-LRB-

-RRB-

One

of

these

the

-LRB-

exon

-RRB-

polymorphism

has

been

associated

with

attenuated

bone

gain

in

prepubertal

boys

-LSB-

-RSB-

decreased

BMD

in

Japanese

-LSB-

-RSB-

American

-LSB-

-RSB-

Australian

-LSB-

-RSB-

and

Dutch

-LSB-

-RSB-

women

as

well

as

in

elderly

Dutch

men

-LSB-

-RSB-

It

has

also

been

associated

with

an

increased

risk

of

fragility

fractures

in

postmenopausal

Australian

women

-LSB-

-RSB-

and

in

elderly

Dutch

men

-LSB-

-RSB-

No

association

however

was

found

between

this

polymorphism

and

bone

loss

in

elderly

men

and

women

-LSB-

-RSB-

Similarly

the

-LRB-

exon

-RRB-

polymorphism

was

associated

with

attenuated

bone

gain

in

prepubertal

boys

but

not

in

girls

participating

in

a

longitudinal

Swiss

study

-LSB-

-RSB-

In

the

same

study

this

polymorphism

was

also

associated

with

decreased

lumbar

spine

bone

mineral

content

and

projected

bone

area

and

there

was

a

trend

toward

lower

BMD

in

the

spine

-LSB-

-RSB-

These

associations

were

mainly

driven

by

men

-LSB-

-RSB-

and

in

haplotype

analysis

including

most

of

the

effect

was

conferred

by

the

polymorphism

Moreover

in

a

case-control

study

by

Ferrari

et

al.

-LSB-

-RSB-

on

idiopathic

osteoporosis

in

males

both

polymorphisms

significantly

increased

the

odds

ratio

for

osteoporosis

Additionally

was

associated

with

low

stature

in

adults

of

both

sexes

-LSB-

-RSB-

Thus

current

evidence

suggests

that

polymorphisms

in

the

gene

may

be

involved

in

the

control

of

peak

bone

mass

and

may

increase

the

risk

of

osteoporosis

in

males

-LSB-

-RSB-

In

the

present

study

we

investigated

the

association

between

the

and

polymorphisms

and

peak

bone

mass

in

men

in

a

population-based

study

Subjects

and

Methods

The

Odense

Androgen

Study

-LRB-

OAS

-RRB-

is

a

population-based

prospective

observational

study

on

the

interrelationship

between

endocrine

status

body

composition

muscle

function

and

bone

metabolism

in

young

men

Details

on

the

design

and

inclusion

of

participants

are

reported

elsewhere

-LSB-

-RSB-

In

brief

men

aged

years

were

randomly

selected

from

the

civil

registration

database

in

Fuenen

County

Denmark

and

invited

by

mail

to

participate

in

the

study

These

subjects

also

received

a

questionnaire

regarding

smoking

medication

chronic

diseases

drug

abuse

etc.

