A

comparison

of

nicotine

dose

estimates

in

smokers

between

filter

analysis

salivary

cotinine

and

urinary

excretion

of

nicotine

metabolites

Rationale

Nicotine

uptake

during

smoking

was

estimated

by

either

analyzing

the

metabolites

of

nicotine

in

various

body

fluids

or

by

analyzing

filters

from

smoked

cigarettes

However

no

comparison

of

the

filter

analysis

method

with

body

fluid

analysis

methods

has

been

published

Introduction

A

number

of

studies

were

published

over

the

last

years

that

attempted

to

determine

the

amount

of

tar

and/or

nicotine

that

smokers

receive

from

their

cigarettes

-LRB-

reviewed

in

Stephen

et

al.

Pritchard

and

Robinson

Scherer

-RRB-

The

methodology

used

falls

into

three

broad

categories

-LRB-

-RRB-

the

analysis

of

biomarkers

in

human

body

fluids

or

expired

breath

-LRB-

-RRB-

the

measurement

of

smoking

behavior

-LRB-

puff

volume

duration

and

frequency

-RRB-

followed

by

a

smoking

machine

set

to

duplicate

human

puffing

conditions

and

-LRB-

-RRB-

the

analysis

of

spent

cigarette

filters

and

the

calculation

of

smoke

yields

from

the

filter

efficiency

Typically

biomarker

measurements

in

blood/plasma

and

smoking

behavior

measurements

require

that

sampling

or

measurements

be

made

in

a

laboratory

environment

There

is

a

possibility

that

smoking

behavior

becomes

atypical

in

this

type

of

environment

-LRB-

Comer

and

Creighton

Ossip-Klein

et

al.

-RRB-

Urine

saliva

and

spent

cigarette

filters

can

be

collected

in

a

smoker

's

everyday

environment

The

analysis

of

urine

samples

for

nicotine

and

major

metabolites

can

provide

quantitative

data

regarding

uptake

of

smoke

constituents

as

the

product

is

used

in

a

smoker

's

everyday

environment

-LRB-

see

Byrd

et

al.

and

references

therein

for

examples

-RRB-

Subject

compliance

can

be

an

issue

when

trying

to

determine

actual

cigarette

yields

because

in

an

unmonitored

environment

the

subject

must

be

relied

upon

to

smoke

only

a

given

brand

not

use

any

other

nicotine-containing

products

provide

an

exact

accounting

of

every

cigarette

smoked

during

the

collection

period

and

collect

all

urinary

output

The

filter

analysis

method

is

one

of

the

least

invasive

of

the

methods

mentioned

above

The

smokers

can

use

their

product

in

their

normal

environment

and

the

only

deviation

from

normal

behavior

is

to

save

the

filters

Compliance

is

not

an

issue

when

trying

to

determine

the

subject

's

cigarette

yield

because

the

filters

from

the

actual

cigarettes

smoked

are

analyzed

In

most

cases

the

returned

filter

can

be

compared

to

those

of

the

subject

's

stated

brand

to

assure

brand

compliance

The

primary

issue

with

this

method

has

been

that

smoking

behavior

can

produce

changes

in

the

filtration

efficiency

of

the

filter

The

filtration

efficiency

can

vary

according

to

the

velocity

of

the

smoke

passing

through

the

filter

and

to

some

extent

the

length

of

the

tobacco

rod

smoked

-LRB-

Overton

Dwyer

and

Abel

Norman

et

al.

-RRB-

The

method

used

for

this

study

was

developed

to

minimize

the

effects

of

smoking

behavior

on

filter

efficiency

by

measuring

only

the

portion

of

the

filter

downstream

of

the

ventilation

holes

-LRB-

i.e.

the

mouth

end

-RRB-

-LRB-

St.Charles

Shepperd

et

al.

