Glycation

does

not

modify

bovine

serum

albumin

-LRB-

BSA

-RRB-

induced

reduction

of

rat

aortic

relaxation

The

response

to

glycated

and

nonglycated

BSA

is

lost

in

metabolic

syndrome

The

effects

of

nonglycated

bovine

serum

albumin

-LRB-

BSA

-RRB-

and

advanced

glycosylation

end

products

of

BSA

-LRB-

AGE-BSA

-RRB-

on

vascular

responses

of

control

and

metabolic

syndrome

-LRB-

MS

-RRB-

rats

characterized

by

hypertriglyceridemia

hypertension

hyperinsulinemia

and

insulin

resistance

were

studied

Albumin

and

in

vitro

prepared

AGE-BSA

have

vascular

effects

however

recent

studies

indicate

that

some

effects

of

in

vitro

prepared

AGEs

are

due

to

the

conditions

in

which

they

were

generated

We

produced

AGEs

by

incubating

glucose

with

BSA

for

days

under

sterile

conditions

in

darkness

and

at

°C

To

develop

MS

rats

male

Wistar

animals

were

given

sucrose

in

drinking

water

since

weanling

Six

month

old

animals

were

used

Blood

pressure

insulin

triglycerides

and

serum

albumin

were

increased

in

MS

rats

Contraction

of

aortic

rings

elicited

with

norepinephrine

was

stronger

There

were

no

effects

of

nonglycated

BSA

or

AGE-BSA

on

contractions

in

control

or

MS

rats

however

both

groups

responded

to

L-NAME

an

inhibitor

of

nitric

oxide

synthesis

Arterial

relaxation

induced

using

acetylcholine

was

smaller

in

MS

rats

Nonglycated

BSA

and

AGE-BSA

significantly

diminished

relaxation

in

a

in

the

control

group

but

the

decrease

was

similar

when

using

nonglycated

BSA

and

AGE-BSA

This

decrease

was

not

present

in

the

MS

rats

and

was

not

due

to

increased

RAGEs

or

altered

biochemical

characteristics

of

BSA

In

conclusion

both

BSA

and

AGE-BSA

inhibit

vascular

relaxation

in

control

artic

rings

In

MS

rats

the

effect

is

lost

possibly

due

to

alterations

in

endothelial

cells

that

are

a

consequence

of

the

illness

Introduction

Reducing

sugars

including

glucose

fructose

trioses

and

glyceraldehyde

alter

proteins

by

nonenzymatic

glycosylation

known

as

glycation

or

Maillard

reaction

This

reaction

occurs

in

several

steps

the

initial

ones

being

relatively

fast

and

reversible

while

the

latter

ones

are

slower

and

irreversible

originating

products

known

as

advanced

glycosylation

end

products

-LRB-

AGEs

-RRB-

AGEs

were

originally

characterized

by

their

yellow-brown

fluorescent

color

and

by

their

ability

to

cross-link

with

and

between

amino

groups

-LRB-

Reynolds

-RRB-

However

the

term

is

now

used

for

a

broad

range

of

advanced

products

of

the

Maillard

reaction

including

N-carboxymethyllysine

-LRB-

CML

-RRB-

and

pyrroline

-LRB-

Reddy

et

al.

Baynes

and

Thorpe

-RRB-

AGEs

exert

their

actions

by

two

different

mechanisms

-LRB-

-RRB-

modifying

structural

intra

and

extracellular

proteins

and

-LRB-

-RRB-

binding

to

their

receptors

-LRB-

RAGEs

-RRB-

that

belong

to

the

immunoglobulin

family

and

are

located

in

the

plasma

membranes

of

monocytes

macrophages

endothelial

cells

and

vascular

smooth

muscle

cells

-LRB-

Brownlee

Basta

et

al.

-RRB-

When

AGEs

bind

to

their

receptors

they

initiate

second

messenger

cascades

They

also

generate

reactive

oxygen

species

which

modulate

cellular

function

and

can

induce

inflammatory

processes

-LRB-

Krieglstein

and

Granger

Basta

et

al.

Ramasamy

et

al.

-RRB-

Albumin

is

one

of

the

main

proteins

undergoing

glycation

reactions

due

to

its

abundance

in

serum

and

to

the

fact

that

it

can

be

glycated

at

multiple

sites

-LRB-

Wautier

and

Guillausseau

-RRB-

Both

bovine

serum

albumin

-LRB-

BSA

-RRB-

and

AGE-BSA

have

been

reported

to

have

vascular

effects

BSA

reacts

with

nitric

oxide

-LRB-

NO

-RRB-

therefore

modulating

its

biological

actions

and

increasing

endothelial

permeability

-LRB-

Predescu

et

al.

-RRB-

Some

forms

of

AGEs

also

diminish

vascular

relaxation

by

decreasing

the

NO

production

of

endothelial

cells

-LRB-

Hogan

et

al.

Xu

et

al.

-RRB-

Nevertheless

there

have

been

recent

reports

that

other

forms

of

in

vitro

prepared

AGEs

have

different

effects

from

the

naturally

produced

AGEs

-LRB-

Valencia

et

al.

-RRB-

AGEs

have

been

implicated

in

aging

processes

and

in

the

development

of

chronic

complications

of

diabetes

such

as

renal

and

vascular

damage

and

could

also

participate

in

complications

of

metabolic

syndrome

-LRB-

Brownlee

Wautier

and

Guillausseau

Valencia

et

al.

-RRB-

There

is

a

significant

association

between

a

high

incidence

of

cardiovascular

events

and

endothelial

damage

in

animal

models

and

diabetic

patients

and

one

of

the

mechanisms

underlying

this

association

is

the

presence

of

AGEs

in

plasma

-LRB-

Tan

et

al.

-RRB-

A

variant

of

fructose-induced

hypertension

by

giving

sucrose

to

rats

has

been

developed

in

our

institution

The

fructose-fed

rat

becomes

hypertensive

hypertriglyceridemic

hyperinsulinemic

and

has

insulin

resistance

it

exhibits

what

is

known

as

metabolic

syndrome

-LRB-

MS

-RRB-

or

Syndrome

X

-LRB-

Hwang

et

al.

-RRB-

Some

of

the

characteristics

of

the

model

developed

in

our

institution

have

already

been

described

-LRB-

Baños

et

al.

-RRB-

and

they

coincide

with

those

of

fructose

fed

animals

i.e.

our

rats

also

develop

hypertension

hypertriglyceridemia

hyperinsulinemia

and

insulin

resistance

They

also

show

increased

oxidative

stress

-LRB-

Baños

et

al.

-RRB-

Some

of

the

characteristics

of

these

animals

are

accentuated

by

age

and

have

been

associated

with

renal

failure

and

cardiovascular

diseases

which

are

related

to

alterations

in

the

structure

and

function

of

endothelial

cells

-LRB-

Rubio

et

al.

-RRB-

AGEs

could

play

a

role

in

the

vascular

damage

found

in

MS

rats

Therefore

the

aim

of

the

present

paper

was

to

determine

the

effect

of

nonglycated

BSA

and

AGE-BSA

on

aortic

rings

from

control

and

MS

rats

Results

Changes

in

body

weight

arterial

pressure

triglycerides

glucose

insulin

and

serum

albumin

Table

I

shows

the

values

of

body

weight

arterial

pressure

glucose

insulin

homeostasis

model

assessment

-LRB-

HOMA

-RRB-

triglycerides

and

serum

albumin

for

control

and

MS

rats

Experimental

animals

developed

metabolic

syndrome

characterized

by

arterial

hypertension

hypertriglyceridemia

hyperinsulinemia

and

insulin

resistance

There

was

a

statistically

significant

increase

in

the

serum

albumin

values

in

MS

rats

Although

there

was

no

significant

change

in

body

weight

the

animals

with

MS

clearly

developed

central

adiposity

Table

I

Clinical

characteristics

and

biochemical

parameters

from

control

and

MS

rats

Control

SM

Body

weight

-LRB-

g

-RRB-

Arterial

pressure

-LRB-

mm

of

Hg

-RRB-

Triglycerides

-LRB-

mg/dL

-RRB-

Glucose

-LRB-

mmol/L

-RRB-

Insulin

-LRB-

μU/mL

-RRB-

HOMA

Albumin

-LRB-

g/dL

-RRB-

Values

are

mean

SEM

n

P

AGE-BSA

preparation

It

was

possible

to

produce

AGE-BSA

under

the

previously

described

condition

After

incubation

with

glucose

the

albumin

solution

took

a

yellow-brown

color

and

showed

the

characteristic

fluorescence

spectrum

of

AGEs

Albumin

incubated

without

glucose

did

not

significantly

change

its

appearance

and

did

not

have

the

characteristic

fluorescence

spectrum

of

AGEs

-LRB-

Figure

-RRB-

Fig.

Fluorescence

spectrum

of

AGE-BSA

and

nonglycated

BSA

Both

solutions

were

tested

at

mg/mL

of

protein

The

peak

of

fluorescence

is

not

present

in

the

nonglycated

BSA

The

interrupted

line

represents

the

saturation

level

of

the

fluorescence

detection

by

the

equipment

employed

The

purification

of

AGE-BSA

on

Affi-Gel-Blue

is

shown

in

Figure

three

peaks

were

observed

which

corresponded

to

the

highly

glycated

BSA

-LRB-

first

one

-RRB-

the

less

glycated

BSA

moderately

and

stronger

bound

to

the

chromatographic

matrix

-LRB-

second

and

third

peaks

respectively

-RRB-

The

first

peak

was

employed

for

subsequent

analysis

and

tested

for

vasoreactivity

Fig.

Purification

of

AGE-BSA

on

Affi-Gel-Blue

Three

peaks

were

observed

the

first

one

corresponds

to

highly

glycated

BSA

which

did

not

bind

to

the

matrix

the

second

one

corresponds

to

the

less

glycated

BSA

which

bound

moderately

and

the

third

peak

corresponds

to

the

stronger

bound

BSA

The

first

peak

was

the

only

one

to

be

further

analyzed

The

arrows

indicate

the

buffer

used

at

each

stage

The

AGE-BSA

produced

had

a

protein

concentration

of

mg/mL

and

a

specific

activity

before

its

purification

in

Affi-Gel

Blue

of

AU/mg

of

protein

and

of

AU/mg

of

protein

after

purification

Fluorescence

was

not

significantly

elevated

when

protein

concentration

of

nonglycated

BSA

was

increased

-LRB-

Figure

-RRB-

The

value

of

specific

activity

was

similar

to

that

obtained

by

other

authors

using

similar

procedures

-LRB-

Verbeke

et

al.