A

total

of

questionnaires

were

returned

and

from

these

men

expressed

interest

in

participation

in

the

clinical

part

of

the

study

Nineteen

withdrew

before

an

appointment

for

inclusion

was

made

Ten

men

did

not

meet

with

the

inclusion

criteria

-LRB-

Caucasian

and

age

between

years

-RRB-

and

another

men

gave

their

notice

after

the

inclusion

had

stopped

Aside

from

these

latecomers

of

the

responders

were

excluded

Of

the

remaining

eligible

subjects

did

not

attend

for

their

appointment

and

did

not

wish

to

reschedule

but

informed

consent

was

obtained

from

men

with

dropout

from

subject

Thus

Caucasian

men

gave

written

informed

consent

and

were

included

in

the

OAS

These

participants

had

similar

age

body

mass

index

-LRB-

BMI

-RRB-

lifestyle

socio-economic

status

and

educational

level

as

the

background

population

as

evaluated

by

questionnaires

and

demographic

data

-LRB-

manuscript

in

preparation

-RRB-

The

protocol

stipulated

that

subjects

with

massive

substance

or

alcohol

abuse

malignant

disease

or

severe

chronic

disease

be

excluded

however

of

the

responders

fulfilled

any

of

these

criteria

The

study

was

approved

by

the

local

Ethics

Committee

of

Fuenen

and

Vejle

Counties

-LRB-

file

number

-RRB-

conducted

according

to

the

Helsinki

Declaration

and

declared

in

ClinicalTrials.gov

registered

September

Genotyping

DNA

was

isolated

from

whole

blood

samples

using

QIAamp

DNA

Blood

Midi

kits

-LRB-

Qiagen

Valencia

CA

USA

-RRB-

The

polymorphism

was

analyzed

using

Taqman

primers

and

probes

that

were

designed

using

Primer

Express

software

-LRB-

Applied

Biosystems

-RRB-

Sense

Antisense

C

probe

T

probe

The

real-time

analysis

was

performed

using

the

ABI

PRISM

Sequence

Detection

System

To

validate

the

method

approximately

of

the

samples

were

repeated

and

in

total

samples

at

least

of

each

genotype

were

sequenced

The

polymorphism

was

analyzed

using

fluorescence

polarization

as

described

previously

-LSB-

-RSB-

using

AcycloPrime-FP

SNP

Detection

kits

-LRB-

Perkin

Elmer

Life

Sciences

Boston

MA

USA

-RRB-

PCR

fragments

containing

the

polymorphisms

were

generated

using

standard

conditions

with

the

following

PCR

primers

Sense

Antisense

Enzymatic

clean-up

and

subsequent

AcycloPrime-FP

reaction

with

SNP

primer

-LRB-

sense

-RRB-

were

performed

according

to

the

instructions

of

the

kit

manufacturer

Fluorescence

polarization

was

read

in

a

Wallac

Victor

multilabel

plate

reader

-LRB-

Perkin

Elmer

Life

Sciences

Boston

MA

USA

-RRB-

Blank

samples

and

samples

with

known

genotype

were

included

as

negative

and

positive

controls

respectively

A

total

of

of

all

samples

were

sequenced

and

genotyping

confirmed

in

all

cases

Anthropometrics

and

Lifestyle

Body

weight

body

height

sitting

height

and

arm

span

were

measured

Data

regarding

exercise

habits

were

collected

using

a

self-administered

questionnaire

Non-sedentary

lifestyle

was

defined

as

participation

in

any

form

of

regular

exercise

Dual-Energy

X-ray

Absorptiometry

-LRB-

DXA

-RRB-

BMD

of

the

lumbar

spine

hip

and

whole-body

was

measured

using

a

Hologic

DXA-scanner

-LRB-

Waltham

MA

USA

-RRB-

The

coefficient

of

variation

-LRB-

CV

-RRB-

for

BMD

is

and

in

the

lumbar

spine

total

hip

and

whole

body

respectively

Lean

body

mass

-LRB-

LBM

-RRB-

and

fat

mass

was

obtained

from

the

whole-body

scans

Biochemistry

Serum

samples

were

drawn

after

an

overnight

fast

between

and

hours

and

stored

for

later

analyses

at

°C

Serum

concentrations

of

osteocalcin

were

measured

by

a

luminoimmunoassay

-LRB-

BRAHMS

Diagnostica