-RRB-

This

results

in

relatively

constant

filtration

efficiency

over

a

wide

range

of

smoking

behavior

The

objective

of

this

study

was

to

compare

the

nicotine

yield

of

human-smoked

cigarettes

-LRB-

mouth

exposure

-RRB-

as

measured

by

the

filter

analysis

method

and

human

smoke

uptake

as

measured

by

biomonitoring

under

strictly

controlled

conditions

The

biomonitoring

measurements

included

salivary

cotinine

and

urinary

nicotine

cotinine

hydroxycotinine

-LRB-

-RRB-

and

their

respective

glucuronide

conjugates

With

a

good

correlation

between

the

methods

future

studies

on

smoker

exposure

can

use

the

simpler

filter

analysis

method

rather

than

resorting

to

human

biomonitoring

techniques

Materials

and

methods

Study

design

The

clinical

portion

of

this

study

was

conducted

by

an

independent

contract

research

organization

in

-LRB-

Covance

Clinical

Research

Unit

Madison

WI

USA

-RRB-

The

analysis

of

salivary

cotinine

and

urinary

nicotine

metabolites

was

performed

at

Covance

Laboratories

-LRB-

Harrogate

North

Yorkshire

UK

-RRB-

Filter

analysis

was

performed

by

the

study

sponsor

-LRB-

Research

and

Development

Brown

Williamson

Tobacco

Company

Macon

GA

USA

-RRB-

The

study

was

approved

by

Covance

's

Institutional

Review

Board

and

performed

in

accordance

with

applicable

federal

regulations

Subjects

who

participated

in

the

study

gave

their

informed

consent

were

told

of

the

purpose

of

the

study

and

could

withdraw

at

any

time

Subject

selection

Habitual

smokers

were

recruited

by

Covance

Enrollment

criteria

included

males

or

nonpregnant

nonlactating

females

between

and

years

of

age

within

to

of

their

ideal

body

weight

who

smoked

at

least

cigarettes

a

day

of

the

same

cigarette

brand

during

the

previous

year

Subjects

were

excluded

if

they

were

under

years

of

age

were

pregnant

or

lactating

participated

in

any

other

clinical

study

within

days

before

study

entry

had

a

history

or

showed

signs

of

a

significant

medical

or

psychiatric

condition

used

prescription

medications

within

days

before

study

entry

had

a

history

of

alcoholism

or

drug

addiction

within

a

year

of

study

entry

or

used

alcohol

or

any

nonprescription

preparations

within

h

of

study

entry

A

few

subjects

deviated

from

the

enrollment

criteria

underweight

-LRB-

-RRB-

overweight

-LRB-

-RRB-

medication

or

alcohol

usage

before

study

entry

-LRB-

-RRB-

low

cigarette

consumption

-LRB-

-RRB-

shortened

brand

loyalty

duration

-LRB-

-RRB-

elevated

clinical

chemistry

-LRB-

-RRB-

abdominal/hernia

surgery

-LRB-

-RRB-

and

positive

drug

screen

before

study

entry

-LRB-

-RRB-

Because

these

deviations

were

considered

minor

and

not

expected

to

interfere

with

the

study

objectives

the

subjects

were

allowed

to

participate

in

the

study

Subjects

were

assigned

into

one

of

four

tar

yield

groups

which

span

the

range

of

Federal

Trade

Commission

-LRB-

FTC

-RRB-

tar

yields

found

in

commercially

available

USA

filtered

cigarettes

mg

-LRB-

ULL

or

ultralights/low

-RRB-

mg

-LRB-

ULH

or

ultralights/high

-RRB-

mg

-LRB-

LTS

or

lights

-RRB-

and

mg

-LRB-

FF

or

full

flavor

-RRB-

The

purpose

was

to

cover

a

wide

range

of

human

nicotine

exposure

to

allow

robust

correlations

between

the

methodologies

The

goal

was

to

enroll

smokers

per

group

however

even

with

additional

recruitment

attempts

only

smokers

enrolled

in

the

ULL

group

-LRB-

market

share

-RRB-

One

subject

in

the

FF

group

withdrew

from

the

study

due

to

illness

Table

summarizes

the

subjects

demographics

and

their

respective

brand

characteristics

by

tar

band

Table

demographics

and

cigarette

brand

characteristics

by

tar

-LRB-

M/F

-RRB-

-LRB-

cm

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Weight

-LRB-

kg

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Menthol

Tar

-LRB-

mg/cig

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Nicotine

-LRB-

mg/cig

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

CO

-LRB-

mg/cig

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Height

weight

and

age

are

shown

as

the

mean

-LRB-

range

-RRB-

Cigarette

yields

are

shown

as

the

mean

-LRB-

SD

-RRB-

for

the

FTC

smoking

method

Subjects

were

confined

to

the

clinic

for

six

calendar

days

to

give

five

consecutive

periods

Nine

confinement

periods

were

staggered

and

limited

to

ten

subjects

or

less

-LRB-

generally

of

the

same

tar

range

group

-RRB-

During

confinement

period

number

one

LTS

smoker

and

two

FF

smokers

were

allowed

to

participate

with

six

ULL

smokers

to

complete

the

LTS

and

FF

cells

Subjects

were

fed

a

standardized

bulk

diet

that

excluded

grilled

smoked

or

barbecued

food

items

Consumption

of

water

and

other

nonalcoholic

beverages

was

unrestricted

Subjects

refrained

from

strenuous

exercise

During

confinement

subjects

smoked

their

usual

cigarette

brand

ad

libitum

in

a

dedicated

smoking

room

equipped

with

ventilation

and

air

filtration

Use

of

any

form

of

nicotine

other

than

the

subject

's

declared

own

brand

was

prohibited

Cigarettes

were

purchased

locally

Urine

and

saliva

collection

and

analysis

Twenty-four-hour

urine

samples

were

collected

from

each

subject

for

five

consecutive

days

Collections

started

at

approximately

hours

-LRB-

first

void

excluded

-RRB-

and

ended

at

approximately

hours

the

following

day

-LRB-

first

void

included

-RRB-

Urine

was

collected

in

amber

plastic

containers

and

kept

refrigerated

throughout

the

collection

period

No

chemical

preservatives

were

used

After

each

sample

collection

volume

and

pH

measurements

were

recorded

a

sample

was

taken

for

creatinine

analysis

and

and

aliquots

were

taken

and

stored

frozen

at

°C

until

shipped

Aliquots

were

shipped

under

dry

ice

to

the

analytical

laboratory

and

stored

at

°C

until

analysis

Urinary

nicotine

nicotine-N-β-glucuronide

cotinine

cotinine-N-β-glucuronide

hydroxycotinine

and

hydroxycotinine-O-β-glucuronide

were

analyzed

using

solid

phase

extraction

-LRB-

SPE

-RRB-

and

liquid

chromatography

with

tandem

mass

spectrometric

detection

using

a

method

developed

and

validated

at

the

analytical

laboratory

-LRB-

Analytical

Procedure

Covance

no.

-RRB-

-LRB-

Sigma-Aldrich

Poole

UK

-RRB-

and

-LRB-

Toronto

Research

Chemicals

North

York

ON

Canada

-RRB-

were

used

as

internal

standards

Nicotine

cotinine

-LRB-

Sigma-Aldrich

-RRB-

hydroxycotinine

and

the

three

corresponding

glucuronides

-LRB-

Toronto

Research

Chemicals

-RRB-

were

used

as

reference

standards

Samples

were

incubated

for

h

at

°C

with

β-glucuronidase

which

enzymatically

deconjugated

the

aglycones

from

their

respective

glucuronides

The

free

and

cleaved

aglycones

and

their

respective

deuterated

internal

standards

were

then

extracted

and

analyzed

Saliva

samples

were

collected

in

sterile

Salivette

tubes

-LRB-

Sarstedt

Newton

NC

USA

-RRB-

from

each

subject

for

five

consecutive

days

at

approximately

hours

On

day

two

additional

saliva

samples

were

collected

at

approximately

and

hours

Clinical

staff

supervised

and

timed

the

process

to

assure

compliance

Collected

saliva

samples

were

immediately

stored

at

°C

shipped

under

dry

ice

to

the

analytical

laboratory

and

stored

at

°C

until

analysis

Salivary

cotinine

was

analyzed

by

a

method

developed

and

validated

at

the

analytical

laboratory

-LRB-

Analytical

Procedure

Covance

no

-RRB-

based

on

a

previously

reported

method

-LRB-

Bentley

et

al.

-RRB-

Filter

collection

and

analysis

To

control

cigarette

brand

accessibility

and

document

cigarette

consumption

cigarettes

were

issued

by

clinical

staff

individually

The

used

filter

had

to

be

returned

before

a

subject

received

their

next

cigarette

Used

filters

were

processed

under

the

supervision

of

clinical

staff

by

having

the

subject

remove

any

tobacco

particles

from

the

used

filter

before

depositing

it

in

an

individually

labeled

glass

container

Used

filters

were

collected

for

five

consecutive

days

with

each

day

starting/ending

at

the

same

time

as

urine

samples

The

filters

generated

daily

by

each

subject

were

shipped

by

overnight

carrier

at

ambient

temperature

to

the

analytical

laboratory

Immediately

upon

receipt

a

portion

of

the

mouth

end

-LRB-

tip

-RRB-

was

cut

from

the

filter

using

a

specially

designed

jig

which

kept

the

cut-length

constant

and

kept

a

single

edge

razor

blade

perpendicular

to

the

filter

Tip-to-tip

length

variation

using

the

jig

was

within

The

tips

were

stored

in

labeled

glass

jars

with

Teflon-lined

lids

at

°C

until

analyzed

Nicotine

yields

from

the

human-smoked

cigarettes

were

estimated

by

analyzing

the

tips

for

nicotine

Five

separate

machine

smoking

regimes

were

used

to

provide

calibration

curves

for

the

filter

tips

of

each

brand

style

tested

Cigarettes

from

the

same

batches

that

the

subjects

smoked

were

used

for

calibration

smoking

The

smoking

regimes

were

chosen

to

give

a

wide

range

of

cigarette

yields

of

approximately

equal

spacing

Table

shows

the

calibration

puffing

conditions

for

each

tar

band

These

machine

puffing

regimes

proved

to

cover

the

spread

of

human

smoking

results

for

all

but

of

the

subject-days

tested

-LRB-

ULH

LTS

and

FF

out

of

total

-RRB-

Six

of

the

involved

extrapolating

by

less

than

mg

of

nicotine/cigarette

and

the

maximum

extrapolation

was

from

to

mg/cigarette

Table

smoking

conditions

by

tar

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T

are

presented

as

puff

volume

-LRB-

ml

-RRB-

puff

interval

-LRB-

s

-RRB-

length

smoked/ventilation

holesO

Open

B

blockedT

indicates

overtipping

plus

distance

-LRB-

mm

-RRB-

P

indicates

number

of

puffs

Calibration

equations

were

calculated

using

a

linear

regression

of

nicotine

yield

as

a

function

of

tip

nicotine

These

equations

were

then

used

to

estimate

human-smoked

cigarette

yield

from

the

measured

tip

nicotine

This

method

was

validated

in

a

separate

study

using

duplicated

human

puffing

profiles

-LRB-

Shepperd

et

al.

