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0022-1 554/82/08082404$02.75

The

Journal

Copyright

©

of

1982

Histochemistry

by

The

and

Histochemical

Cytochemistry

Society,

Study

MELVIN

of

H.

Serotonin

VAN

WOERT,

lB

Inc.

in

II

Neuropsychiatric

MAGNUSSEN,

YOUNG

HEE

Vol.

(),

No.

8,

Disorders’

LOWE,

and

pp.

824-82 .

1982

Printed in U.S.A.

 

EUNYONG

 

CHUNG

 

HWANG

 

Departments

 

ofNeurology

(M.H.V.W.:

 

Y.H.L.;

E.C.H.).

and

Pharmacology

 

(M.H.V.W.).

 

Mount

 

Sinai

School

ofMedicine.

Nezi

York.

Neu

York

10029

 

and

Department

of

Neurology

(1.M.).

Aarhus

 

Kommunehospital.

 

DK-8000.

Aarhus.

 

C.

Denmark

 

Received

for

publication

March

16,

1982;

accepted

March

25,

1982

(OA

82-157SA)

 

KEY

WORDS:

Senotonin,

Humans;

High-pressure

 

liquid

chno-

 

matognaphy;

 

Cenebnospinal

fluid

; Plasma;

Myoclonus.

 

Disturbances

 

of

serotonin

(S-HT)

metabolism

have

been

de-

2.

Cerebrospinal

 

fluid

 

(CSF)

5-hydroxyindoleacetic

acid

(5-

tected

 

in

numerous

 

human

diseases.

Perhaps

the

best

known

 

HIAA),

the

major

metabolite

of

5-HT,

reflects

the

synthesis

and

understood

 

serotonin

 

disorder

is

the

carcinoid

syndrome,

and

turnover

 

of

5-HT

 

in

the

central

nervous

system.

It

is

caused

 

by

the

rapid

synthesis

of

S-HT

by

a

carcinoid

tumor

 

possible

to

increase

 

the

sensitivity

of

this

index

of

S-HT

turn-

originating

from

the

entenochnomaffin

cells

in

the

small

in-

over

by

pretreatment

 

of

the

patient

with

probenecid,

which

testine.

 

Platelets

 

possess

 

specific

subcellular

storage

sites

for

blocks

the

secretion

of

S-HIAA

out

of

the

CSF

and

brain,

5-HT

and

preferentially

 

accumulate

5-HT

synthesized

in

the

thus

giving

a

measure

 

of

the

accumulation

of

CSF

5-HIAA

gut.

Abnormalities

 

of

platelet

accumulation

of

5-HT

are

found

 

over

a given

period

of

time.

CSF

5-HIAA

levels

are

lower

in

in

several

 

hemotological

 

disorders

such

as

storage

pool

disease

some

patients

 

with

depression

and

also

in

patients

with

certain

and

Hermansky-Pudlak

 

syndrome.

types

of

myoclonus

 

before

and

after

the

administration

of

 

There

is

considerable

 

evidence

that

5-HT

 

abnormalities

probenecid

 

(3,18,19).

 

also

may

be

present

 

in

a number

of

neuropsychiatnic

diseases.

3.

Platelet

S-HT

and

 

plasma

S-HIAA

also

have

been

re-

However,

 

the

relationship

of

5-HT

 

to

the

pathogenesis

of

ported

to

be

abnormal

in

some

neurological

diseases.

5-HT

is

these

 

central

nervous

 

system

disorders

is

less

well

understood

usually

measured

 

in

platelets

instead

of

in

plasma

because

it

compared

 

to

peripheral

S-HT

diseases.

Several

techniques

have

 

is

concentrated

 

about

 

25,000

times

more

in

platelets

than

been

 

used

to

uncover

abnormalities

 

of

5-HT

metabolism

in

plasma

in

man

(9).

During

 

migraine

headache

attacks

platelet

neunopsychiatric

 

disorders:

 

5-HT

levels

decrease

 

significantly

as

compared

with

headache-

 

I

.

Evaluation

of

the

effect

of

S-HT

agonists

and

antagonists

free

intervals

(15).

On

the

other

hand,

platelet

S-HT

is

often

on

clinical

signs

and

symptoms

 

in

patients

 

with

neurological

increased

 

in

patients

 

with

infantile

autism

(6).

and

psychiatric

diseases.

 

The

hypothesis

 

that

brain

S-HT

de-

4.

Platelets

contain

 

a

high

affinity

S-HT

uptake

system

ficiencies

play

a

role

in

the

pathogenesis

of

a

subcategory

of

which

is

similar

to

that

present

in

serotonergic

neurons.

Fur-

mental

depression

and

certain

 

types

ofmyoclonus

 

is supported

thermore,

 

the

5-HT

uptake

system

in

both

platelets

and

brain

by

studies

with

5-HT

precursors.