Berlin

Germany

-RRB-

with

an

intra-assay

and

inter-assay

CV

of

and

respectively

Serum

levels

of

type

collagen

C-terminal

telopeptide

-LRB-

-RRB-

were

measured

using

radioimmunoassay

-LRB-

RIA

-RRB-

-LRB-

Orion

Diagnostica

Espoo

Finland

-RRB-

with

an

intra-assay

and

inter-assay

CV

of

and

respectively

Bone-specific

alkaline

phosphatase

-LRB-

bone-AP

-RRB-

concentration

in

serum

was

determined

enzymatically

after

monoclonal

antibody

capture

-LRB-

Metra

Biosystems

Mountain

View

CA

USA

-RRB-

The

intra-assay

CV

was

and

the

inter-assay

CV

for

this

analysis

Serum

levels

of

D

were

measured

using

a

competitive

radioimmunoassay

-LRB-

DiaSorin

Stillwater

MN

USA

-RRB-

with

an

intra-assay

CV

of

and

an

inter-assay

CV

of

Serum

IGF-I

was

determined

in

acid

ethanol

extracts

as

described

previously

-LSB-

-RSB-

Intra

and

inter-assay

variation

averaged

less

than

and

less

than

respectively

Statistics

Data

are

reported

as

the

mean

SD

median

-LRB-

range

-RRB-

or

median

-LSB-

CI

-RSB-

as

appropriate

BMD

values

are

shown

in

absolute

values

Regarding

the

allele

dose-analyses

BMD

values

were

transformed

to

Z-scores

using

the

SD

observed

in

our

population

Hardy-Weinberg

equilibrium

was

checked

using

chi-square

analysis

Linkage

disequilibrium

-LRB-

D'

-RRB-

between

polymorphisms

was

evaluated

by

using

GOLD

software

-LSB-

-RSB-

Haplotype

analysis

was

undertaken

using

FAMHAP

software

for

constructing

haplotypes

-LSB-

-RSB-

Comparisons

between

groups

were

performed

using

one-way

ANOVA

unpaired

t-test

or

similar

nonparametric

tests

Associations

between

parameters

were

analyzed

using

Pearson

's

regression

analysis

and

multiple

linear

regression

analysis

Dichotomies

were

tested

using

the

chi-square

test

A

p

value

of

less

than

was

considered

significant

Statistical

analyses

were

performed

using

SPSS

and

SPSS

Results

Genotype

Frequencies

For

the

polymorphism

the

CC

genotype

was

found

in

-LRB-

-RRB-

the

CT

genotype

in

-LRB-

-RRB-

and

the

TT

genotype

in

-LRB-

-RRB-

respectively

-LRB-

Table

-RRB-

allele

frequencies

of

and

for

the

C

and

T-alleles

respectively

Table

BMD

and

lifestyle

-LRB-

sedentary/non-sedentary

-RRB-

by

genotypes

-LRB-

and

polymorphisms

-RRB-

values

-LRB-

ANOVA

-RRB-

GGGAAAp

values

-LRB-

ANOVA

-RRB-

Age

-LRB-

years

-RRB-

NSBody

height

-LRB-

cm

-RRB-

Arm

span

-LRB-

cm

-RRB-

Body

weight

-LRB-

kg

-RRB-

NSBody

mass

index

-LRB-

-RRB-

NSTotal

fat

mass

-LRB-

kg

-RRB-

NSLean

body

mass

-LRB-

kg

-RRB-

NSUnits

alcohol/week

-LRB-

n

-RRB-

NSCigarettes/day

-LRB-

n

-RRB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

-LSB-

-RSB-

NSSerum

-LRB-

nmol/l

-RRB-

NSSerum

IGF-I

-LRB-

μg/l

-RRB-

NSSerum

osteocalcin

-LRB-

mmol/l

-RRB-

NSSerum

-LRB-

μg/l

-RRB-

-LRB-

U/l

-RRB-

NSBMDspine

-LRB-

-RRB-

NSBMDhip

-LRB-

-RRB-

BMDWB

-LRB-

-RRB-

NSSedentary/non-sedentary

-LRB-

n

-RRB-

significant

differences

were

found

between

the

groups

-LRB-

ANOVA

-RRB-

This

was

true

also

if

the

CT

and

TT

genotypes

were

pooled

-LRB-

t-test

-RRB-

Genotypes

were

in

Hardy-Weinberg

equilibrium

-LRB-

χ2

p

χ2

p

-RRB-

p

comparing

the

CC

genotype

with

CT

TT

pooled

NS

not

significant

Similarly

the

GG

GA

AA

genotypes

of

the

polymorphism

were

found

in

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

respectively

This

corresponds

to

allele

frequencies

of

and

for

the

G

and

A-alleles

respectively

Both

allele

distributions

were

compatible

with

Hardy-Weinberg

equilibrium

-LRB-

χ2

p

and

χ2

p

-RRB-

The

two

polymorphisms

were

not

in

complete

linkage

disequilibrium

-LRB-

D'