-RRB-

Aging

tests

by

the

study

sponsor

have

shown

that

tip

nicotine

values

were

constant

when

whole

filters

were

stored

in

glass

jars

at

ambient

temperature

for

up

to

days

For

this

study

tips

were

cut

from

the

whole

filters

within

days

One

to

nine

tips

per

sample

were

extracted

using

either

or

ml

of

methanol

containing

mg/ml

decanol

internal

standard

Tips

were

extracted

for

min

using

a

flatbed

orbital

shaker

at

rpm

All

available

tips

were

divided

into

at

least

three

separate

batches

which

were

extracted

and

analyzed

on

different

days

to

average

analytical

variation

For

samples

from

days

and

extract

absorbance

at

nm

was

also

measured

to

audit

the

nicotine

analysis

using

an

independent

method

Absorbance

gives

a

measure

of

the

tar

deposited

on

the

filter

-LRB-

Sloan

and

Curran

Shepperd

et

al.

-RRB-

and

we

have

found

that

absorbance

per

tip

correlates

linearly

with

nicotine

per

tip

For

days

and

replicate

vials

of

the

extract

were

stored

at

°C

and

subsequently

analyzed

for

nicotine

to

audit

the

nicotine

analysis

further

The

root

mean

square

difference

between

the

nicotine

replicates

was

μg/ml

-LRB-

-RRB-

A

backup

set

of

replicate

vials

was

kept

for

use

when

outlying

data

points

were

identified

by

either

of

the

auditing

methods

Samples

were

retested

when

they

were

outside

of

the

confidence

interval

of

the

absorbance

per

tip

vs

nicotine

per

tip

correlation

-LRB-

days

and

-RRB-

or

when

nicotine

replicates

differed

by

more

than

-LRB-

days

and

-RRB-

The

extract

was

analyzed

for

nicotine

by

gas

chromatography

using

an

Agilent

Series

II

with

a

flame

ionization

detector

and

a

J&W

Scientific

m

Megabore

mm

ID

DB-Wax

-LRB-

μm

film

-RRB-

fused

silica

capillary

column

-LRB-

Agilent

Palo

Alto

CA

USA

-RRB-

The

UV

absorbance

of

the

extract

was

measured

using

an

Ocean

Optics

-LRB-

Dunedin

FL

USA

-RRB-

spectrometer

equipped

with

a

fiber

optic

dip

probe

with

a

path

length

This

gave

absorbance

values

within

the

linear

range

of

less

than

without

further

dilution

Absorbance

was

measured

on

the

same

day

the

tips

were

extracted

because

it

was

found

that

extract

absorbance

declines

with

even

overnight

storage

Results

Measured

urinary

concentrations

for

each

metabolite

were

multiplied

by

their

respective

daily

urine

volumes

and

converted

based

on

molecular

weights

to

yield

recovery

results

in

nicotine

equivalents

The

sum

of

the

nicotine

equivalents

for

nicotine

cotinine

and

were

calculated

for

each

subject

per

day

to

give

total

daily

urinary

nicotine

equivalents

-LRB-

UNE

-RRB-

in

milligrams

per

day

Nicotine

yield

per

cigarette

was

calculated

from

the

mean

nicotine

per

tip

values

for

each

subject-day

and

the

appropriate

calibration

equation

for

the

brand

smoked

This

was

multiplied

by

the

number

of

cigarettes

smoked

by

the

subject

that

day

to

give

nicotine

yield

in

milligrams

per

day

This

study

design

allowed

us

to

achieve

a

wide

range

of

nicotine

exposure

suitable

for

the

correlation

of

the

three

methods

of

nicotine

uptake

estimation

Nicotine

yield

from

the

human-smoked

cigarettes

-LRB-

mouth

exposure

-RRB-

as

measured

by

filter

analysis

ranged

from

to

mg/day

UNE

ranged

from

to

mg/day

and

saliva

cotinine

ranged

from

to

ng/ml

The

mean

-LRB-

SD

-RRB-

proportions

of

urinary

metabolites

including

the

glucuronides

were

-LRB-

-RRB-

for

nicotine

-LRB-

-RRB-

for

cotinine

and

-LRB-

-RRB-

for

of

the

total

UNE

measured

When

expressed

as

a

percentage

of

nicotine

entering

the

mouth

the

proportions

were

-LRB-

-RRB-

for

nicotine

-LRB-

-RRB-

for

cotinine

-LRB-

-RRB-

for

and

-LRB-

-RRB-

for

total

nicotine

equivalents

Statistical

correlations

Observed

values

from

the

three

methodologies

were

correlated

with

each

other

Figure

shows

the

three

plots

and

correlations

for

all

data

points

Figure

is

a

graph

of

the

UNE

vs

daily

nicotine

yield

estimated

from

filter

analysis

Fig.

shows

UNE

vs

saliva

cotinine

and

Fig.

shows

daily

nicotine

yield

from

filter

analysis

vs

saliva

cotinine

Linear

regressions

were

significant

for

both

the

slope

and

intercept

-LRB-

p

-RRB-

for

all

three

correlations

The

best

correlation

was

obtained

with

UNE

vs

daily

nicotine

yield

-LRB-

Fig.

-RRB-

The

slope

indicates

that

of

the

variation

in

nicotine

mouth

exposure

calculated

from

filter

analysis

appeared

as

variation

in

the

six

urinary

compounds

measured

The

standard

error

of

the

regression

-LRB-

SER

-RRB-

was

mg

nicotine/day

Saliva

cotinine

did

not

correlate

as

well

with

either

UNE

-LRB-

Fig.

SER

mg/day

-RRB-

or

nicotine

yield

-LRB-

Fig.

SER

mg/day

-RRB-

Because

the

amount

of

nicotine

entering

the

mouth

was

greater

than

the

sum

of

the

UNE

the

standard

error

of

the

nicotine

yield

would

be

greater

than

that

of

the

UNE

due

to

scaling

even

if

the

correlations

were

equivalent

To

allow

an

equal

comparison

the

standard

error

of

the

percent

of

the

difference

between

calculated

-LRB-

using

the

regression

equation

-RRB-

and

measured

values

was

calculated

for

the

three

correlations

The

standard

error

calculated

in

this

manner

was

and

for

UNE

vs

nicotine

yield

and

saliva

cotinine

respectively

and

for

nicotine

yield

vs

saliva

cotinine

Summary

statistics

for

all

correlations

are

in

Table

Fig.

of

the

three

methodologies

-LRB-

individual

measurements

-RRB-

Table

of

the

statistics

for

linear

correlationsCorrelation

-LRB-

y

x

-RRB-

Intercept

-LRB-

CI

-RRB-

Slope

-LRB-

CI

-RRB-

error

-LRB-

-RRB-

Urine

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

mg/day

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Urine

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

mg/day

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Filter

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

mg/day

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Filter

urine

-LRB-

creatinine

normalized

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

mg/day

mg/day/mmol

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Urine

FTCmg/cig

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Filter

FTCmg/cig

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Saliva

FTCng/ml

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Urine

Urinary

nicotine

equivalents

Filter

daily

nicotine

yield

estimated

from

filter

analysis

Saliva

saliva

cotinine

concentration

FTC

FTC

nicotine

yield

CI

confidence

interval

The

means

of

the

five

daily

data

values

per

subject

are

shown

in

Fig.

using

the

same

format

as

Fig.