-RRB-

Fig.

Evaluation

of

the

specific

activity

-LRB-

SA

-RRB-

of

AGE-BSA

The

increase

of

specific

activity

obtained

by

the

purification

process

on

Affi-Gel-Blue

is

observed

Bold

circles

represent

the

nonglycated

BSA

Bold

squares

represent

the

unpurified

AGE-BSA

and

bold

triangles

represent

the

purified

AGE-BSA

Specific

activity

increased

in

approximately

by

the

purification

process

Changes

in

mass

and

isoelectric

point

of

BSA

and

glycated

BSA

Table

II

shows

the

results

of

the

analysis

of

the

main

glycation

sites

of

the

BSA

structure

The

analysis

was

done

using

the

sequence

of

bovine

serum

albumin

reported

in

the

GenBank

with

number

-LRB-

gi

-RRB-

and

the

PyMOL

program

-LRB-

DeLano

Scientific

LLC

CA

-RRB-

The

disposition

of

the

lysine

and

arginine

residues

in

the

three-dimensional

structure

was

evaluated

The

analysis

shows

that

a

maximum

of

residues

from

the

present

in

the

molecule

can

be

glycated

The

rest

of

the

residues

are

in

the

inside

of

the

structure

of

BSA

Table

II

Distribution

of

the

amino

acids

lysine

and

arginine

in

the

three-dimensional

structure

of

BSA

Amino

acid

position

in

the

three-dimensional

structure

Amino

acid

Totally

exposed

Partially

exposed

Nonexposed

Lysine

261,273,275,285,312,316,322,350,362,375,377,388,396

Arginine

Total

The

amino

acids

localized

in

depression

areas

are

considered

as

partially

exposed

Figure

shows

a

molecular

mass

of

Da

for

nonglycated

BSA

-LRB-

Figure

-RRB-

After

glycation

the

molecular

mass

increased

to

Da

-LRB-

Figure

-RRB-

The

net

increase

of

Da

observed

corresponds

to

the

addition

of

glucose

molecules

considering

that

the

molecular

mass

of

glucose

is

Da

and

that

during

each

reaction

of

Amadori

product

a

water

molecule

is

liberated

-LRB-

Da

-RRB-

The

amount

of

glucose

bound

to

BSA

in

our

preparation

was

very

close

to

the

theoretically

possible

therefore

we

consider

that

our

AGE-BSA

is

a

highly

glycated

product

Fig.

Analysis

of

BSA

and

AGE-BSA

by

mass

spectrometry

The

mass

increase

of

protein

after

glycation

is

observed

The

mass

of

nonglycated

BSA

was

Da

-LRB-

A

-RRB-

and

that

of

AGE-BSA

was

Da

-LRB-

B

-RRB-

The

net

increase

of

mass

was

Da

corresponding

to

the

addition

of

molecules

of

glucose

to

each

BSA

molecule

Inset

change

of

isoelectric

point

of

BSA

after

glycation

Nonglycated

BSA

had

a

pI

of

while

AGE-BSA

had

a

pI

of

Line

is

the

broad

range

pI

standard

kit

-LRB-

pH

-RRB-

Nonglycated

BSA

had

an

isoelectric

point

of

The

glycation

process

changed

the

isoelectric

point

to

-LRB-

Figure

inset

-RRB-

Vascular

responses

Norepinephrine-induced

vasoconstriction

was

higher

in

MS

rats

than

in

control

rats

-LRB-

Table

III

-RRB-

nevertheless

vasoconstriction

was

not

significantly

modified

in

aortic

rings

from

control

or

MS

rats

in

the

presence

of

nonglycated

BSA

or

AGE-BSA

L-NAME

significantly

increased

vascular

contraction

in

both

groups

-LRB-

versus

respectively

-RRB-

since

inhibition

of

NOS

induces

an

imbalance

in

vasoconstriction

and

vasodilation

which

is

greater

in

MS

than

in

control

rats

-LRB-

Figure

-RRB-

Fig.

Effect

of

AGE-BSA

on

vascular

contraction

in

aortic

rings

from

control

-LRB-

solid

bars

-RRB-

and

MS

rats

-LRB-

open

bars

-RRB-

The

contractions

were

induced

by

NE

μM

and

basal

tension

was

normalized

to

in

control

and

MS

rats

Tension

values

in

grams

are

shown

in

Table

III

Results

are

the

means

SEM

of

six

independent

experiments

P

between

MS

and

control

P

between

the

Tyrode

solution

and

L-NAME

Table

III

Values

of

contraction

-LRB-

g

-RRB-

and

relaxation

-LRB-

-RRB-

of

aortic

rings

in

control

and

experimental

rats

Control

MS

Vascular

contraction

Vascular

relaxation

The

results

are

expressed

as

relaxation

percentage

of

the

initial

precontraction

level

with

NE

-LRB-

μM

-RRB-

Final

relaxation

with

Ach

-LRB-

M

-RRB-

is

reported

Values

are

mean

SEM

n

P

Basal

relaxation

was

diminished

in

MS

rats

when

compared

to

control

rats

-LRB-

Table

III

-RRB-

Figure

shows

the

effect

of

nonglycated

BSA

and

AGE-BSA

upon

vascular

relaxation

in

aortic

rings

from

control

and

MS

rats

In

control

rings

nonglycated

BSA

and

AGE-BSA

at

AU/mL

-LRB-

mg/mL

-RRB-

significantly

inhibited

the

endothelium-dependent

vasorelaxation

in

a

This

effect

was

not

present

when

a

lower

dose

was

used

-LRB-

AU/mL

mg/mL

-RRB-

Fresh

albumin

fatty

acid

free

diminished

vasorelaxation

in

aortic

rings

from

control

rats

in

the

same

proportion

as

pre-incubated

control

BSA

and

AGE-BSA

at

a

concentration

of

mg/mL

-LRB-

data

not

shown

-RRB-

Fig.

Effect

of

nonglycated

BSA

and

AGE-BSA

at

AU/mL

-LRB-

mg/mL

-RRB-

on

endothelium-dependent

vasorelaxation

in

aortic

rings

from

control

-LRB-

A

-RRB-

and

MS

rats

-LRB-

B

-RRB-

Results

are

expressed

as

relaxation

percentage

from

the

initial

precontraction

level

with

NE

μM

P

between

the

Tyrode

solution

and

nonglycated

BSA

and

AGE-BSA

P

between

the

Tyrode

solution

in

MS

and

control

rats

In

MS

aortic

rings

vasorelaxation

was

not

significantly

affected

by

BSA

or

AGE-BSA

at

any

of

the

doses

tested

Immunofluorescence

of

RAGEs

Results

of

expression

of

RAGE

are

shown

in

Figure

There

is

an

increased

RAGE

immunoreactivity

in

aortas

from

MS

rats

when

compared

to

control

rats

In

aortas

from

C

rats

fluorescence

is

observed

mainly

in

the

endothelium

while

in

MS

aortas

both

the

endothelium

and

muscular

fibers

fluoresce

Damage

due

to

the

development

of

the

metabolic

syndrome

can

also

be

appreciated

by

the

presence

of

buckles

and

a

loss

of

continuity

in

elastic

fibers

Fig.

Immunohistochemistry

for

RAGE

in

aortas

of

control

-LRB-

Panel

A

-RRB-

and

MS

-LRB-

Panel

B

-RRB-

rats

Negative

control

-LRB-

Panel

C

-RRB-

Magnification

Asterisk

indicates

internal

elastic

lamina

There

is

an

increased

RAGE

immunoreactivity

in

MS

rats

Discussion

The

aim

of

the

present

paper

was

to

determine

the

effect

of

nonglycated

BSA

and

AGE-BSA

on

vascular

aortas

from

control

and

MS

rats

in

which

the

condition

is

induced

by

a

high

ingestion

of

sucrose

and

where

the

structure

and

function

of

endothelial

cells

are

altered

Both

albumin

and

AGEs

have

been

reported

to

have

vascular

effects

-LRB-

Vlassara

et

al.

-RRB-

However

the

vascular

effects

of

AGEs

might

not

be

produced

by

all

types

of

AGEs

since

AGEs

may

have

very

different

structures

depending

on

the

reactions

generated

during

the

in

vitro

glycation

process

of

the

protein

by

reducing

sugars

-LRB-

Katchalsky

and

Sharon

-RRB-

Some

of

the

AGEs

generated

are

capable

of

cross-linking

to

proteins

and

others

have

the

capacity

to

interact

with

specific

receptors

-LRB-

RAGEs

-RRB-

present

in

some

cellular

types

-LRB-

Valencia

et

al.

-RRB-

There

have

also

been

recent

reports

that

some

of

the

effects

of

in

vitro

prepared

AGEs

are

due

to

metal

ions

which

are

contaminants

commonly

found

in

AGE

preparations

Various

metal

ions

have

been

shown

to

drive

inflammatory

responses

via

generation

of

oxidative

stress

-LRB-

Valencia

et

al.

-RRB-

The

use

of

a

chelator

such

as

EDTA

to

facilitate

the

reaction

could

induce

some

of

the

effects

reported

to

be

produced

by

AGEs

-LRB-

Al-Abed

et

al.

-RRB-

Our

results

show

that

body

weight

was

not

significantly

modified

in

MS

rats

even

if

the

diet

of

the

sucrose-fed

rats

was

hypercaloric

-LRB-

Baños

et

al.

-RRB-

-LRB-

Table

I

-RRB-

However

sucrose-fed

animals

showed

increased

central

adiposity

Insulin

was

increased

in

MS

rats

and

this

increase

accompanies

an

increase

in

blood

pressure

Triglycerides

were

also

elevated

Therefore

the

MS

rats

show

many

of

the

characteristics

of

metabolic

syndrome

Serum

albumin

concentration

was

significantly

higher

in

MS

rats

when

compared

to

control

rats

However

Rumble

et

al.