R

²

-RRB-

Covariates

Data

on

covariates

in

relation

to

genotypes

are

shown

in

Table

No

significant

differences

regarding

covariates

-LRB-

age

body

height

body

weight

BMI

sitting

height

-LRB-

not

shown

-RRB-

total

fat

mass

lean

body

mass

alcohol

consumption

smoking

serum

or

serum

IGF-I

-RRB-

were

seen

between

the

genotypes

of

the

two

polymorphisms

Regarding

the

genotypes

however

subjects

with

the

AA

genotype

had

significantly

increased

body

height

-LRB-

vs.

and

cm

p

-RRB-

and

arm

span

-LRB-

vs.

and

cm

p

-RRB-

BMD

in

Relation

to

Age

In

the

spine

no

relationship

between

BMD

and

age

could

be

detected

Maximum

BMD

in

the

total

hip

was

observed

around

the

age

of

years

In

subjects

above

this

age

BMD

was

significantly

lower

-LRB-

ANOVA

p

-RRB-

In

subjects

aged

years

BMD

was

lower

that

the

observed

maximum

Genotypes

in

Relation

to

BMD

Regarding

the

polymorphism

no

significant

differences

between

BMD

in

the

spine

hip

or

whole

body

were

found

between

subjects

with

the

CC

CT

and

TT

genotypes

When

the

CT

and

TT

genotypes

were

pooled

whole-body

BMD

was

significantly

lower

in

the

CT

TT

compared

with

the

CC

genotype

-LRB-

vs.

p

-RRB-

Regarding

the

Val

polymorphism

a

significant

difference

in

hip

BMD

was

observed

since

subjects

with

the

AA

genotype

had

lower

BMD

-LRB-

vs.

and

p

-RRB-

Table

also

shows

the

distribution

of

genotypes

in

relation

to

sedentary/non-sedentary

lifestyle

but

no

significant

difference

in

lifestyle

was

found

between

any

of

the

genotypes

Table

shows

the

gene-dose

effect

on

BMD

expressed

as

the

change

in

Z-score

of

each

copy

of

the

T

or

A-allele

respectively

The

analysis

was

performed

both

with

and

without

adjustment

for

BMI

smoking

-LRB-

-RRB-

any

continuous

medication

-LRB-

-RRB-

and

serum

in

the

overall

study

population

-LRB-

multiple

regression

analysis

-RRB-

Regarding

the

polymorphism

whole-body

BMD

tended

to

decrease

with

each

copy

of

the

T-allele

-LRB-

p

-RRB-

in

the

overall

study

population

-LRB-

n

-RRB-

No

significant

association

between

number

of

T-alleles

and

BMD

at

the

other

skeletal

sites

was

found

Regarding

the

polymorphism

no

significant

gene-dose

effect

could

be

demonstrated

in

the

overall

population

Table

regression

analysis

determining

the

change

in

BMD

Z-score

for

each

copy

of

the

T

or

population

-LRB-

n

-RRB-

BMDspine

-LRB-

-RRB-

NS

-LRB-

-RRB-

NS

-LRB-

-RRB-

NS

-LRB-

-RRB-

NSBMDhip

-LRB-

-RRB-

NS

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

NSBMDWB

-LRB-

-RRB-

p

-LRB-

-RRB-

p

-LRB-

-RRB-

NS

-LRB-

-RRB-

NSNon-sedentary

-LRB-

n

-RRB-

BMDspine

-LRB-

-RRB-

p

-LRB-

-RRB-

p

-LRB-

-RRB-

p

-LRB-

-RRB-

p

BMDhip

-LRB-

-RRB-

NS

-LRB-

-RRB-

NS

-LRB-

-RRB-

NS

-LRB-

-RRB-

NSBMDWB

-LRB-

-RRB-

p

-LRB-

-RRB-

p

-LRB-

-RRB-

NS

-LRB-

-RRB-

NSData

are

shown

as

mean

effect

and

confidence

interval

Z-scores

were

derived

from

the

study

population

itselfaAdjusted

for

BMI

-LRB-

ln-transformed

-RRB-

lean

body

mass

-LRB-

ln-transformed

-RRB-

smoking

-LRB-

-RRB-

any

continuous

medication

-LRB-

-RRB-

and

serum

When

analysis

was

restricted

to

non-sedentary

men

-LRB-

n

-RRB-

however

a

significant

correlation

between

the

number

of

T-alleles

at

the

polymorphism

and

BMD

in

the

spine

was

found

in

both

the

unadjusted

-LRB-

-LSB-

CI

-RSB-

p

-RRB-

-LRB-

Fig.