Both

tryptophan

and

L-5-

contains

specific

binding

 

sites

for

5H-imipramine,

an

antide-

hydroxytryptophan

 

(L-5-HTP),

 

as

well

as

specific

S-HT

uptake

pressant

drug

that

blocks

S-HT

uptake

(5,16).

Investigation

blockers,

have

been

reported

to

improve

depression

and

al-

of

platelet

5-HT

uptake

 

and

H-imipramine

binding

may

in-

leviate

myoclonus

 

(18,19).

The

methodology

 

and

specific

pro-

dicate

abnormalities

 

of

these

systems

in

neuropsychiatnic

dis-

tocols

used

in

human

pharmacological

 

investigation

are

reg-

orders.

In

fact,

platelet

5-HT

uptake

( 1 7)

and

5H-imipramine

ulated

and

monitored

by

the

Food

and

Drug

Administration

binding

to

platelet

 

membranes

(2)

from

depressed

patients

(FDA)

(see

Appendix).

 

were

lower

than

normal.

Platelet

5-HT

and

5-HT

uptake

 

have

been

shown

to

be

decreased

in

levels Down’s

syndrome

as

well

(7,14).

 
 

5.

The

most

direct

 

method

of

looking

for

brain

S-HT

ab-

 

‘Supported

by

U.S.

Public

Health

Senvicegnants

NS

12341,

Clin-

normalities

is

the

measurement

of

5-HT

and

S-HIAA

con-

ical

Center

for

Research

on

Parkinson’s

 

and

Allied

Diseases

NS

I

1 63

1,

RR

0007

1 from

the

Division

of

Research

Resources,

General

Clinical

centrations

and

3H-5-HT,

 

H-lysergic

acid

diethylamide

(LSD),

Research

Center

Branch,

and

a

Merck

International

Fellowship

in

and

5H-imipramine

 

receptor

binding

in

autopsy

tissue.

This

Clinical

Pharmacology

 

(to

I.M.).

approach

 

has

been

particularly

productive

in

providing

new

824

SEROTONIN

IN

NEUROPSYCHIATRIC

DISORDERS

information

 

regarding

defects

in

S-HT

metabolism

in

mental

depression.

 

Materials

 

and

Methods

Quantitative

Determination

 

of

5-HIAA

and

Homovanillic

Acid

(HVA)

in

CSF

In

order

to

measure

simultaneously

CSF

5-HIAA

and

HVA

(dopa-

mine

metabolite)

by

high-performance

 

liquid

chromatography

 

(HPLC),

we

developed

 

the

following

method.

 

The

LC

system

consists

of

a model

 

6000

A

solvent

delivery

system

and

model

U6K

loop

injector

from

Waters

Associates

(Milford,

MA)

and

LC-4A

Ampenometnic

 

detector

coupled

 

to

a TL-5

glassy

carbon

electrode

 

from

Bioanalytical

 

Systems

 

(W.

Lafayette,

IN).

The

chno-

matographic

column

 

is

a

Waters

uBondapak

 

C-18

reverse-phase

 

col-

umn

(3.9

mm

inner

diameter

 

x

30

cm).

 

The

mobile

plase

consists

of

potassium

phosphate

buffer

(0.

1

M,

pH

3.7)

containing

1

mM

ethylenediamine

 

tetnaacetic

acid

(EDTA)

and

7Yc

acetonitnile.

 

The

flow

rate

is

2.0

mI/mm

and

the

column

is

operated

at

ambient

temperature.

The

electrochemical

detector

is

set

at

a

potential

 

of

0.85

V

and

a

sensitivity

of

2

nA/V.

 
 

CSF

is

obtained

 

by

lumbar

tap

in

the

lateral

decubitus

 

position,

and

0.05

ml

of

6

N

HC1

is

added

promptly

to

each

ml

of

CSF

and

the

sample

stoned

at

-

20#{176}Cuntil

assayed

(within

2

weeks).

 

Since

there

is

a

concentration

 

gradient

for

monoamine

 

metabolites

 

in

the

CSF,

the

same

fraction

of CSF

removed

during

 

lumbar

puncture

should

be

assayed

 

in

each

patient.

The

spinal

tap

is

performed

 

at

9:00

AM.

after

a

12

hr

period

of

bed

nest

and

fasting,

since

both

exercise

 

and

diet

can

alter

CSF

monoamine

metabolite

levels.

 

0.5

ml

of

0.1

M

sodium

acetate

buffer

(pH

1.6)

containing

 

1%

ascorbic

acid,

and

10

jal

(containing

 

50

ng)

of

internal

standard

(4-

hydnoxy-3-methoxyphenylpropnionic

 

acid

(HMPPA)

are

added

to

each

1

ml

sample

ofCSF

(duplicate)

in

a

13

ml

stoppered

centrifuge

 

tube.