Again

for

all

correlations

the

slopes

and

intercepts

were

significant

-LRB-

p

-RRB-

The

correlation

for

UNE

vs

nicotine

yield

-LRB-

Fig.

-RRB-

improved

significantly

as

shown

in

Table

The

slope

increased

to

while

the

intercept

moved

closer

to

zero

The

saliva

cotinine

correlations

-LRB-

Fig.

c

-RRB-

also

improved

but

not

as

dramatically

as

the

urinary

nicotine

The

correlation

for

UNE

vs

saliva

cotinine

-LRB-

Fig.

-RRB-

had

an

value

of

with

a

standard

error

of

mg/day

-LRB-

-RRB-

The

correlation

for

nicotine

yield

vs

saliva

cotinine

-LRB-

Fig.

-RRB-

had

an

value

of

with

a

standard

error

of

mg/day

-LRB-

-RRB-

Fig.

of

the

three

methodologies

-LRB-

average

per

subject

-RRB-

Other

findings

Although

the

primary

purpose

of

this

study

was

to

determine

the

statistical

correlations

between

the

three

methods

of

estimating

nicotine

uptake

data

collected

during

the

experiment

allowed

for

additional

observations

Creatinine

normalization

Normalization

by

urinary

creatinine

is

useful

when

only

a

single

or

partial

daily

urine

collection

is

taken

To

test

the

effect

of

creatinine

normalization

observed

daily

UNE

were

divided

by

their

respective

millimole

creatinine

A

correlation

was

calculated

with

nicotine

yield

as

the

dependent

variable

and

UNE/mmol

creatinine

as

the

independent

variable

The

results

are

summarized

in

Table

Both

the

intercept

and

slope

are

significant

-LRB-

p

-RRB-

but

normalization

with

creatinine

clearly

degrades

the

correlation

compared

to

the

correlation

without

normalization

The

findings

suggest

that

normalization

with

creatinine

adds

another

factor

of

variability

which

has

a

degrading

effect

on

and

therefore

indicates

that

the

use

of

urine

samples

without

normalization

is

expected

to

provide

more

accurate

results

than

the

analysis

of

partial

-LRB-

h

-RRB-

urine

samples

with

normalization

This

is

in

agreement

with

Heavner

et

al.

-LRB-

-RRB-

who

showed

mechanistically

that

creatinine

normalization

may

be

appropriate

for

some

but

not

all

of

the

urinary

metabolites

of

nicotine

and

that

other

methods

of

normalization

may

be

more

appropriate

for

spot

urine

samples

FTC

smoke

yields

Because

this

was

a

confined

clinical

study

subjects

would

not

necessarily

be

expected

to

behave

as

if

they

were

in

their

normal

environment

However

because

a

wide

range

of

FTC

cigarette

yields

were

tested

it

was

considered

worthwhile

to

test

the

correlation

between

the

measured

FTC

yields

and

the

three

methods

of

nicotine

estimation

Correlation

results

are

summarized

in

Table

for

the

mean

values

for

the

three

methods

vs

the

FTC

values

on

a

per

subject

basis

All

correlations

gave

significant

slopes

and

intercepts

-LRB-

p

-RRB-

Differences

between

tar

band

groupings

The

results

for

mouth

exposure

to

nicotine

-LRB-

from

filter

analysis

-RRB-

UNE

saliva

cotinine

and

cigarettes

smoked

per

day

were

grouped

by

tar

band

and

the

mean

standard

deviation

and

one-way

analysis

of

variance

-LRB-

ANOVA

-RRB-

were

calculated

on

a

per

subject

basis

-LRB-

Altman

and

Bland

-RRB-

The

results

are

shown

in

Table

Group

means

increased

in

rank

order

with

increasing

group

yield

except

for

saliva

cotinine

and

cigarettes

smoked

per

day

All

measurements

except

cigarettes

smoked

per

day

gave

a

significant

-LRB-

p

-RRB-

effect

of

group

by

ANOVA

For

each

measurement

results

with

different

letter

assignments

are

significantly

different

at

the

confidence

level

-LRB-

Fisher

test

-RRB-

Normalizing

urinary

nicotine

metabolites

with

creatinine

resulted

in

an

increase

in

p

value

for

effect

of

group

and

no

significant

differences

between

the

FF

LTS

and

ULH

groups

All

three

groups

were

significantly

higher

than

the

ULL

group

This

again

shows

that

creatinine

normalization

degrades

the

discriminating

power

Table

SD

for

measurements

grouped

by

tar

band

ULLULHLTSFFANOVA

pNo

of

Mouth

exposure

-LRB-

mg

nicotine/cigarette

-RRB-

c

Mouth

exposure

-LRB-

mg

nicotine/day

-RRB-

c

Urinary

nicotine

-LRB-

mg/cigarette

-RRB-

c

Urinary

nicotine

-LRB-

mg/day

-RRB-

a

c

Creatinine

normalized

urinary

nicotine

-LRB-

mg/day/mmol

-RRB-

Saliva

cotinine

-LRB-

ng/ml

-RRB-

Cigarettes/day

-LRB-

self-reported

minus

measured

-RRB-

Results

with

different

letters

are

significantly

different

at

the

confidence

level

Self-reported

vs

actual

cigarette

usage

Table

also

shows

the

difference

in

self-reported

and

measured

cigarette

usage

Self-reported

cigarette

usage

was

questioned

and

compliance

with

respect

to

truthfulness

can

be

a

concern

-LRB-

Byrd

et

al.