-RRB-

and

adjusted

analysis

-LRB-

-LSB-

CI

-RSB-

p

-RRB-

-LRB-

Table

-RRB-

Similarly

each

copy

of

the

A-allele

in

the

polymorphism

decreased

the

lumbar

spine

BMD

in

unadjusted

-LRB-

-LSB-

CI

-RSB-

p

-RRB-

and

adjusted

-LRB-

-LSB-

CI

-RSB-

p

-RRB-

analysis

Fig.

of

the

lumbar

spine

total

hip

and

whole

body

in

relation

to

the

genotype

regarding

the

-LRB-

upper

panel

-RRB-

and

the

-LRB-

lower

panel

-RRB-

polymorphisms

in

the

gene

Participants

with

a

non-sedentary

-LRB-

left

-RRB-

and

sedentary

lifestyle

-LRB-

right

-RRB-

are

displayed

Data

are

shown

as

the

mean

SE

-LRB-

-RRB-

p

p

Genotypes

in

Relation

to

Bone

Markers

No

significant

differences

were

observed

between

genotypes

of

the

or

polymorphisms

regarding

bone

markers

-LRB-

Table

-RRB-

Haplotype

Analysis

The

effect

of

haplotypes

of

the

and

polymorphisms

on

BMD

adjusted

for

BMI

lean

body

mass

smoking

drugs

and

vitamin

D

are

shown

in

Table

No

significant

association

between

BMD

and

haplotypes

was

found

This

was

also

the

case

when

sedentary

and

non-sedentary

men

were

analyzed

separately

-LRB-

not

shown

-RRB-

Table

effect

of

haplotypes

of

and

polymorphisms

on

BMD

adjusted

for

BMI

lean

body

mass

smoking

drugs

and

vitamin

-LRB-

-RRB-

NSBMDhip

-LRB-

-RRB-

NSBMDWB

-LRB-

-RRB-

NSData

are

shown

as

the

mean

SD

No

significant

associations

between

BMD

and

haplotypes

were

found

when

sedentary

and

non-sedentary

men

were

analyzed

separately

-LRB-

not

shown

-RRB-

Discussion

We

found

a

significant

association

between

the

and

the

polymorphisms

in

the

gene

and

BMD

in

young

men

especially

when

physical

activity

was

taken

into

account

as

confounder

This

finding

supports

a

number

of

recent

papers

reporting

significant

associations

between

polymorphisms

in

this

gene

and

BMD

-LSB-

-RSB-

Some

of

these

studies

involved

or

were

restricted

to

men

-LSB-

-RSB-

Moreover

Ferrari

et

al.

-LSB-

-RSB-

suggested

that

the

polymorphisms

in

the

gene

are

associated

with

bone

gain

in

prepubertal

boys

Only

one

study

-LSB-

-RSB-

failed

to

demonstrate

an

association

between

variations

in

the

gene

and

BMD

but

most

likely

this

is

due

to

their

restricted

sample

size

-LRB-

n

-RRB-

Major

disruption

of

the

gene

-LRB-

homozygous

nonsense

or

frame-shift

mutations

-RRB-

resulting

in

loss-of-function

has

been

demonstrated

to

be

responsible

for

the

osteoporosis

pseudoglioma

syndrome

-LSB-

-RSB-

Conversely

a

number

of

dominantly

inherited

gain-of-function

mutations

result

in

high

bone

mass

phenotypes

-LSB-

-RSB-

and

autosomal

dominant

osteopetrosis

type

-LSB-

-RSB-

Minor

changes

in

this

gene

i.e.

frequently

occurring

polymorphisms

may

also

be

of

importance

in

the

regulation

of

bone

mass

as

suggested

by

the

association

studies

mentioned

above

Further

support

comes

from

mapping

a

quantitative

trait

locus

for

BMD

to

chromosome

in

some

-LSB-

-RSB-

but

not

all

studies

-LSB-

-RSB-

In

a

case-control

study

comprising

men

with

idiopathic

osteoporosis

and

controls

Ferrari

et

al.