-LRB-

-RRB-

reported

that

diabetic

and

hypertensive

rats

had

lower

serum

albumin

levels

than

the

normotensive

Sprague

Dawley

rats

Vascular

contraction

to

norepinephrine

increased

MS

rats

in

this

study

This

has

been

previously

described

for

this

model

-LRB-

Baños

et

al.

-RRB-

and

additionally

we

have

previously

found

that

contraction

to

KCl

in

MS

rats

is

also

increased

and

that

this

is

due

to

an

elevated

response

to

endothelin

-LRB-

Nava

et

al.

Rubio

et

al.

-RRB-

The

increase

in

the

circulating

levels

of

albumin

in

the

MS

group

may

contribute

to

the

increased

vascular

tone

observed

in

this

group

Indeed

an

increase

in

circulating

albumin

levels

has

been

observed

in

some

forms

of

hypertensive

pathology

due

to

the

hemoconcentration

induced

by

edema

which

positively

correlates

with

the

hypertension

levels

-LRB-

Allen

and

Patterson

-RRB-

AGEs

could

also

participate

in

the

increase

in

vascular

contraction

in

MS

rats

since

they

increase

the

production

of

by

endothelial

cells

-LRB-

Tan

et

al.

-RRB-

Our

results

show

that

vasoconstriction

was

not

modified

by

nonglycated

BSA

or

by

AGE-BSA

-LRB-

Figure

-RRB-

This

is

in

accordance

with

the

results

of

Xu

et

al.

-LRB-

-RRB-

Vasorelaxation

in

MS

rats

was

significantly

diminished

in

comparison

to

control

rats

and

could

be

attributed

to

an

imbalance

of

constricting

and

relaxing

factors

as

has

been

previously

reported

-LRB-

Baños

et

al.

-RRB-

Furthermore

as

our

model

of

MS

rats

is

caused

by

the

chronic

ingestion

of

sucrose

vascular

damage

could

be

a

probable

consequence

of

AGEs

increased

production

AGEs

deposit

inside

smooth

muscle

and

endothelial

cells

as

well

as

in

extracellular

matrix

proteins

increasing

rigidity

Hogan

et

al.

-LRB-

-RRB-

have

reported

that

AGEs

react

with

NO

produced

by

endothelial

cells

They

also

reported

that

AGEs

reduce

the

antiproliferative

effect

of

NO

on

aortic

smooth

muscle

vascular

and

renal

mesangial

cells

Insulin

which

is

also

increased

in

our

model

stimulates

collagen

synthesis

in

vascular

smooth

muscle

cells

-LRB-

Ruiz

Torres

et

al.

-RRB-

and

could

contribute

to

diminished

vasorelaxation

in

MS

rats

Arterial

endothelium-dependent

relaxation

is

also

diminished

hypertensive

rats

-LRB-

Ibarra

et

al.

Küng

and

Lüscher

Challah

et

al.

Freitas

et

al.

Shipley

and

Muller-Delp

-RRB-

These

authors

have

proposed

that

the

reduced

response

to

Ach

could

be

a

consequence

of

an

impairment

of

either

the

generation

-LRB-

synthesis

or

release

-RRB-

of

relaxant

factors

or

the

cellular

response

to

them

In

our

study

both

nonglycated

BSA

and

AGE-BSA

decreased

vasorelaxation

in

the

same

proportion

in

control

rats

In

arterial

rings

incubated

in

the

presence

of

glycated

and

nonglycated

BSA

the

response

to

acetylcholine

was

reduced

in

comparison

to

control

rings

incubated

in

the

absence

of

BSA

-LRB-

Figure

-RRB-

The

reduction

in

vascular

relaxation

in

the

presence

of

nonglycated

BSA

is

in

accordance

with

the

report

by

Predescu

et

al.

-LRB-

-RRB-

These

authors

suggested

that

BSA

reacts

with

NO

therefore

modulating

its

biological

actions

and

increasing

endothelial

permeability

Although

in

our

experiments

we

also

found

a

decrease

by

AGE-BSA

it

seems

to

be

due

to

BSA

since

there

was

not

a

significant

difference

in

the

reduction

produced

by

AGE-BSA

and

by

nonglycated

BSA

Previously

Xu

et

al.

-LRB-

-RRB-

studied

the

vascular

effects

of

AGEs

produced

in

vitro

testing

vasoreactivity

in

rings

of

thoracic

aorta

from

New

Zealand

white

rabbits

and

in

vitro

production

of

NO

in

endothelial

cells

from

human

umbilical

veins

These

authors

reported

that

there

was

not

a

significant

effect

of

AGEs

upon

vasoconstriction

induced

by

NE

and

our

results

are

in

accordance

with

this

result

However

they

found

that

AGEs

diminished

the

NO

production

of

isolated

endothelial

cells

and

that

vasodilation

was

reduced

in

thoracic

aortic

rings

while

we

found

that

AGEs

did

not

modify

vasodilation

Our

data

suggest

that

it

is

BSA

that

exerts

the

diminished

vasorelaxing

effect

and

not

AGEs

In

their

study

the

authors

produced

AGEs

incubating

BSA

with

glucose

instead

of

glucose

which

is

a

faster

reaction

Glucose

is

a

reducing

sugar

of

great

intracellular

importance

and

can

glycate

easier

than

glucose

since

it

is

present

in

a

higher

proportion

in

the

open-chain

form

-LRB-

Hogan

et

al.

-RRB-

Their

reaction

was

facilitated

by

the

use

of

EDTA

which

could

induce

the

effect

reported

by

them

-LRB-

Al-Abed

et

al.

-RRB-

AGEs

derived

from

a

long

period

of

incubation

of

BSA

with

glucose

as

was

done

in

this

paper

conduces

mainly

to

fluorescent

cross-linking

AGE

production

Using

specific

antibodies

these

authors

found

high

levels

of

carboximetillysine

-LRB-

CML

-RRB-

in

their

preparation

and

identified

CML

as

the

AGE

responsible

of

the

NO

synthesis

inhibition

Therefore

the

kind

of

AGEs

used

by

them

could

explain

the

contradiction

between

their

results

and

what

we

observed

in

the

present

paper

Furthermore

the

difference

of

the

species

used

for

testing

vasodilation

in

both

papers

-LRB-

New

Zealand

rabbits

and

Wistar

rats

-RRB-

might

account

for

the

contradicting

results

found

The

same

authors

-LRB-

Xu

et

al.

-RRB-

did

not

find

the

expected

previously

reported

decrease

in

vascular

relaxation

when

they

added

nonglycated

BSA

The

absence

of

a

vascular

effect

of

nonglycated

BSA

in

the

above-mentioned

study

is

probably

due

to

the

very

low

doses

assayed

in

the

order

of

μg/mL

It

is

known

that

albumin

has

a

physiologic

effect

at

higher

concentration

Indeed

our

own

results

show

that

the

inhibitory

effect

of

BSA

or

AGE-BSA

was

only

observed

when

the

protein

concentration

assayed

was

about

of

mg/mL

but

not

when

it

was

only

of

mg/mL

The

response

to

nonglycated

BSA

and

AGEs

disappeared

in

the

MS

rats

-LRB-

Figure

-RRB-

Alterations

in

the

structure

and

function

of

the

vascular

structures

caused

by

the

metabolic

syndrome

could

be

the

underlying

cause

of

the

lack

of

response

in

these

animals

This

physiological

adaptation

could

be

considered

as

a

protector

effect

to

counteract

the

increased

basal

vasoconstriction

observed

in

metabolic

syndrome

rats

and

must

be

better

studied

We

evaluated

the

presence

of

AGEs

receptors

-LRB-

RAGEs

-RRB-

from

aortas

in

MS

rats

to

better

explore

the

mechanism

by

which

AGEs

participate

in

this

pathology

Many

of

the

deleterious

effects

of

AGEs

are

mediated

by

binding

to

their

receptors

which

are

present

in

various

cellular

types

In

endothelial

cells

the

interaction

AGE

RAGE

modulates

the

expression

of

citocynes

and

adhesion

molecules

and

increases

the

production

of

oxygen

reactive

species

-LRB-

ROS

-RRB-

generating

oxidative

stress

-LRB-

Ramasamy

et

al.

-RRB-

Koyama

et

al.

-LRB-

-RRB-

demonstrated

that

an

alternative

form

of

RAGE

the

endogenous

secretory

RAGE

-LRB-

esRAGE

-RRB-

is

associated

with

the

metabolic

syndrome

or

atherosclerosis

in

the

absence

of

diabetes

Inflammation

might

be

the

factor

linking

the

RAGE

system

with

MS.

Although

a

higher

number

of

RAGEs

were

observed

in

the

aortas

from

MS

rats

AGE-BSA

did

not

have

any

effect

on

arterial

relaxation

in

this

group

Moreover

the

absence

of

differences

in

arterial

relaxation

induced

by

AGE-BSA

or

BSA

in

the

control

group

suggests

that

the

possible

effect

of

AGE-BSA

on

vascular

function

in

not

mediated

by

RAGEs

The

exposition

of

the

vascular

tissue

to

AGE-BSA

during

the

development

of

MS

could

also

cause

the

loss

of

the

relaxation

capacity

of

aortas

The

changes

in

the

charge

and

composition

of

the

BSA

molecule

produced

by

glycation

increase

its

aggregation

capacity

When

the

isoelectric

point

is

closer

to

physiological

pH

hydrophobic

interactions

are

favored

These

changes

could

increase

the

cross-linking

activity

of

AGEs

to

proteins

and

the

capacity

to

deposit

in

the

extracellular

matrix

of

the

tissues

Correspondingly

we

observed

an

increased

aggregation

capacity

of

AGE-BSA

during

alcohol

precipitation

or

desalting

However

the

loss

of

the

relaxing

capacity

due

to

biochemical

changes

of

AGE-BSA

is

not

favored

by

our

results

since

there

were

no

differences

in

the

relaxation

in

the

presence

of

BSA

or

AGE-BSA

We

speculate

that

the

cause

could

be

the

altered

albumin

internalization

to

endothelial

cells

and

therefore

a

modified

neutralization

of

NO

This

should

be

further

evaluated

The

molecular

structure

and

charge

of

albumin

facilitate

the

cotransport

of

a

number

of

hydrophobic

molecules

enzymes

and

hormones

across

the

endothelium

and

the

maintenance

of

vascular

integrity

and

transvascular

oncotic

pressure

gradient

-LRB-

Metha

and

Malik

-RRB-

Endothelial

permeability

to

plasma

proteins

and

liquid

is

increased

in

inflammation

a

condition

manifested

by

protein-rich

edema

-LRB-

Metha

and

Malik

-RRB-

Inflammation

is

present

in

metabolic

syndrome

and

therefore

alterations

in

albumin

transport

across

endothelial

cells

are

likely

to

be

present

Bevers

et

al.