-RRB-

In

this

study

subjects

were

asked

to

estimate

their

cigarette

usage

as

part

of

the

recruiting

process

This

gave

a

basis

to

compare

with

the

measured

usage

during

the

study

-LRB-

overall

mean

cigarettes/day

SD

range

to

-RRB-

The

mean

value

for

the

-LRB-

average

self-reported

-RRB-

usage

per

subject

was

cigarettes/day

-LRB-

SD

range

to

-RRB-

Even

when

broken

down

to

the

smaller

tar

band

groups

of

to

subjects

the

mean

difference

in

measured

and

reported

usage

was

less

than

cigarettes/day

Within-subject

variation

Because

the

trial

took

place

over

five

consecutive

days

within-subject

day-to-day

variation

was

calculated

for

each

of

the

measured

variables

In

addition

on

day

saliva

samples

were

taken

at

approximately

and

hours

for

measurement

of

saliva

cotinine

to

estimate

within-day

variation

Results

are

summarized

in

Table

and

are

expressed

as

a

pooled

-LRB-

root-mean-squared

-RRB-

coefficient

of

variation

The

pooled

day-to-day

variations

for

each

variable

were

similar

ranging

from

for

cigarettes

smoked

per

day

to

for

UNE

per

day

The

within-day

variation

of

for

saliva

cotinine

was

approximately

half

the

day-to-day

variation

of

When

a

single

factor

ANOVA

was

calculated

using

saliva

cotinine

for

the

three

time

periods

sampled

on

day

the

variation

between

time

periods

was

insignificant

-LRB-

p

-RRB-

However

this

could

have

been

overwhelmed

by

the

subject-to-subject

variation

in

nicotine

uptake

This

factor

was

removed

by

dividing

the

individual

measurement

by

the

daily

average

per

subject

for

all

three

time

periods

A

single

factor

ANOVA

was

significant

for

time

of

day

-LRB-

p

-RRB-

with

the

mean

-LRB-

confidence

interval

-RRB-

saliva

cotinine

values

being

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

times

the

average

daily

value

per

subject

for

the

and

hour

samples

respectively

Even

though

the

time

of

day

had

a

statistically

significant

effect

the

practical

differences

were

small

Table

variation

expressed

as

a

pooled

coefficient

of

variation

-LRB-

CV

-RRB-

AnalyteTypePooled

CV

-LRB-

range

-RRB-

Filter

-LRB-

nicotine

yield/day

-RRB-

-LRB-

-RRB-

Urine

-LRB-

nicotine

equiv

day

-RRB-

-LRB-

-RRB-

Saliva

cotinine

-LRB-

-RRB-

Saliva

cotinine

-LRB-

-RRB-

Cigarettes/dayDay-to-day

-LRB-

-RRB-

Discussion

All

three

of

the

estimation

methods

correlated

significantly

with

each

other

but

the

best

overall

correlation

was

between

the

filter

analysis

method

and

UNE

The

slopes

for

the

correlations

of

UNE

as

a

function

of

mouth

exposure

to

nicotine

were

and

for

the

individual

and

average

regressions

respectively

implying

that

about

of

the

difference

in

the

UNE

measured

in

this

study

was

due

to

a

difference

in

mouth

exposure

to

nicotine

However

the

mean

of

the

total

UNE

expressed

as

a

percentage

of

the

nicotine

entering

the

mouth

was

The

mean

value

falls

within

the

reported

range

of

-LRB-

Benowitz

et

al.

-RRB-

to

-LRB-

Curvall

et

al.

-RRB-

but

the

slopes

fall

below

this

range

The

difference

between

the

two

methods

is

due

to

the

significant

intercept

calculated

using

the

linear

regression

The

intercept

using

the

average

results

was

lower

than

the

intercept

using

the

individual

results

and

the

slope

was

greater

The

intercept

could

represent

compartmental

nicotine

and/or

metabolite

storage

with

subsequent

carryover

from

storage

before

entering

the

study

This

influence

should

diminish

with

a

average

compared

to

using

single

day

results

An

example

of

this

is

demonstrated

in

Fig.

where

there

is

a

single

circled

data

point

that

appears

to

be

an

outlier

Urinary

output

of

nicotine

equivalents

was

approximately

two

times

the

mouth

exposure

of

nicotine

for

that

day

However

this

was

a

day

measurement

for

a

single

subject

For

subsequent

days

the

data

points

for

this

subject

are

buried

within

all

the

other

data

points

One

possibility

is

that

the

subject

had

a

much

larger

exposure

to

nicotine

before

participating

in

the

study

and

the

unusually

high

urinary

output

was

due

to

clearance

of

the

prior

exposure

Averaging

the

results

over

days

would

take

out

much

of

the

metabolic

influence

and

result

in

a

much

better

correlation

as

demonstrated

with

these

results

For

the

saliva

cotinine

correlations

the

values

only

improved

slightly

with

averaging

Part

of

the

reason

for

the

improvement

with

all

correlations

can

be

attributed

to

averaging

out

measurement

variation

This

should

be

similar

for

all

correlations

However

the

additional

improvement

in

the

filter

vs

urine

correlation

can

be

explained

by

metabolism

Nicotine

has

a

relatively

short

serum

elimination

half-life

of

h

-LRB-

Scherer

et

al.

Benowitz

and

Jacob

Benowitz

et

al.

-RRB-

which

means

that

the

urinary

nicotine

should

be

from

the

nicotine

taken

in

that

day

Cotinine

has

a

serum

elimination

half-life

typically

reported

at

h

-LRB-

Scherer

et

al.

Benowitz

et

al.

De

Schepper

et

al.

-RRB-

and

a

similar

urine

elimination

half-life

-LRB-

Benowitz

and

Jacob

De

Schepper

et

al.

-RRB-

which

is

downstream

metabolically

from

cotinine

has

a

serum

elimination

half-life

of

h

with

a

similar

urine

elimination

half-life

-LRB-

Scherer

et

al.

Benowitz

and

Jacob

-RRB-

Given

the

pharmacokinetic

information

these

urinary

metabolites

must

have

originated

from

nicotine

exposure

on

multiple

days

as

has

been

demonstrated

directly

using

nicotine

infusion

-LRB-

Scherer

et

al.

-RRB-

Because

measurements

were

taken

over

sequential

days

of

input

-LRB-

filter

analysis

-RRB-

the

temporal

source

of

urinary

and

salivary

metabolites

could

be

estimated

by

multiple

regression

of

the

metabolites

vs

the

daily

nicotine

exposure

This

analysis

was

performed

using

the

nicotine

exposure

for

the

current

day

and

the

two

previous

days

according

to

the

equation

where

-LRB-

N

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

equals

the

nicotine

exposure

for

the

current

day

day

before

and

days

before

the

urine

or

saliva

sample

respectively

Metabolite

measurements

from

days

and

were

used

of

the

coefficients

for

nicotine

exposure

from

days

before

the

urine

collection

were

significant

-LRB-

p

-RRB-

For

saliva

cotinine

the

coefficient

for

days

before

was

not

significant

at

the

confidence

but

the

p

value

of

was

small

enough

to

warrant

consideration

Given

this

the

regressions

were

recalculated

using

-LRB-

N

-RRB-

and

-LRB-

-RRB-

and

including

the

urinary

metabolite

measurements

from

day

as

well

The

results

of

this

regression

are

shown

in

Table

Table

regression

results

for

source

of

urinary

and

salivary

metabolites

as

a

function

of

nicotine

exposure

from

current

and

previous

dayCoefficientSum

of

urinaryUrinary

nicotineUrinary

cotinineUrinary

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Previous

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Current

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Results

expressed

as

regression

coefficient

-LRB-

p

value

-RRB-

For

the

sum

of

all

urinary

metabolites

the

coefficients

were

significant

for

the

current

day

and

prior

day

's

exposure

This

implies

that

the

total

urinary

metabolites

originated

from

nicotine

exposure

over

days

The

only

coefficient

that

was

significant

for

the

urinary

nicotine

regression

was

for

the

current

day

's

exposure

as

would

be

expected

The

coefficient

of

implies

that

of

the

nicotine

entering

the

mouth

appeared

as

urinary

nicotine

plus

the

glucuronide

This

agrees

closely

with

the

estimates

of

-LRB-

Benowitz

et

al.

-RRB-

and

-LRB-

Curvall

et

al.

-RRB-

For

the

urinary

cotinine

regression

coefficients

for

the

current

day

and

previous

day

were

significant

with

the

current

day

being

about

twice

that

of

the

previous

day

The

two

coefficients

imply

that

of

the

nicotine

entering

the

mouth

appears

as

urinary

cotinine

plus

the

glucuronide

This

also

agrees

closely

with

estimates

of

-LRB-

Benowitz

et

al.

-RRB-

and

-LRB-

Curvall

et

al.

-RRB-

For

the

regression

the

coefficients

for

the

current

day

and

previous

day

were

significant

with

the

current

day

being

slightly

smaller

than

the

previous

day

The

sum

of

the

coefficients

-LRB-

-RRB-

was

smaller

than

the

reported

to

of

the

total

urinary

metabolites

-LRB-

Benowitz

et

al.

Curvall

et

al.