-LSB-

-RSB-

found

that

and

conferred

an

odds

ratio

for

osteoporosis

of

-LRB-

CI

-RRB-

and

-LRB-

CI

-RRB-

respectively

Also

a

single

prospective

study

-LSB-

-RSB-

comprising

elderly

Australian

women

demonstrated

that

the

c.

G

-LRB-

-RRB-

polymorphism

was

associated

with

decreased

BMD

and

increased

incidence

of

fractures

This

polymorphism

located

in

exon

is

synonymous

but

was

found

to

be

in

linkage

disequilibrium

with

-LSB-

-RSB-

Similarly

Mizuguchi

et

al.

-LSB-

-RSB-

found

a

linkage

equilibrium

block

comprising

exons

to

In

our

study

no

significant

effect

of

or

on

BMD

was

found

apart

from

the

ANOVA

analysis

which

suggested

that

the

AA

genotype

at

was

associated

with

low

BMD

of

the

hip

and

increased

body

height

The

number

of

participants

with

this

genotype

however

was

low

-LRB-

n

-RRB-

and

we

found

no

significant

gene-dose

effect

to

back

this

finding

In

non-sedentary

subjects

however

we

found

a

significant

association

between

genotype

and

the

and

Z-score

BMD

in

the

spine

both

in

unadjusted

analysis

and

when

adjusted

for

BMI

smoking

continuous

medication

and

serum

levels

of

Further

we

observed

an

association

between

and

whole-body

BMD

in

the

unadjusted

analysis

This

interaction

between

lifestyle

-LRB-

sedentary/non-sedentary

-RRB-

genotype

and

BMD

has

not

previously

been

reported

Such

an

association

is

fully

in

line

with

the

proposed

role

of

as

a

player

in

adapting

bone

to

weight-bearing

acts

as

a

co-receptor

for

Wnt

proteins

and

is

expressed

in

the

osteoblast

and

a

number

of

other

cell

types

The

-LRB-

exon

-RRB-

polymorphism

results

in

a

substitution

of

alanine

with

the

chemically

similar

valine

No

functional

studies

have

been

performed

on

this

polymorphism

however

the

polymorphism

is

located

within

the

second

LDL-repeat

of

-LSB-

-RSB-

and

this

region

is

involved

in

the

ligand

binding

of

the

LDL-receptor

-LSB-

-RSB-

Also

preliminary

results

have

shown

that

drugs

that

inhibit

Dkk

binding

to

increase

bone

formation

in

vitro

-LSB-

-RSB-

and

seems

to

be

involved

in

the

adaptive

response

of

bone

to

mechanical

load

and

may

be

partly

responsible

for

the

mechanostat

-LSB-

-RSB-

It

is

presently

unclear

why

association

between

the

and

genotypes

and

BMD

was

found

only

in

the

spine

and

not

in

the

hip

in

the

non-sedentary

group

Differences

between

the

skeletal

sites

regarding

loading

pattern

-LRB-

compression

vs.

bending

-RRB-

and

bone

architecture

-LRB-

trabecular

vs.

cortical

-RRB-

may

be

important

In

our

study

the

numbers

of

T-alleles

and

A-alleles

of

the

and

polymorphisms

respectively

were

associated

with

a

decrease

in

BMD

of

the

lumbar

spine

of

SD

in

the

non-sedentary

participants

-LRB-

i.e.

approximately

-RRB-

Previous

studies

have

assessed

this

impact

to

be

from

-LSB-

-RSB-

to

-LSB-

-RSB-

in

women

and

men

and

women

respectively

While

a

number

of

genes

have

been

associated

with

bone

mass

in

women

-LRB-

e.g.