-LRB-

-RRB-

reported

that

in

bEnd

endothelial

cells

which

only

produce

eNOS

and

have

higher

levels

of

NO

than

other

endothelial

cells

incubation

with

low

albumin

levels

increased

eNOs

activity

and

the

production

of

NO

In

conclusion

both

BSA

and

AGE-BSA

inhibit

vascular

relaxation

mediated

by

NO

in

control

artic

rings

in

the

same

proportion

This

inhibitory

effect

is

likely

to

be

due

to

the

structure

of

BSA

and

not

to

the

fluorescent

AGEs

synthesized

by

the

employed

procedure

However

in

MS

rats

this

inhibitory

vascular

relaxation

effect

is

lost

possibly

due

to

alterations

in

endothelial

cells

that

are

a

consequence

of

the

illness

Materials

and

methods

Animals

and

arterial

pressure

determination

Experiments

in

animals

were

approved

by

the

Laboratory

Animal

Care

Committee

of

our

institution

and

were

conducted

in

compliance

with

our

institution

's

ethical

guidelines

for

animal

research

Weanling

male

Wistar

rats

aged

days

and

weighing

g

were

separated

into

two

groups

group

control

rats

-LRB-

C

-RRB-

were

given

tap

water

for

drinking

and

group

MS

rats

were

given

sucrose

in

drinking

water

during

a

period

All

animals

were

fed

Purina

rat

chow

-LRB-

Purina

Mills

Inc.

Richmond

IN

-RRB-

ad

libitum

which

provides

kJ/g

with

protein

fat

and

carbohydrate

Animals

were

kept

at

controlled

temperature

and

a

light-dark

cycle

Systolic

arterial

blood

pressure

was

measured

in

conscious

animals

using

the

tail

cuff

method

the

cuff

was

connected

to

a

pneumatic

pulse

transducer

-LRB-

Narco

Bio-Systems

Inc.

Healthdyne

Co.

Austin

TX

-RRB-

and

a

programmed

electrosphyngomanometer

The

mean

of

five

independent

determinations

was

calculated

AGE-BSA

preparation

AGE-BSA

were

prepared

purified

and

characterized

in

the

reproductive

biology

laboratory

of

Instituto

Nacional

de

Ciencias

Médicas

y

Nutrición

Salvador

Zubirán

following

the

technique

previously

described

-LRB-

Makita

et

al.

-RRB-

AGE-BSA

was

prepared

by

incubating

of

bovine

serum

albumin

-LRB-

BSA

fraction

V

Sigma

Chemical

St.

Louis

MO

-RRB-

in

mM

of

sodium

phosphate

buffer

-LRB-

pH

-RRB-

with

mM

glucose

for

days

under

sterile

conditions

in

darkness

and

at

°C

After

incubation

the

product

was

reduced

with

mM

sodium

borohydride

-LRB-

Sigma

Chemical

-RRB-

for

min

at

room

temperature

to

stabilize

the

Schiff

bases

formed

-LRB-

Eble

et

al.

Fluckiger

and

Gallop

Verbeke

et

al.

-RRB-

Then

the

unbound

glucose

and

unreacted

were

removed

by

extensive

dialysis

against

a

mM

sodium

phosphate

buffer

-LRB-

pH

-RRB-

Less

glycosylated

BSA

was

removed

from

AGE-BSA

by

affinity

chromatography

on

Affi-Gel

Blue

-LRB-

Bio-Rad

Laboratories

Richmond

CA

-RRB-

-LRB-

Travis

et

al.

Verbeke

et

al.

-RRB-

The

Affi-Gel

matrix

has

a

blue

dye

named

Cibacron

Blue

with

natural

affinity

for

native

albumin

A

column

with

mL

of

matrix

with

a

binding

capacity

of

mg

of

BSA

was

employed

mL

of

AGE-BSA

solution

with

a

concentration

of

mg/mL

was

applied

in

each

purification

process

using

a

flow

rate

of

mL/min

The

chromatogram

was

performed

evaluating

a

dilution

of

each

tube

Protein

concentration

of

AGE-BSA

was

evaluated

by

the

Lowry

method

using

BSA

as

standard

-LRB-

Lowry

et

al.

-RRB-

Finally

AGE-specific

fluorescence

determination

was

performed

by

measuring

emission

at

nm

upon

excitation

at

nm

using

an

Aminco

fluorescence

spectrometer

-LRB-

Aminco-Bowman

Series

SLM

Instruments

Inc.

Rochester

NY

-RRB-

-LRB-

Monnier

and

Cerami

Makita

et

al.

-RRB-

Fluorescence

value

of

AGE-BSA

was

measured

at

a

protein

concentration

of

mg/mL

and

expressed

in

arbitrary

units

-LRB-

AU

-RRB-

The

specific

activity

-LRB-

SA

-RRB-

was

expressed

as

arbitrary

units

of

fluorescence

per

mg

of

protein

-LRB-

AU/mg

-RRB-

The

control

BSA

sample

-LRB-

nonglycated

BSA

-RRB-

was

incubated

under

identical

conditions

but

without

glucose

AGE-BSA

and

its

control

-LRB-

nonglycated

BSA

-RRB-

were

maintained

at

°C

until

used

Mass

increase

analysis

of

glycated

BSA

The

mass

change

of

BSA

after

glycation

was

analyzed

by

matrix-assisted

laser

desorption

ionization

mass

spectrometry

-LRB-

MALDI-TOF

-RRB-

-LRB-

Voyager

DE-PRO

from

Applied

Biosystems

CA

-RRB-

equipped

with

a

nitrogen

laser

-LRB-

nm

-RRB-

operating

in

a

positive

high-energy

linear

mode

This

analysis

was

made

in

the

Analysis

and

Molecular

Diagnose

Unit

of

the

Insituto

Nacional

de

Salud

Pública

de

México

Aliquots

of

μL

containing

mg/mL

of

BSA

or

AGE-BSA

were

diluted

to

mL

with

deionized

water

and

centrifuged

in

Amicon

K

-LRB-

Millipore

Corporation

MA

-RRB-

at

g

for

min

to

a

final

volume

of

μL

Washing

was

repeated

three

times

A

μL

aliquot

was

taken

for

MALDI-TOF

analysis

The

matrix

solution

employed

for

BSA

analysis

contained

mg/mL

acid

-LRB-

sinapinic

acid

-RRB-

in

vol/vol

acetonitrile

in

vol/vol

aqueous

trifluoroacetic

acid

For

AGE-BSA

the

matrix

solution

contained

acid

-LRB-

gentisic

acid

which

is

employed

for

analysis

of

glycoproteins

-RRB-

to

mg/mL

in

vol/vol

acetonitrile

A

sample

protein

solution

-LRB-

μL

-RRB-

of

a

vol/vol

mixture

of

mg/mL

of

total

protein

and

the

matrix

solution

were

applied

on

a

sample

spot

in

a

steel

plate

slide

and

dried

in

a

warm

air

stream

Bovine

's

insulin

-LRB-

Da

-RRB-

E.

coli

's

Thioredoxin

-LRB-

Da

-RRB-

and

horse

's

Apomyoglobyn

-LRB-

Da

-RRB-

were

used

as

standard

for

calibration

Isoelectric

point

of

glycated

BSA

The

change

in

the

isoelectric

point

of

BSA

after

glycation

was

analyzed

by

isoelectric

focusing

in

polyacrylamide

gels

using

a

FastSystem

-LRB-

Pharmacia

Uppsala

Sweden

-RRB-

The

bands

were

dyed

with

silver

nitrate

and

the

isoelectric

point

was

evaluated

using

the

analytical

unit

of

FastSystem

employing

a

broad

range

standard

kit

-LRB-

pH

-RRB-

-LRB-

Amersham

Biosciences

Buckinghamshire

United

Kingdom

-RRB-

as

a

protein

calibrator

Blood

sample

collection

and

determination

of

glucose

insulin

triglycerides

and

serum

albumin

After

overnight

fasting

-LRB-

h

-RRB-

the

animals

were

killed

by

decapitation

and

blood

was

collected

It

was

spun

and

the

serum

was

separated

by

centrifugation

at

g

during

min

at

room

temperature

and

stored

at

°C

until

needed

Serum

insulin

was

determined

using

a

commercial

radioimmunoassay

-LRB-

RIA

-RRB-

specific

for

rat

-LRB-

Linco

Research

Inc.

Missouri

-RRB-

its

sensitivity

was

ng/mL

and

intra

and

interassay

coefficients

of

variation

were

and

respectively

Glucose

concentration

was

assayed

using

an

enzymatic

SERA-PAKR

Plus

from

Bayer

Corporation

-LRB-

Sées

France

-RRB-

Homeostasis

model

assessment

-LRB-

HOMA

-RRB-

was

used

as

an

index

to

measure

the

degree

of

insulin

resistance

and

was

calculated

by

the

formula

-LSB-

insulin

-LRB-

μU/mL

-RRB-

glucose

-LRB-

mmol/L

-RRB-

-RSB-

-LRB-

Matthews

et

al.

Pickavance

et

al.

Nandhini

et

al.

-RRB-

Triglycerides

-LRB-

TGs

-RRB-

were

determined

by

commercially

available

procedures

-LRB-

Randox

Laboratories

LTD

Antrim

United

Kingdom

-RRB-

Serum

albumin

was

evaluated

in

both

groups

of

animals

using

a

kit

commercialized

by

Hycel

of

Mexico

which

uses

bromocresol

green

-LRB-

IL-test

Instrumentation

Laboratory

Company

Lexington

MA

-RRB-

Samples

were

read

using

an

automatic

system

Sample

preparation

and

tension

recording

The

animals

were

killed

by

decapitation

and

aortas

were

immediately

dissected

and

placed

in

an

oxygenated

normal

Tyrode

solution

-LRB-

mM

NaCl

KCl

Hepes

and

glucose

-RRB-

pH

Arteries

were

carefully

cleaned

from

connective

and

adipose

tissue

taking

care

not

to

damage

the

endothelium

Tension

measurements

were

made

as

previously

described

-LRB-

Nava

et

al.