-RRB-

It

is

possible

that

because

is

the

third

step

in

the

metabolism

of

nicotine

there

was

too

much

of

a

smoothing

effect

for

the

change

in

daily

mouth

exposure

to

fully

capture

For

saliva

cotinine

all

coefficients

were

significant

suggesting

that

saliva

cotinine

results

are

an

amalgam

of

at

least

days

of

nicotine

exposure

Saliva

cotinine

correlations

suffer

because

results

are

expressed

as

concentration

rather

than

an

absolute

value

and

as

such

can

be

influenced

by

body

size

In

addition

other

variables

come

into

play

that

are

not

easily

explained

An

example

of

this

is

shown

in

Figs.

and

by

the

data

points

with

boxes

around

them

These

were

from

one

subject

that

appeared

to

have

unusually

low

saliva

cotinine

values

for

both

UNE

-LRB-

Figs.

and

-RRB-

and

mouth

exposure

of

nicotine

-LRB-

Figs.

and

-RRB-

This

subject

had

the

highest

mouth

exposure

of

nicotine

of

all

the

subjects

studied

yet

the

saliva

cotinine

values

were

only

slightly

above

midrange

There

was

nothing

unusual

about

this

subject

and

average

urinary

output

was

within

SD

-LRB-

ml

-RRB-

of

the

average

for

all

subjects

-LRB-

ml

-RRB-

It

is

unlikely

that

nicotine

yields

were

overestimated

because

Fig.

shows

that

the

data

points

are

scattered

about

the

regression

line

for

UNE

vs

nicotine

yield

It

is

also

unlikely

that

the

saliva

cotinine

concentrations

were

in

error

because

the

saliva

cotinine

for

each

day

was

analyzed

in

separate

batches

along

with

other

samples

The

subject

did

not

rapidly

metabolize

cotinine

to

because

cotinine

accounted

for

of

the

urinary

metabolites

measured

for

this

subject

compared

to

an

average

of

for

all

subjects

indicating

the

converse

Therefore

it

must

be

concluded

that

this

is

an

anomaly

characteristic

of

this

individual

As

shown

in

Table

with

many

of

the

estimation

methods

the

correlations

are

only

slightly

better

than

simply

correlating

with

FTC

nicotine

yield

of

the

cigarette

The

average

nicotine

mouth

exposure

as

measured

by

the

filter

method

correlated

with

the

FTC

nicotine

yield

with

a

standard

error

of

and

an

value

of

The

correlation

of

nicotine

mouth

exposure

with

saliva

cotinine

gave

a

standard

error

of

and

creatinine-normalized

urinary

metabolites

gave

a

standard

error

of

with

values

approximately

higher

Correlations

of

FTC

nicotine

with

biomarkers

in

this

study

were

stronger

than

that

reported

in

other

studies

-LRB-

Byrd

et

al.

Jarvis

et

al.

Ueda

et

al.

Hecht

et

al.

Bernert

et

al.

-RRB-

We

believe

that

there

are

valid

reasons

for

this

One

is

that

the

correlations

were

performed

using

the

averages

per

subject

instead

of

a

single

sample

per

subject

In

addition

exact

compliance

to

brand

and

cigarette

consumption

was

assured

in

the

current

study

whereas

all

but

one

-LRB-

Bernert

et

al.

-RRB-

of

the

referenced

studies

used

self-reported

brand

identification

and

cigarette

consumption

Self-reported

brand

information

was

reported

to

have

about

a

error

rate

when

compared

with

packs

returned

-LRB-

Peach

et

al.

-RRB-

or

in

a

test

retest

comparison

-LRB-

Eisenhower

et

al.

-RRB-

This

and

the

potential

for

use

of

alternate

brands

during

a

study

can

further

confound

a

correlation

with

biomarkers

Other

confounding

factors

are

the

use

of

creatinine

normalized

spot

urine

samples

instead

of

urine

samples

and

analyzing

for

a

subset

of

the

nicotine

metabolites

used

in

the

current

study

Simple

creatinine

normalization

is

only

biologically

valid

for

xenobiotics

that

have

the

same

excretion

mechanism

and

urinary

flow

rate

dependence

as

creatinine

Heavner

et

al.

-LRB-

-RRB-

have

shown

that

-LRB-

free

and

glucuronide

-RRB-

and

cotinine

glucuronide

have

urinary

flow

rate

dependence

similar

to

creatinine

while

nicotine

-LRB-

free

and

glucuronide

-RRB-

free

cotinine

and

the

free

and

glucuronide

forms

of

-LRB-

methylnitrosamino

-RRB-

-LRB-

-RRB-

do

not

In

conclusion

two

methods

stand

out

as

superior

One

is

the

filter

method

which

estimates

mouth

level

exposure

directly

on

a

per

cigarette

basis

Filter

collection

need

not

be

quantitative

the

filter

returned

can

be

compared

to

the

brand

it

is

supposed

to

be

and

it

can

be

readily

determined

if

it

was

smoked

or

not

Thus

brand

compliance

and

smoking

status

can

be

assured

even

if

the

subject

happened

to

occasionally

use

a

different

brand

or

other

form

of

nicotine

during

the

study

With

self-reported

daily

cigarette

use

from

groups

of

at

least

subjects

the

exposure

per

cigarette

can

be

converted

accurately

to

daily

exposure

In

addition

mouth

exposure

to

tar

can

also

be

estimated

using

the

filter

method

-LRB-

Shepperd

et

al.