PTH

-LSB-

-RSB-

chain

of

type

collagen

-LSB-

-RSB-

vitamin

D

receptor

-LSB-

-RSB-

transforming

growth

factor

-LSB-

-RSB-

-LSB-

-RSB-

and

osteoprotegerin

-LSB-

-RSB-

-RRB-

only

a

few

genes

have

been

shown

to

be

associated

with

peak

bone

mass

in

men

We

have

previously

reported

that

the

MTHFR

gene

is

associated

with

peak

bone

mass

in

the

same

series

of

men

as

reported

here

-LSB-

-RSB-

Each

of

these

polymorphisms

including

the

gene

only

accounts

for

a

fraction

of

the

variability

in

BMD

however

the

impact

suggested

by

our

study

is

not

trivial

We

found

no

association

between

either

projected

bone

area

at

the

spine

or

hip

or

body

weight

and

any

of

the

polymorphisms

however

our

data

demonstrated

a

significant

impact

of

the

AA

genotype

of

the

polymorphism

and

body

height

as

previously

reported

by

Ferrari

et

al.

-LSB-

-RSB-

and

Koay

et

al.

-LSB-

-RSB-

In

our

study

the

number

of

subjects

with

the

AA

genotype

was

however

very

low

-LRB-

n

-RRB-

Thus

this

finding

clearly

needs

confirmation

in

other

study

designs

The

allele

frequencies

found

in

our

study

corresponded

closely

to

those

reported

in

Dutch

Australian

Japanese

and

Korean

populations

-LSB-

-RSB-

Our

study

has

some

limitations

We

only

tested

two

polymorphisms

in

the

gene

We

can

not

therefore

rule

out

the

possibility

that

the

associations

found

in

our

study

could

be

due

to

other

polymorphisms

in

the

gene

or

even

in

neighboring

genes

in

linkage

with

the

polymorphisms

tested

Indeed

Bollerslev

et

al.

-LSB-

-RSB-

found

that

several

of

the

polymorphisms

in

the

gene

were

in

linkage

disequilibrium

with

each

other

in

Australian

women

In

our

study

haplotype

analyses

were

negative

This

might

be

due

to

the

limited

number

of

participants

Testing

large

arrays

of

polymorphisms

or

haplotypes

in

relation

to

many

outcome

variables

-LRB-

i.e.

bone

mass

at

several

skeletal

sites

-RRB-

however

carry

an

increased

risk

of

detecting

spurious

associations

Also

our

study

had

a

limited

size

Given

the

observed

genotype

frequency

and

BMD

values

our

study

had

the

power

to

detect

differences

in

BMDof

the

spine

between

CC

and

CT

TT

of

-LRB-

alpha

and

beta

-RRB-

Similarly

our

study

had

the

power

to

detect

differences

of

between

GG

and

GA

AA

in

the

overall

analysis

Our

study

also

has

several

strengths

First

it

was

population-based

In

contrast

to

most

similar

studies

-LSB-

-RSB-

that

recruited

participants

by

advertisements

we

recruited

our

participants

by

direct

mailing

on

the

basis

of

the

National

Personal

Registry

that

includes

all

Danish

citizens

This

enabled

us

to

account

meticulously

for

the

recruitment

of

our

participants

Thus

our

sample

was

not

skewed

with

respect

to

key

socio-economic

parameters

-LSB-

-RSB-

Second

our

population

had

a

relatively

homogeneous

genetic

background

both

due

to

a

small

influx

to

our

population

and

since

participants

with

a

first

or

second-generation

immigrant

background

were

excluded

on

the

basis

on

data

from

our

national

registry

Third

we

selected

young

men

aged

years

since

these

were

expected

to

have

reached

their

peak

bone

mass

This

was

confirmed

by

our

data

demonstrating

an

infinitesimal

decrease

in

BMD

by

age

Moreover

very

few

were

afflicted

with

chronic

diseases

or

had

long-term

exposure

of

environmental

factors

that

may

affect

bone

mass

We

conclude

that

the

-LRB-

exon

-RRB-

and

-LRB-

exon

-RRB-

polymorphisms

of

the

gene

are

significantly

associated

with

lumbar

spine

peak

bone

mass

in

physically

active

men

potentially

accounting

for

of

BMD

in

the

spine

This

gene-environment

interaction

provides

support

for

as

a

mediator

of

load-induced

bone

formation

and

suggests

that

this

gene

is

involved

in

the

pathogenesis

of

osteoporosis

in

men

B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O

association
bone mineral density
men
polymorphisms

low-density lipoprotein receptor-related protein 5