-RRB-

Briefly

segments

of

about

to

mm

long

were

cut

and

two

S-shaped

silver

wires

-LRB-

Medwire

Corp

Mount

Vernon

NY

-RRB-

were

inserted

into

the

lumen

to

measure

tension

developed

transversely

by

rings

of

the

vessel

One

of

the

silver

wires

was

fixed

to

the

bottom

of

an

in

vitro

chamber

and

the

other

was

attached

to

a

tension

transducer

that

was

connected

to

a

Grass

polygraph

model

The

chamber

was

filled

with

a

Tyrode

solution

and

termorregulated

and

bubbled

with

carbogen

-LRB-

oxygen

carbon

dioxide

-RRB-

A

basal

passive

tension

of

g

was

applied

after

determination

in

preliminary

tests

that

this

was

the

optimal

resting

tension

under

our

experimental

conditions

Arteries

were

allowed

to

rest

for

h

and

the

solution

was

changed

every

min

Contraction

was

induced

twice

by

the

addition

of

norepinephrine

-LRB-

NE

-RRB-

-LRB-

μM

-RRB-

Arteries

were

washed

by

adding

a

fresh

Tyrode

solution

to

the

chamber

allowing

the

rings

to

return

to

their

basal

tension

-LRB-

g

-RRB-

The

mean

contraction

value

was

considered

as

of

response

The

vasodilator

activity

was

studied

by

cumulative

concentration

response

curves

to

acetylcholine

-LRB-

Ach

-RRB-

-LRB-

M

-RRB-

on

precontracted

aortic

rings

in

the

following

experimental

groups

-LRB-

A

-RRB-

incubated

only

with

the

Tyrode

solution

-LRB-

B

-RRB-

incubated

during

min

with

AGE-BSA

at

AU/mL

-LRB-

C

-RRB-

incubated

with

AGE-BSA

at

AU/mL

-LRB-

D

-RRB-

incubated

with

nonglycated

BSA

at

a

protein

concentration

equivalent

to

UA/mL

of

AGE-BSA

-LRB-

mg/mL

-RRB-

-LRB-

E

-RRB-

incubated

with

nonglycated

BSA

at

a

protein

concentration

equivalent

to

AU/mL

of

AGE-BSA

-LRB-

mg/mL

-RRB-

and

-LRB-

F

-RRB-

incubated

with

L-NAME

at

μM

in

both

control

and

MS

rats

The

percentage

of

the

response

in

each

experimental

group

was

calculated

in

relation

to

the

tension

developed

by

the

same

ring

during

its

basal

contraction

Immunohistochemistry

of

RAGEs

Aortic

rings

of

both

MS

and

C

rats

were

quickly

frozen

in

Tissue-Tek

-LRB-

Sakura

Finetek

USA

Inc.

Torrance

CA

-RRB-

Sections

were

fixed

with

acetone

and

were

blocked

with

PBS/Azide

BSA

for

min

Subsequently

sections

were

incubated

during

h

at

room

temperature

with

a

rabbit

polyclonal

antibody

against

RAGE

-LRB-

Santa

Cruz

Biotechnology

Inc.

Santa

Cruz

CA

-RRB-

Primary

antibodies

were

detected

by

using

goat

anti-rabbit

FITC

-LRB-

Jackson

ImmunoResearch

Laboratories

Inc.