-RRB-

The

other

measurement

which

is

considered

by

many

to

be

the

gold

standard

is

the

measurement

of

urinary

nicotine

and

metabolites

from

urine

samples

without

creatinine

normalization

This

method

appears

to

reflect

the

mean

daily

nicotine

uptake

of

the

last

days

O O O B O O O O O B O O O O O O O O O B B O B 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 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 B 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 B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O 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 B 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 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 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 I 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 B I 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 B I 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 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 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 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 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 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 B O O O O O O O O O O O O O O O O B 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 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 O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 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 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 B 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 B 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 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 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 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 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 B O O O O O O O B 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 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 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 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 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 B O O B 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 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 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 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 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 B O O B O B O O O B O O B 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 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 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 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 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 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 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 B 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 O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B O O O O O O O O B O B 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 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 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 O O O O O O O O O O O O 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 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 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 O O O O O O O O O O O O O O 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 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 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 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 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 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 B 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 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 B 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 B O O O O O O O O O B 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 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 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 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 B 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Modification of neuropathic pain sensation through microglial ATP receptors Neuropathic pain that typically develops when peripheral nerves are damaged through surgery , bone compression in cancer , diabetes , or infection is a major factor causing impaired quality of life in millions of people worldwide . Recently , there has been a rapidly growing body of evidence indicating that spinal glia play a critical role in the pathogenesis of neuropathic pain . Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors . Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia through P2 purinoceptors expressing on microglia . It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion . In this review , we describe the modification of neuropathic pain sensation through microglial P2X4 and P2X7 , with the possibility of P2Y6 and P2Y12 involvement . Introduction There is a type of pain that does not go away even though the tissue has already healed . One type of this pain is called neuropathic pain that typically develops when peripheral nerves are damaged such as through surgery , bone compression in cancer , diabetes , or infection . Neuropathic pain is a major factor causing impaired quality of life in millions of people worldwide and is frequently resistant to all known analgesic drugs . Over the last decade , accumulating evidence concerning how peripheral nerve injury creates neuropathic pain has suggested that nerve injury produces molecular and cellular alterations that result in multiple forms of neuronal plasticity and anatomical reorganization in the dorsal horn of the spinal cord . These alterations have been proposed to be crucial in the pathogenesis of neuropathic pain -LSB- 1 , 2 -RSB- . While the dominant theme in research on neuropathic pain has been to understand the roles of neurons in the peripheral nervous system and the dorsal horn , there is a rapidly growing body of evidence indicating that spinal glial cells play a critical role in the pathogenesis of neuropathic pain . Recently , growing evidence has indicated that neuron-glia interaction is a key idea to understand functions of the central nervous system -LRB- CNS -RRB- . Especially glia play important roles in pathophysiological situations of the CNS including psychiatric disorders , physical trauma , and infections -LSB- 3 -RSB- . Glia consist of three members : astrocytes , oligodendrocytes , and microglia . Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors , even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells . Microglia , which are thought to be residential macrophages in the CNS , express P2 purinoceptors , mainly P2X4 and P2X7 as well as P2Y2 , P2Y6 , and P2Y12 -LSB- 4 -RSB- . Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia -LSB- 5 , 6 -RSB- . It is clear that these nucleotides trigger the release of various neurotoxic and neuroprotective cytokines and growth factors via different purine receptors -LSB- 4 , 7 -RSB- or induce P2Y12-dependent chemotaxis in cultured cells -LSB- 8 , 9 -RSB- . It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion -LSB- 10 -- 12 -RSB- . Other purine receptors are upregulated in microglia in response to neuronal injury , as was recently demonstrated for P2Y6 -LSB- 6 -RSB- . The P2Y6 receptor triggers microglial phagocytosis -LSB- 6 -RSB- . In this paper , we review the modification of pain sensation through microglial P2X4 and P2X7 , and also point out the possibility of P2Y6 and P2Y12 involvement in pain signaling . P2X4 in neuropathic pain We found that the marked tactile allodynia that develops following nerve injury was reversed by acutely administering TNP-ATP intrathecally but was unaffected by administering PPADS -LSB- 10 -RSB- . From the pharmacological profiles of TNP-ATP -LRB- blocking P2X4 at high concentration -RRB- and PPADS -LRB- not blocking P2X4 -RRB- , it was suggested that tactile allodynia depends upon P2X4 in the spinal cord . The expression of P2X4 protein , normally low in the naïve spinal cord , progressively increased in the days following nerve injury with a time course parallel to that of the development of tactile allodynia . Double immunolabeling analysis demonstrated that not neurons or astrocytes but activated microglia in the dorsal horn were intensely positive for P2X4 protein -LSB- 10 -RSB- . Moreover , intrathecally administered antisense oligodeoxynucleotide for P2X4R reduced the expression of P2X4 protein in spinal microglia and prevented the development of the nerve injury-induced tactile allodynia . In naïve rats , intrathecal administration of cultured microglia that were preincubated with ATP to activate P2X4 produced tactile allodynia over the 3 -- 5 h after the administration -LSB- 10 -RSB- . Since it was already reported that the nerve injury-induced tactile allodynia depends on a depolarizing shift in the Eanion of spinal lamina I -LRB- LI -RRB- neurons in the dorsal spinal cord , resulting in converting the GABAA receptor - and glycine receptor-mediated inhibition to excitation -LSB- 13 -RSB- , it was considered that microglia may affect Eanion in LI neurons . To investigate this possibility , microglia were administered to the lumbar spinal level of naïve rats by an intrathecal catheter as described -LSB- 11 -RSB- . Administering microglia stimulated with ATP caused a progressive tactile allodynia over the 5 h after injection . Eanion in LI neurons from rats administered ATP-stimulated microglia was shifted to − 61.6 mV from − 68.3 mV of normal rats . In addition , we found that GABA response switched from hyperpolarizing in control rats to depolarizing in microglia-treated rats . Activated microglia secrete various biologically active molecules , one of which , BDNF , was implicated in the hypersensitivity of dorsal horn neurons that follows sensitization and inflammation -LSB- 14 -- 16 -RSB- and in anion gradient shifts in the hippocampus -LSB- 17 -RSB- . Indeed , intrathecal administration of recombinant BDNF produced tactile allodynia comparable to that produced by ATP-stimulated microglia -LSB- 11 -RSB- . Eanion of LI neurons in slices treated with BDNF -LRB- > 90 min , in vitro -RRB- was significantly less negative than that of LI neurons from control slices . The rise in -LSB- Ca2 + -RSB- i was prevented by the GABAA receptor blocker bicuculline , confirming that the effect was mediated by GABAA receptors . Thus , acute administration of BDNF in slices caused a depolarizing shift in Eanion and caused GABA to produce net excitation -LSB- 11 -RSB- . Moreover , a function-blocking antibody against the TrkB receptor -LRB- anti-TrkB -RRB- and a BDNF-sequestering fusion protein -LRB- TrkB-Fc -RRB- acutely inhibited the allodynia and the shift of Eanion of LI neurons -LSB- 11 -RSB- . The administration of ATP-stimulated microglia with either anti-TrkB or TrkB-Fc did not develop tactile allodynia . After pretreatment of microglia with double-stranded short interfering RNA directed against BDNF -LRB- BDNF siRNA -RRB- , the ATP-stimulated microglia injected intrathecally into normal rats did not cause the allodynia -LSB- 11 -RSB- . Anti-TrkB and BDNF siRNA prevented the shift in Eanion induced by ATP-stimulated microglia . ATP stimulation caused release of BDNF from microglia in culture . This effect of ATP was blocked by treating the cultures with the P2X receptor blocker TNP-ATP . In addition , pretreatment of the microglia with BDNF siRNA prevented release of BDNF by ATP stimulation . By bath application of TNP-ATP to spinal slices taken from allodynic rats 2 weeks after nerve injury , Eanion of LI neurons was returned to normal value -LSB- 11 -RSB- . These findings indicate that P2X4-dependent release of BDNF from microglia is necessary to sustain both the tactile allodynia and the depolarizing shift in Eanion in LI neurons that result from nerve injury -LRB- Fig. 1 -RRB- . Fig. 1Schematic illustration of potential mechanisms by which P2X/Y receptors in activated microglia modulate neuropathic pain signaling in the dorsal horn . Activated microglia in the spinal cord after nerve injury express ionotropic ATP receptors -LSB- e.g. , P2X4 receptor -LRB- P2X4R -RRB- or P2X7R -RSB- . P2X4R or P2X7R activation leads to the release of bioactive diffusible factors such as BDNF and other proinflammatory factors -LRB- cytokines and chemokines -RRB- . BDNF causes a collapse of transmembrane anion gradient in dorsal horn lamina I neurons presumably through the downregulation of KCC2 , which in turn renders GABA and glycine effects depolarizing , rather than hyperpolarizing , in these neurons . Microglial factors may also interact with excitatory synapses of neighboring dorsal horn neurons and enhance the excitability in dorsal horn neurons . The net hyperexcitability in the dorsal horn pain network by these factors from activated microglia may be responsible for neuropathic pain . Microglia also express G protein-coupled ATP receptors -LSB- e.g. , P2Y6 receptor -LRB- P2Y6R -RRB- and P2Y12R -RSB- . Activating P2Y12R and P2Y6R leads to chemotaxis and phagocytosis , respectively , but their functional relevance to neuropathic pain remained to be determined P2X7 in neuropathic pain P2X7 purinoceptors are a ligand-gated nonselective cationic channel and are expressed predominantly on immune cells -LSB- 18 -RSB- . Stimulation of the P2X7 receptors on microglia is associated with release of cytokines including tumor necrosis factor-α -LRB- TNF-α -RRB- -LSB- 5 , 19 -RSB- , interleukin-6 -LRB- IL-6 -RRB- -LSB- 20 -RSB- , and interleukin-1β -LRB- IL-1β -RRB- -LSB- 21 -- 23 -RSB- . Several cytokines such as IL-1β , IL-6 , and TNF-α in the dorsal horn are increased after nerve lesion -LSB- 24 -- 26 -RSB- and have been implicated in contributing to neuropathic pain -LSB- 24 -- 27 -RSB- . Recent evidence indicates the relationship between TNF-α and neuropathic pain -LSB- 28 -- 32 -RSB- , and TNF-α released after injury is proposed as an initiator of abnormal pain sensation . TNF-α is upregulated after nerve injury in both dorsal root ganglion -LRB- DRG -RRB- and spinal cord -LSB- 33 -RSB- . The inhibition of TNF-α reduces the hyperalgesia in neuropathic pain models -LSB- 34 -RSB- . Recent evidence indicates the relationship between inflammatory cytokines including IL-1β and neuropathic pain -LSB- 25 , 26 , 28 -RSB- . The expression of IL-1β is upregulated in the spinal cord of several rat neuropathy models -LSB- 24 -- 26 -RSB- . These findings further support a role for central IL-1β in the development and maintenance of neuropathic pain through induction of a proinflammatory cytokine cascade -LRB- Fig. 1 -RRB- . Recently , it was reported that in mice lacking P2X7 inflammatory and neuropathic hypersensitivity is completely absent to both mechanical and thermal stimuli , whilst normal nociceptive processing is preserved -LSB- 12 -RSB- . Contribution of P2X7 receptor to neuropathic pain is also demonstrated by using the recently developed selective antagonist for P2X7 receptor A-740003 -LSB- 35 -RSB- . The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1β , and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggested a selective effect of the gene deletion on release of IL-1β and IL-10 , with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1 . In addition , P2X7 receptors were upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients -LSB- 12 -RSB- . It was hypothesized that the P2X7 receptor plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin via regulation of mature IL-1β production . In addition , a recent study has shown that activation of P2X7 receptors expressed on satellite glia that enwrap each DRG neuron leads to a release of TNF-α , which in turn increases the excitability of DRG neurons -LSB- 36 -RSB- . P2X7 receptors are upregulated in surrounding satellite glial cells in the DRG in humans -LSB- 12 -RSB- . Implication of TNF-α has been reported in neuropathic pain -LSB- 28 -- 32 -RSB- . Therefore , the contribution of P2X7 receptor to neuropathic pain might be related not only to the activation of microglial P2X7 receptors , but also to P2X7 receptors on other cell types such as satellite glia . P2Y6 and P2Y12 in neuropathic pain ? When neurons are injured or dead , microglia are activated , resulting in their interaction with immune cells , active migration to the site of injury , release of proinflammatory substances , and the phagocytosis of damaged cells or debris . For such activation of microglial motilities , extracellular nucleotides have a central role . Extracellular ATP functions as a chemoattractant . Microglial chemotaxis by ATP via P2Y12 receptors was originally found by Honda et al. -LSB- 8 -RSB- and has recently been confirmed in vivo in P2Y12 receptor knockout animals -LSB- 37 -RSB- . Neuronal injury results in the release or leakage of ATP that appears to be a `` find-me '' signal from damaged neurons to microglia to cause chemotaxis . In addition to microglial migration by ATP , another nucleotide , UDP , an endogenous agonist of the P2Y6 receptor , greatly activates the motility of microglia and orders microglia to eat damaged neurons . UDP does not cause chemotaxis , but instead causes phagocytosis by microglia -LSB- 6 -RSB- . Phagocytosis , a specialized form of endocytosis , is the uptake by the cell of relatively large particles -LRB- > 1.0 μm -RRB- into vacuoles and has a central role in tissue remodeling , inflammation , and the defense against infectious agents -LSB- 38 -RSB- . Phagocytosis is initiated by the activation of various cell surface phagocytosis receptors , including Fc receptors , complement receptors , integrins , endotoxin receptors -LRB- CD18 , CD14 -RRB- , mannose receptors , and scavenger receptors -LSB- 39 -RSB- , which are activated by corresponding extracellular ligands . In the CNS , a full innate immune system , i.e. , Fc receptors , complement system , scavenger receptors , and Toll-like receptors etc. , has been described , and microglia reveal related roles as dedicated phagocytes . Since recognition is the first and the most important step for phagocytosis , extensive studies on phagocytosis receptors have been reported . It is well-known that dying cells express so-called eat-me signals such as phosphatidylserine on their surface membrane -LSB- 39 -RSB- , by which microglia recognize the apoptotic cells to catch and remove them -LSB- 39 -RSB- . We first found that exogenously applied UDP caused microglial phagocytosis in a concentration-dependent manner , which was P2Y6 receptor-dependent . We found that neuronal injury caused by kainic acid -LRB- KA -RRB- upregulated P2Y6 receptors in microglia , the KA-evoked neuronal injury resulted in an increase in extracellular UTP , which was immediately metabolized into UDP in vivo and in vitro . Moreover , UDP leaked from injured neurons caused P2Y6 receptor-dependent phagocytosis in vivo and in vitro . These results suggest that UDP could be a molecule that signals the crisis of damaged neurons to microglia , triggering phagocytosis . It should be noted that nucleotides could be both `` find-me '' and `` eat-me '' signals . Cells release ATP , and we also found that KA caused an increase in extracellular UTP/UDP . Therefore microglia might be attracted by ATP/ADP -LSB- 8 , 40 , 41 -RSB- and subsequently recognize UDP , leading to the removal of the dying cells and their debris . Are P2Y6 and P2Y12 receptors involved in neuropathic pain ? For this question , there is a report indicating that the UDP-sensitive P2Y6 receptor produces inhibitory effects on spinal pain transmission in a neuropathic pain model -LSB- 42 -RSB- . In the neuropathic pain model , in which the sciatic nerves of rats were partially ligated , UDP -LRB- 30 and 100 nmol/rat -RRB- produced significant antiallodynic effects . UDP -LRB- 100 nmol/rat -RRB- caused no motor deficit in the inclined plane test -LSB- 42 -RSB- . The mechanism of this UDP-evoked inhibitory effect on neuropathic pain is unknown . There is no evidence to show the site -LRB- neuron or glia -RRB- of action of UDP . We are now investigating the involvement of P2Y12 in pain signaling and have obtained evidence suggesting the role of P2Y12 in the neuropathic pain state . Conclusion Neuropathic pain is a major factor causing impaired quality of life in millions of people worldwide . We have to try to reveal the mechanism of this pain in order to develop effective drugs against the pain . Recently , there has been a rapidly growing body of evidence indicating that spinal glia , especially microglia , play a critical role in the pathogenesis of neuropathic pain and that nucleotides play an important role in neuron-glia communication through P2 purinoceptors . We described the modification of neuropathic pain signaling through microglial P2X4 and P2X7 , with the possibility of P2Y6 and P2Y12 involvement -LRB- Fig. 1 -RRB- . It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion . Microglial P2Y6 and P2Y12 play very interesting roles for phagocytosis and chemotaxis , respectively . Besides , there is a possibility that these receptors are involved in pain sensation . Additional experiments are needed to clarify the possibility .

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

allodynia
atp
microglia
neuropathic pain
p2x4
spinal cord