West

Grove

PA

-RRB-

at

room

temperature

for

min

Negative

controls

were

prepared

by

substituting

the

primary

antibody

with

an

irrelevant

antibody

Staining

was

analyzed

using

fluorescence

microscopy

Statistical

analysis

Results

are

expressed

as

mean

standard

errors

of

the

mean

-LRB-

SEM

-RRB-

from

to

different

artery

preparations

The

percentage

of

contraction

in

each

experiment

was

calculated

and

the

mean

was

then

determined

When

applicable

-LRB-

comparisons

between

two

values

MS

and

controls

-RRB-

statistical

analysis

was

done

by

Student

's

t-test

Comparisons

between

groups

were

done

by

analysis

of

variance

-LRB-

ANOVA

-RRB-

or

Anova

on

ranks

followed

by

Student

Newman

Keuls

or

Dunn

's

tests

depending

on

whether

the

data

were

normally

distributed

or

not

using

the

SigmaStat

program

-LRB-

Jandel

Scientific

San

Rafael

CA

-RRB-

Differences

were

considered

statistically

significant

when

P

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

bsa
metabolic syndrome
vascular relaxation


Characterization

of

anticoagulant

heparinoids

by

immunoprofiling

Heparinoids

are

used

in

the

clinic

as

anticoagulants

A

specific

pentasaccharide

in

heparinoids

activates

antithrombin

III

resulting

in

inactivation

of

factor

Xa

and

when

additional

saccharides

are

present

inactivation

of

factor

IIa

Structural

and

functional

analysis

of

the

heterogeneous

heparinoids

generally

requires

advanced

equipment

is

time

consuming

and

needs

-LRB-

extensive

-RRB-

sample

preparation

In

this

study

a

novel

and

fast

method

for

the

characterization

of

heparinoids

is

introduced

based

on

reactivity

with

nine

unique

anti-heparin

antibodies

Eight

heparinoids

were

biochemically

analyzed

by

electrophoresis

and

their

reactivity

with

domain-specific

anti-heparin

antibodies

was

established

by

ELISA

Each

heparinoid

displayed

a

distinct

immunoprofile

matching

its

structural

characteristics

The

immunoprofile

could

also

be

linked

to

biological

characteristics

such

as

the

anti-Xa/anti-IIa

ratio

which

was

reflected

by

reactivity

of

the

heparinoids

with

antibodies

-LRB-

indicative

for

-RRB-

and

-LRB-

indicative

for

domains

allowing

anti-factor

IIa

activity

-RRB-

In

addition

the

immunoprofile

could

be

indicative

for

heparinoid-induced

side-effects

such

as

heparin-induced

thrombocytopenia

as

illustrated

by

reactivity

with

antibody

which

defines

a

very

high

sulfated

domain

In

conclusion

immunoprofiling

provides

a

novel

fast

and

simple

methodology

for

the

characterization

of

heparinoids

and

allows

high-throughput

screening

of

-LRB-

new

-RRB-

heparinoids

for

defined

structural

and

biological

characteristics

Introduction

Glycosaminoglycans

-LRB-

GAGs

-RRB-

are

long

unbranched

negatively

charged

polysaccharide

chains

The

precursor

of

heparin

and

heparan

sulfate

-LRB-

HS

-RRB-

is

composed

of

alternating

N-acetylglucosamine

-LRB-

GlcNAc

-RRB-

and

d-glucuronic

acid

-LRB-

GlcA

-RRB-

residues

In

chondroitin

sulfate

-LRB-

CS

-RRB-

and

dermatan

sulfate

-LRB-

DS

-RRB-

the

aminosugar

is

a

N-acetylgalactosamine

-LRB-

GalNAc

-RRB-

instead

of

GlcNAc

The

polysaccharide

chain

precursors

are

modified

by

a

sequential

series

of

enzymatic

reactions

including

N-deacetylation/N-sulfation

of

GlcA

to

l-iduronic

acid

-LRB-

IdoA

-RRB-

and

O-sulfation

at

various

positions

-LSB-

-RSB-

Heparin

is

widely

used

as

an

anticoagulant

A

specific

pentasaccharide

sequence

-LRB-

GlcNAc/NS

-LRB-

-RRB-

GlcA

GlcNS

-LRB-

OS

-RRB-

IdoA

-LRB-

-RRB-

GlcNS

-LRB-

-RRB-

-RRB-

-LSB-

-RSB-

in

heparin

binds

with

high

affinity

to

the

enzyme

inhibitor

antithrombin

III

causing

a

conformational

change

resulting

in

the

exposition

of

its

active

sites

and

in

inactivation

of

factor

Xa

For

the

inactivation

of

factor

IIa

an

additional

site

in

heparin

proximal

to

the

pentasaccharide

is

necessary

The

formation

of

a

ternary

complex

between

antithrombin

III

factor

IIa

and

heparin

which

requires

at

least

saccharide

residues

for

efficient

formation

subsequently

results

in

the

inactivation

of

factor

IIa

-LSB-

-RSB-

Next

to

heparin

other

heparinoids

are

used

including

preparations

containing

other

GAGs

such

as

HS

and

DS

low

molecular

weight

heparins

-LRB-

LMWHs

-RRB-

and

fondaparinux

a

synthetic

pentasaccharide

identical

in

structure

to

the

antithrombin

III-binding

pentasaccharide

Heparinoids

are

used

to

treat

patients

with

venous

thrombosis

and

pulmonary

embolism

-LSB-

-RSB-

In

addition

they

are

used

to

prevent

thrombosis

in

patients

that

undergo

surgery

and

to

prevent

clotting

in

the

extracorporeal

circulation

during

hemodialysis

-LSB-

-RSB-

Due

to

their

lower

molecular

weight

LMWHs

have

a

high

anti-factor

Xa

activity

compared

to

their

anti-factor

IIa

activity

resulting

in

a

more

refined

regulation

of

coagulation

-LSB-

-RSB-

In

addition

the

biological

half-life

of

LMWHs/fondaparinux

is

longer

in

comparison

to

heparin

and

the

incidence

of

heparin-induced

thrombocytopenia

-LRB-

HIT

-RRB-

is

substantially

reduced

-LSB-

-RSB-

LMWHs

are

synthesized

from

heparin

by

controlled

chemical

or

enzymatic

depolymerization

processes

resulting

in

the

production

of

oligosaccharides

of

various

lengths

-LRB-

Table

-RRB-

Dalteparin

and

nadroparin

are

produced

by

deamination

using

nitrous

acid

resulting

in

the

formation

of

an

unnatural

residue

at

the

reducing

end

of

the

resulting

oligosaccharides

Enoxaparin

is

produced

by

chemical

β-elimination

at

alkaline

pH

whereas

tinzaparin

is

produced

by

enzymatic

β-elimination

by

heparinase

Both

processes

result

in

the

formation

of

an

unsaturated

uronic

acid

residue

at

the

non-reducing

end

of

the

GAG

fragments

-LSB-

-RSB-

Table

of

therapeutic

heparinoidsHeparinoid

generic

name

-LRB-

brand

name

-RRB-

Composition

-LRB-

and

method

of

heparin

modification

-RRB-

Molecular

weight

Dalteparin

-LRB-

Fragmin

-RRB-

LMWH

-LRB-

deaminative

cleavage

with

nitrous

acid

-RRB-

Nadroparin

-LRB-

Fraxiparine

-RRB-

LMWH

-LRB-

deaminative

cleavage

with

nitrous

acid

-RRB-

Enoxaparin

-LRB-

Clexane

Lovenox

-RRB-

LMWH

-LRB-

β-eliminative

cleavage

by

alkaline

treatment

-RRB-

Tinzaparin

-LRB-

Innohep

Logiparin

-RRB-

LMWH

-LRB-

β-eliminative

cleavage

by

heparinase

-RRB-

Sulodexide

-LRB-

Vessel

Due

F

-RRB-

fast-moving

heparina

DS

Danaparoid

-LRB-

Orgaran

-RRB-

HS

DS

Fondaparinux

-LRB-

Arixtra

-RRB-

Pentasaccharide

All

drugs

are

derived

from

porcine

intestinal

mucosa

with

the

exception

of

the

chemically

synthesized

pentasaccharide

fondaparinux.LMWH

Low-molecular

weight

heparin

HS

heparan

sulfate

DS

dermatan

sulfate

CS

chondroitin

sulfateaHeparin

that

is

less

sulfated

and

has

a

lower

molecular

weight

-LSB-

-RSB-

All

heparinoids

with

the

exception

of

fondaparinux

are

heterogeneous

mixtures

of

different

GAG

chains

Structural

analysis

of

heparinoids

is

generally

done

by

nuclear

magnetic

resonance

spectroscopy

high

pressure

liquid

chromatography

mass

spectrometry

capillary

electrophoresis

or

polyacrylamide

gel

electrophoresis

-LSB-

-RSB-

In

general

these

techniques

require

sophisticated

instrumentation

and

sometimes

extensive

sample

preparation

Alternative

methodologies

that

are

fast

and

simple

are

needed

Immunoprofiling

of

heparinoids

could

be

a

fast

and

simple

method

to

characterize

the

complex

heparinoids

Recently

a

number

of

phage

display-derived

antibodies

were

generated

reactive

with

various

domain

structures

within

heparin

In

this

study

nine

antibodies

all

defining

different

heparin

epitopes

were

applied

to

study

their

reactivity

with

heparinoids

Materials

and

methods

Heparinoids

The

following

heparinoids

were

included

in

the

study

heparin

-LRB-

Sigma

St.

Louis

MO

-RRB-

dalteparin

-LRB-

Pharmacia

Woerden

The

Netherlands

-RRB-

nadroparin

-LRB-

Sanofi-Synthélabo

Maassluis

The

Netherlands

-RRB-

enoxaparin

-LRB-

Aventis

Pharma

Hoevelaken

The

Netherlands

-RRB-

tinzaparin

-LRB-

LEO

Pharma

Breda

The

Netherlands

-RRB-

sulodexide

-LRB-

Alfa

Wassermann

S.p.A.

Bologna

Italy

-RRB-

danaparoid

-LRB-

Organon

Oss

The

Netherlands

-RRB-

and

fondaparinux

-LRB-

Sanofi-Synthélabo

Maassluis

The

Netherlands

-RRB-

Characteristics

are

given

in

Table

Quantification

of

glycosaminoglycans

by

Farndale

assay

A

Farndale

assay

was

done

to

determine

the

concentration

of

GAGs

in

the

preparations

-LSB-

-RSB-

To

μl

of

the

standard

-LRB-

heparin

μg/ml

-RRB-

and

heparinoids

ml

Farndale

reagent

-LRB-

mmol/l

glycine

mmol/l

NaCl

and

mmol/l

blue

pH

-RRB-

was

added

and

absorbance

was

measured

directly

at

nm

Determination

of

glycosaminoglycan

classes

by

agarose

gel

electrophoresis

The

different

classes

of

GAGs

in

the

heparinoids

were

separated

by

agarose

gel

electrophoresis

fixed

in

-LRB-

w/v

-RRB-

azure

A

in

mmol/l

mmol/l

formiate

and

-LRB-

w/v

-RRB-

azide

-LRB-

pH

-RRB-

and

stained

by

combined

azure

A

silver

treatment

-LSB-

-RSB-

Enzymatic

digestion

of

heparinoids

Heparinoids

were

diluted

in

heparinase

I

digestion

buffer

-LRB-

mmol/l

NaAc

and

mmol/l

Ca

-LRB-

Ac

-RRB-

pH

-RRB-

and/or

chondroitinase

ABC

digestion

buffer

-LRB-

mmol/l

Tris

and

mmol/l

Mg

-LRB-

Ac

-RRB-

pH

-RRB-

to

a

final

content

of

mg/ml

The

enzymes

were

added

to

a

final

concentration

of

IU/ml

for

heparinase

I

-LRB-

E.C.

from

Flavobacterium

heparinum

Seikagaku

Tokyo

Japan

-RRB-

and

IU/ml

for

chondroitinase

ABC

-LRB-

E.C.

from

Proteus

vulgaris

Sigma

-RRB-

Heparinase

I

cleaves

the

linkages

GlcNS

predominantly

present

in

heparin

and

to

a

minor

extent

in

HS

-LSB-

-RSB-

Chondroitinase

ABC

cleaves

CS

and

DS

-LSB-

-RSB-

The

solutions

were

incubated

for

h

at

°C

Determination

of

glycosaminoglycan

size

by

polyacrylamide

gel

electrophoresis

Heparinoids

and

their

enzymatic

digestion

products

were

separated

by

polyacrylamide

gel

electrophoresis

using

a

gel

-LSB-

-RSB-

fixed

in

-LRB-

w/v

-RRB-

alcian

blue

in

-LRB-

v/v

-RRB-

HAc

and

stained

by

combined

azure

A

silver

treatment

-LSB-

-RSB-

Production

of

anti-heparin

antibodies

Antibodies

-LRB-

Table

-RRB-

were

obtained

by

biopanning

against

HS

or

heparin

using

bovine

kidney

HS

-LRB-

antibodies

-LSB-

-RSB-

-LSB-

-RSB-

and

-LSB-

-RSB-

-RRB-

porcine

intestinal

mucosa

heparin

-LRB-

antibodies

and

-LSB-

-RSB-

-RRB-

human

lung

HS

-LRB-

antibody

Smits

et

al.

unpublished

results

-RRB-

and

mouse

and

human

skeletal

muscle

GAG

-LRB-

antibodies

and

-LSB-

-RSB-

respectively

-RRB-

Large

scale

production

of

soluble

antibodies

was

performed

as

described

before

-LSB-

-RSB-

Table

of

epitope-specific

anti-heparin

antibodiesAntibodyVH

sequenceVH

familyDP

geneGAG

used

for

selectionEssential

modificationsInhibitory

kidney

HS

-LSB-

-RSB-

N-sulfation

-LRB-

minor

importance

-RRB-

kidney

HS

-LSB-

-RSB-

N-sulfation

C5-epimerization2-O-sulfation6-O-sulfation

-LRB-

likely

-RRB-

kidney

HS

-LSB-

-RSB-

kidney

HS

-LSB-

-RSB-

N-sulfation6-O-sulfationC5-epimerization

-LRB-

likely

-RRB-

intestinal

mucosa

heparin

-LSB-

-RSB-

Sulfation

required

position

-LRB-

s

-RRB-

intestinal

mucosa

heparin

-LSB-

-RSB-

Sulfation

required

position

-LRB-

s

-RRB-

lung

HScN-sulfation

-LRB-

high

-RRB-

-LRB-

high

-RRB-

skeletal

muscle

GAG

-LSB-

-RSB-

N-sulfation

C5-epimerization2-O-sulfationd6-O-sulfation

-LRB-

high

-RRB-

skeletal

muscle

GAG

-LSB-

-RSB-

N-acetylation

-LRB-

control

-RRB-

Given

are

the

antibody

name

amino

acid

sequence

of

the

VH

complementary

determining

region

-LRB-

-RRB-

VH

germ

line

gene

family

DP

gene

number

selection

moiety

and

relevant

modification

reactions

required

for

antibody

binding

Table

derived

from

Wijnhoven

et

al.

unpublished

results.GAG

Glycosaminoglycan

HS

heparan

sulfateaAntibody

possibly

requires

as

yet

unspecified

O-sulfated

residues

-LSB-

-RSB-

bAlthough

the

chemical

nature

of

the

specific

heparin/HS

structure

recognized

is

not

exactly

known

the

antibody

defines

a

unique

sulfated

heparin/HS

structure

as

demonstrated

by

its

specific

staining

pattern

on

renal

cryosections

-LSB-

-RSB-

cSmits

et

al.

unpublished

resultsdAntibody

requires

an

internal

iduronic

acid

residue

-LSB-

-RSB-

Enzyme-linked

immunosorbent

assay

Reactivity

of

the

anti-heparin

antibodies

with

the

heparinoids

was

evaluated

by

enzyme-linked

immunosorbent

assay

-LRB-

ELISA

-RRB-

Wells

of

microtiter

plates

were

coated

overnight

by

incubation

with

the

heparinoids

that

bound

by

passive

adsorption

-LSB-

-RSB-

After

blocking

with

PBS

containing

-LRB-

w/v

-RRB-

BSA

and

-LRB-

v/v

-RRB-

for

h

anti-heparin

antibodies

-LSB-

in

PBS

containing

-LRB-

w/v

-RRB-

BSA

and

-LRB-

v/v

-RRB-

-RSB-

were

added

for

h

As

a

negative

control

antibody

was

used

which

is

identical

to

most

antibodies

used

but

does

not

bind

any

GAG

Bound

antibodies

were

detected

using

mouse

IgG

anti-VSV

tag

antibody

-LRB-

Boehringer

Mannheim

Mannheim

Germany

-RRB-

followed

by

incubation

with

alkaline

phosphatase-conjugated

rabbit

anti-mouse

IgG

-LRB-

Dako

Glostrup

Denmark

-RRB-

both

for

h.

Enzyme

activity

was

detected

using

μl

mg

p-nitrophenyl

phosphate

-LRB-

ICN

Aurora

OH

-RRB-

ml

M

mM

-LRB-

pH

-RRB-

as

a

substrate

Absorbance

was

measured

at

nm

-LSB-

-RSB-

Background

reactivity

measured

with

the

control

antibody

was

substracted

The

absorbance

with

control

antibody

was

comparable

to

the

absorbance

measured

when

primary

antibody

was

omitted

Immunofluorescence

staining

Human

kidney

cryosections

-LRB-

μm

-RRB-

were

incubated

with

anti-heparin

antibodies

in

the

absence

or

presence

of

μg/ml

of

the

heparinoids

Bound

antibodies

were

detected

as

described

-LSB-

-RSB-

Results

General

biochemical

analysis

of

heparinoids

The

different

classes

of

GAGs

in

the

heparinoids

were

separated

by

agarose

gel

electrophoresis

-LRB-

Fig.

-RRB-

Heparin

showed

a

band

at

about

the

same

position

as

the

HS

standard

whereas

the

LMWHs

dalteparin

nadroparin

enoxaparin

and

tinzaparin

migrated

somewhat

further

into

the

gel

Danaparoid

displayed

bands

at

the

level

of

HS

and

DS

whereas

the

CS

band

which

only

represents

of

the

prepration

-LRB-

Table

-RRB-

could

not

be

detected

Sulodexide

clearly

showed

two

bands

representing

the

fast-moving

heparin

-LRB-

heparin

that

is

less

sulfated

and

has

a

lower

molecular

weight

-LSB-

-RSB-

-RRB-

and

DS

fractions

The

chemically

synthesized

pentasaccharide

fondaparinux

which

has

a

much

lower

molecular

weight

compared

to

the

other

heparinoids

migrated

further

into

the

gel

Fig.

gel

electrophoresis

of

heparinoids

The

different

classes

of

GAGs

in

the

drugs

were

visualized

by

a

combined

azure

A

silver

staining

procedure

The

standard

contains

ng

HS

ng

DS

and

ng

CS

To

analyze

the

molecular

range

of

the

heparinoids

gel

electrophoresis

using

polyacrylamide

was

applied

The

LMWHs

clearly

displayed

distinct

fragments

with

a

lower

molecular

weight

compared

to

heparin

-LRB-

Fig.

-RRB-

The

smaller

fragments

of

enoxaparin

compared

to

tinzaparin

-LRB-

both

prepared

by

β-elimination

-RRB-

can

be

explained

by

a

preference

of

heparinase

for

iduronic

acid

in

contrast

to

chemical

alkaline

treatment

which

results

in

cleavage

at

both

sulfated

and

non-sulfated

iduronic

acid

residues

Sulodexide

and

danaparoid

showed

a

broad

smear

with

less

distinct

fragments

in

line

with

their

methods

of

preparation

which

does

not

include

a

specific

depolymerization

step

The

pentasaccharide

fondaparinux

demonstrated

primarily

one

individual

band

To

further

establish

the

nature

of

the

heparinoids

they

were

subjected

to

enzymatic

digestion

using

heparinase

I

and/or

chondroitinase

ABC

-LRB-

Fig.

for

dalteparin

enoxaparin

and

sulodexide

-RRB-

Heparin

dalteparin

nadroparin

enoxaparin

tinzaparin

and

fondaparinux

were

only

cleaved

by

heparinase

I

in

accordance

with

their

derivation

from

heparin

Sulodexide

was

cleaved

by

heparinase

I

as

well

as

chondroitinase

ABC

which

is

in

line

with

the

heparin/DS

nature

of

the

preparation

Danaparoid

was

primarily

cleaved

by

chondroitinase

ABC

reflecting

the

presence

of

DS/CS

whereas

heparinase

I

was

less

effective

Fig.

percent

polyacrylamide

gel

electrophoresis

of

undigested

-LRB-

a

-RRB-

and

enzymatically

digested

-LRB-

b

-RRB-

heparinoids

Heparinoids

were

visualized

by

a

combined

alcian

blue-silver

staining

procedure

Treatment

with

heparinase

I

resulted

in

cleavage

of

dalteparin

and

enoxaparin

whereas

treatment

with

chondroitinase

ABC

did

not

Sulodexide

was

cleaved

by

heparinase

I

as

well

as

chondroitinase

ABC

Characterization

of

heparinoids

using

anti-heparin

antibodies

Reactivity

of

anti-heparin

antibodies

with

immobilized

heparinoids

was

analyzed

by

ELISA

-LRB-

Table

-RRB-

For

reference

reactivity

of

antibodies

with

heparin

was

set

at

A

distinct

antibody-binding

profile

could

be

observed

for

all

antibodies

although

also

similar

reactivity

with

a

number

of

antibodies

was

noticed

For

instance

enoxaparin

and

tinzaparin

both

prepared

by

β-elimination

reacted

differently

with

antibodies

and

whereas

they

reacted

similarly

with

the

other

antibodies

Nadroparin

reacted

very

poorly

with

all

the

antibodies

tested

which

may

be

in

line

with

its

overall

low

amount

of

sulfated

disaccharides

in

comparison

with

other

LMWHs

-LSB-

-RSB-

In

contrast

sulodexide

and

danaparoid

reacted

strongly

with

antibody

which

defines

a

low-sulfated

and

N-acetylated

epitope

in

comparison

to

the

LMWHs

This

is

in

line

with

the

presence

of

low-sulfated

heparin

-LRB-

HS

-RRB-

and

HS

in

sulodexide

and

danaparoid

respectively

and

which

contain

a

substantial

amount

of

N-acetylated

glucosamine

Compared

to

heparin

the

other

heparinoids

reacted

poorly

with

antibody

which

defines

a

very

highly

sulfated

oligosaccharide

Table

of

anti-heparin

antibodies

with

heparinoids

-LRB-

ELISA

-RRB-

antibodies

were

applied

to

heparinoids

immobilized

on

microtiter

plates

Given

is

the

reactivity

of

the

antibodies

with

the

heparinoids

relative

to

unmodified

heparin

Values

are

given

as

mean

SEM

-LRB-

n

-RRB-

Each

hepainoid

shows

a

distinct

immunoprofile

Heparinoids

could

be

ranked

according

to

their

reactivity

with

antibodies

For

instance

reactivity

of

antibody

was

highest

with

heparin

and

sulodexide

followed

by

tinzaparin

dalteparin

and

enoxaparin

Reactivity

with

the

other

heparinoids

was

very

low

or

absent

Antibody

reacted

strongly

with

heparin

followed

by

sulodexide

and

tinzaparin

whereas

other

heparinoids

reacted

poorly

Reactivity

of

antibody

was

highest

with

heparin

and

sulodexide

followed

by

danaparoid

and

enoxaparin/tinzaparin/dalteparin

whereas

reactivity

with

nadroparin

was

absent

These

data

indicate

that

distinct

heparin

domain

structures

are

present

in

different

commercially

available

heparinoids

The

only

chemically

defined

heparinoid

studied

was

the

pentasaccharide

fondaparinux

-LRB-

GlcNS

-LRB-

-RRB-

GlcA

GlcNS

-LRB-

OS

-RRB-

IdoA

-LRB-

-RRB-

GlcNS

-LRB-

-RRB-

-RRB-

Due

to

its

small

size

-LRB-

kDa

-RRB-

it

was

assayed

using

a

competition

ELISA

-LRB-

Table

-RRB-

Heparin

was

included

as

a

reference

and

values

obtained

for

fondaparinux

were

related

to

those

obtained

for

heparin

Antibodies

and

both

requiring

N-acetylation

in

their

epitopes

did

not

react

with

fondaparinux

Antibody

which

reacts

with

an

extremely

high

sulfated

oligosaccharide

was

also

not

reactive

These

results

are

in

line

with

the

chemical

composition

of

fondaparinux

which

contains

no

N-acetylated

glucosamine

and

a

non-sulfated

glucuronic

acid

residue

Reactivity

with

antibody

which

reacts

poorly

with

epitopes

was

very

weak

and

reflects

the

high

amount

of

in

fondaparinux

Reactivity

with

antibody

which

needs

N-sulfation

high

and

an

internal

iduronic

acid

residue

was

only

moderate/poor

reflecting

the

lack

of

an

internal

iduronic

acid

-LRB-

i.e.

flanked

by

other

iduronic

acid

residues

-RRB-

Reactivity

with

antibody

which

requires

N

and

and

antibody

which

requires

at

least

and

N

and

sulfation

was

high

which

corresponds

with

the

chemical

structure

of

fondaparinux

Reactivity

was

also

high

with

antibodies

and

both

defining

an

as

yet

undefined

but

sulfated

oligosaccharide

These

data

indicate

that

reactivity

with

antibodies

may

provide

some

chemical

information

of

heparinoids

Table

of

anti-heparin

antibodies

with

heparin

and

fondaparinux

-LRB-

competition

ELISA

-RRB-

Ratio

are

the

amounts

of

heparinoids

-LRB-

micrograms

per

milliliter

-RRB-

which

result

in

a

inhibition

of

the

binding

of

the

anti-heparin

antibodies

to

immobilized

HS/heparin

-LRB-

values

-RRB-

and

the

ratio

between

antibody

reactivity

with

fondaparinux

and

heparin

Values

are

given

as

mean

SEM

-LRB-

n

-RRB-

Antibody

showed

a

strong

reactivity

with

fondaparinux

followed

by

antibodies

and

The

other

antibodies

showed

a

relatively

weak

or

no

reactivity

Reactivity

of

the

anti-heparin

antibodies

with

immobilized

heparinoids

was

supported

by

immunofluorescence

analysis

-LRB-

Fig.

-RRB-

When

human

renal

cryosections

were

simultaneously

incubated

with

anti-heparin

antibodies

and

heparinoids

staining

was

completely

abolished

with

those

heparinoids

which

reacted

strongly

with

the

antibodies

in

ELISA

-LRB-

e.g.

heparin

and

sulodexide

for

antibodies

and

-RRB-

An

obvious

decreased

staining

was

noticed

for

those

heparinoids

which

reacted

moderately

with

the

antibodies

-LRB-

e.g.

nadroparin

for

antibody

-RRB-

whereas

no

major

differences

in

staining

were

observed

for

those

heparinoids

which

did

not

bind

to

antibodies

-LRB-

e.g.

nadroparin

for

antibody

-RRB-

Fig.

staining

of

human

renal

cryosections

with

anti-heparin

antibodies

and

in

the

absence

and

presence

of

heparinoids

Staining

was

abolished

when

renal

cryosections

were

incubated

with

or

in

the

presence

of

heparin

or

sulodexide

staining

was

decreased

in

the

presence

of

nadroparin

whereas

staining

was

unaffected

Bar

represents

μm

magnification

is

identical

for

each

photograph

G

Glomerulus

BC

Bowman

's

capsule

T

renal

tubule

Discussion

In

this

study

eight

heparinoids

were

characterized

by

their

reactivity

with

nine

different

anti-heparin

antibodies

All

heparinoids

showed

a

distinct

antibody-binding

profile

in

line

with

differences

in

GAG

composition

-LRB-

heparin

HS

CS

DS

-RRB-

molecular

weight

and

method

of

preparation

A

number

of

antibodies

reacted

strongly

with

certain

heparinoids

but

not

with

others

It

may

therefore

be

possible

to

deduce

chemical

and

biological

information

of

a

specific

heparinoid

by

virtue

of

its

antibody

profile

For

instance

antibody

showed

reactivity

with

only

heparin

This

antibody

defines

a

stretch

of

trisulfated

-LRB-

N

and

-RRB-

and

epimerized

disaccharides

and

therefore

defines

a

highly

sulfated

heparin

domain

-LRB-

Smits

et

al.

unpublished

results

-RRB-

The

lack

of

reactivity

of

the

other

heparinoids

for

indicates

the

absence

or

the

destruction

of

the

domain

during

their

preparation

Absence

may

hold

for

sulodexide

and

danaparoid

which

contain

HS

rather

than

heparin

Destruction

by

the

depolymerization

processes

may

hold

for

the

LMWHs

In

this

respect

reactivity

with

antibody

may

be

indicative

for

the

degree

of

depolymerization

The

antibody

profile

of

the

chemically

synthesized

pentasaccharide

fondaparinux

is

compatible

with

its

chemical

structure

GlcNS

-LRB-

-RRB-

GlcA

GlcNS

-LRB-

OS

-RRB-

IdoA

-LRB-

-RRB-

GlcNS

-LRB-

-RRB-

Reactivity

with

antibodies

and

indicates

it

to

contain

iduronic

acid

and

N

and

residues

whereas

poor/no

reactivity

with

antibodies

and

indicates

the

lack

of

internal

iduronic

acid

residues

-LRB-

i.e.

flanked

by

other

iduronic

acids

-RRB-

and

N-acetylated

glucosamine

residues

Lack

of

reactivity

with

antibody

indicates

that

fondaparinux

does

not

contain

a

stretch

of

tri-sulfated

disaccharides

Next

to

information

on

the

chemical

properties

of

the

heparinoids

the

antibody

profile

may

be

indicative

for

their

biological

activity

e.g.

the

anti-Xa/anti-IIa

ratio

This

ratio

is

of

essential

importance

since

it

is

associated

with

a

more

predictable

anticoagulant

effect

The

anti-Xa/anti-IIa

ratio

is

for

unfractionated

heparin

for

LMWHs

for

sulodexide

and

infinite

for

fondaparinux

-LSB-

-RSB-

Antibodies

such

as

and

-LRB-

Tables

and

-RRB-

which

are

highly

reactive

with

heparin

and

sulodexide

but

poorly/not

with

LMWHs

and

fondaparinux

likely

bind

to

a

heparin

domain

structure

at

a

site

proximal

to

the

pentasaccharide

sequence

and

may

be

indicative

for

a

high

anti-factor

IIa

activity

These

results

indicate

that

when

a

heparinoid

is

recognized

by

antibodies

and

-LRB-

which

showed

a

high

reactivity

with

fondaparinux

Table

-RRB-

as

well

as

by

antibodies

and

the

heparinoid

is

able

to

form

a

ternary

complex

with

antithrombin

III

and

factor

IIa

resulting

in

inactivation

of

both

factors

Xa

and

IIa

-LSB-

-RSB-

On

the

other

hand

when

a

heparinoid

is

recognized

by

antibodies

and

but

not

by

antibodies

and

it

is

does

not

contain

the

heparin

domain

structure

at

the

site

proximal

to

the

pentasaccharide

required

for

factor

IIa

binding

In

this

case

only

factor

Xa

will

be

inactivated

When

a

ratio

is

calculated

from

reactivity

of

heparinoids

with

antibodies

and

-LRB-

indicative

for

and

for

the

presence

of

domains

allowing

IIa

activity

respectively

-RRB-

and

heparin

is

assigned

a

ratio

of

the

following

numbers

are

found

dalteparin

nadroparin

enoxaparin

tinzaparin

sulodexide

and

fondaparinux

-LRB-

no

antibody

reactivity

observed

-RRB-

These

numbers

are

in

line

with

the

above

mentioned

anti-Xa/anti-IIa

ratios

which

were

determined

by

enzymatic

assays

-LSB-

-RSB-

and

indicate

that

biological

characteristics

are

associated

with

antibody

profiles

Heparin-induced

thrombocytopenia

-LRB-

HIT

-RRB-

is

a

major

complication

in

patients

administered

heparin

Heparin

binding

to

platelet

factor

-LRB-

-RRB-

causes

a

conformational

change

in

the

protein

rendering

it

antigenic

Susceptible

patients

develop

antibodies

to

the

complex

and

binding

of

the

complex

to

its

receptor

on

platelets

results

in

platelet

activation

and

aggregation

In

addition

platelets

are

removed

by

activated

immune

cells

resulting

in

thrombocytopenia

-LSB-

-RSB-

Highly

sulfated

domains

within

heparin/HS

are

involved

in

the

binding

to

-LSB-

-RSB-

and

an

increase

in

sulfation

and

size

is

associated

with

an

increased

risk

of

HIT

-LSB-

-RSB-

This

may

explain

why

the

incidence

of

HIT

is

lower

in

patients

treated

with

LMWHs

danaparoid

or

fondaparinux

compared

to

unfractionated

heparin

-LSB-

-RSB-

Antibody

which

defines

a

highly

sulfated

domain

reacted

strongly

with

heparin

but

not

with

the

other

heparinoids

Reactivity

with

this

antibody

may

thus

be

used

to

obtain

information

about

the

risk

of

a

heparinoid

to

induce

HIT

Although

we

have

focussed

here

on

the

immunoprofiling

of

heparinoids

in

the

context

of

anticoagulation

this

technique

may

also

be

useful

to

evaluate

heparinoids

in

other

settings

Heparin

and

LMWHs

have

been

used

to

treat

patients

with

kidney

disease

since

an

ameliorating

effect

on

a.o.

proteinuria

has

been

found

-LSB-

-RSB-

Likewise

heparinoids

are

reported

to

have

anti-tumor

and

anti-metastatic

effects

-LSB-

-RSB-

the

degree

of

sulfation

being

of

crucial

importance

Heparinase

III-digested

heparin

-LRB-

highly

sulfated

fragments

-RRB-

inhibited

tumor

growth

whereas

heparinase

I-digested

heparin

-LRB-

low

sulfated

fragments

-RRB-

promoted

tumor

growth

-LSB-

-RSB-

underscoring

the

necessity

to

characterize

the

preparation

used

In

addition

to

the

implementation

of

these

antibodies

to

characterize

the

therapeutic

potential

of

heparinoids

they

may

be

used

to

stage

a

disease

-LRB-

e.g.

kidney

disease

cancer

-RRB-

characterized

by

a

specific

HS

profile

in

tissue

blood

and/or

urine

Since

the

antibody

profile

of

the

heparinoids

used

in

these

studies

may

be

correlated

to

a

specific

biological

effect

antibodies

may

be

used

for

the

identification

and

subsequent

characterization

of

the

active

structures

within

the

heparinoids

which

may

result

in

the

development

of

more

specific

drugs

The

antibodies

described

here

may

also

be

used

to

counteract

bleeding

in

patients

administered

an

overdose

of

heparinoids

This

is

generally

accomplished

by

treatment

with

protamine

sulfate

-LSB-

-RSB-

but

this

rather

unspecific

polycation

may

cause

hypotension

bradycardia

and

thrombocytopenia

In

addition

it

may

cause

other

severe

side

effects

including

anaphylactic

and

anaphylactoid

reactions

resulting

in

respiratory

distress

circulatory

collapse

capillary

leak

and

pulmonary

hypertension

-LSB-

-RSB-

There

is

a

need

for

safer

agents

that

inhibit

the

anticoagulant

activity

of

heparinoids

and

some

antibodies

described

here

may

be

useful

especially

those

that

also

react

with

fondaparinux

which

is

difficult

to

inhibit

in

vivo

It

has

already

been

described

that

antibodies

and

strongly

inhibited

heparin-induced

anticoagulation

-LRB-

APTT

clotting

assay

-RRB-

-LSB-

-RSB-

Antibody

also

blocked

the

anticoagulant

activities

of

heparin

and

fondaparinux

in

an

anti-factor

Xa

assay

-LSB-

-RSB-

These

studies

indicate

that

anti-heparin

antibodies

may

react

with

heparinoids

thereby

neutralizing

their

anticoagulant

effect

Finally

our

antibodies

may

be

used

for

fast

industrial

screening

of

heparinoids

A

quality

check

of

heparinoid

batches

may

be

performed

using

the

antibodies

since

each

heparinoid

shows

a

specific

immunoprofile

In

conclusion

this

study

presents

a

novel

way

of

characterizing

heparinoids

using

immunoprofiling

Reactivity

with

antibodies

was

found

indicative

for

chemical

and

biological

aspects

of

the

heparinoids

and

may

therefore

be

used

for

a

fast

and

simple

screening

of

industrial

batches

for

defined

characteristics

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

antibody anticoagulation heparinoid immunoprofiling