Four

milliliters

of

ethyl

acetate

is

added,

the

mixture

shaken

on

a

Vortex

mixer

for

2

mm,

and

centrifuged.

 

The

ethyl

acetate

layer

is

transferred

to

a

test

tube

and

extraction

 

with

ethyl

acetate

(4

ml)

is

repeated.

The

combined

ethyl

acetate

 

fractions

are

taken

down

to

dryness

under

nitrogen

and

the

residue

dissolved

in

200

jal

methanol,

and

5-10

jal

injected

into

the

HPLC.

The

recovery

ofHVA

was

86 A

and

5-HIAA

was

467c.

Normal

CSF

5-HIAA

levels

range

from

12-

38

ng/ml

and

HVA

from

18-105

ng/ml.

 
 

825

centrifuge,

and

add

6

ml

of

n-hexane

to

the

supennatant

and

vortex.

After

centnifugation,

apply

25

jal ofextnacted

plasma

to HPLC

column

containing

the

cation-exchange

 

resin,

Aminex

A-S

(Bio-Rad

 

Labona-

tories,

Richmond,

 

CA).

 

The

column

buffer

 

consists

of

0.

1

M

sodium

penchlonate

and

0.

1

M

sodium

acetate

 

(adjusted

 

to

pH

5.0

with

acetic

acid)

with

a

flow

rate

of

0.3

mI/mm

and

temperature

of

70#{176}C.The

effluent

of

the

column

is

reacted

with

the

fluorogen

 

reagent

 

(3

mmol

ofOPT,

20

mg

FC-134

and

150

mg

Bnij

in

1

liter

of9

N

HCI),

and

the

fluorescence

detected

with

a fluoronephalometen

 

(excitation

wave-

length:

360

nm;

emission

measured

 

at

480

nm).

5-HT

and

5-HIAA.

Thirty-six

milliliters

of

venous

blood

is col-

lected

in

a

50

ml

plastic

tube

containing

 

4

ml

of0.7%

 

saline

with

1 /

EDTA

as

anticoagulant,

and

centrifuged

at

1300

rpm

for

10

mm

at

26#{176}Cin

an

International

Centrifuge.

 

Six

milliliters

of

platelet-rich

 

plasma

(PRP)

are

transferred

to

 

another

plastic

tube

and

 

an

aliquot

taken

for

determination

of

the

platelet

count

by

an

automated

 

elec-

tronic

counter

(Coulten

Counter,

Model

S).

Three

milliliters

 

of

PRP

are

centrifuged

 

at

 

4

100

rpm

for

20

mm

to

remove

the

platelets,

and

this

platelet-free

plasma

(PFP)

is

frozen

and

stored

at

-

20#{176}Cuntil

assayed

for

5-HIAA.

 

Both

5-HT

and

5-HIAA

are

measured

 

within

1

week

after

collection

of

the

blood

sample.

 

Three

milliliters

 

of

PRP

or

PFP

is thoroughly

mixed

 

with

7.5

ml

of

ice-cold

0.4

N

penchlonic

acid

 

containing

0.15

ml

of

10

f

EDTA

and

0.07

ml

of

5

f

sodium

metasulfite

 

solution.

The

mixture

is

cen-

tnifuged

at

5000

x

g

for

10

mm

at

4#{176}C.The

pH

of

the

supennatant

is

adjusted

with

2N

 

potassium

carbonate,

to

5-6

for

5-HT

 

or

2.2

for

5-HIAA

before

 

chromatography.

 

5-HT

is purified

 

on

a weak

cation-exchange

 

column

 

(Amberlite

CG5O,

100-200

 

mesh,

0.5

cm

in

diameter

and

5.5

cm

long

column)

and

eluted

with

2.5

ml

of2

N

HCI

(1).

0.2

ml

aliquots

are

mixed

with

1.2

ml

0.004

OPT

in

10

N

HCI

containing

0.17

cysteine

as

mod-

ified

from

the

method

ofCurzon

 

and

Green

(8).

The

mixture

is heated

to

Bowman

fluorescence

70#{176}Cfor

5-HIAA

20

mm

and

cooled

to

is used

spectrofluonometer

product

is isolated

(activation:

using

360

a strong

room

for

nm;

temperature.

detection

emission

of

cation-exchange

the

An

Aminco-

indole-OPT

470

nm).

column

(Bionad

AG

SOW-X4,

200-400

mesh,

 

0.5

cm

in

diameter

and

5.5

cm

long

column)

and

eluted

in

6

ml

of

60c

methanol

of

which

0.4

ml

is

processed

with

OPT

for

fluonometry

 

as

described

by

Curzon

and

Green

(8)

and

Atack

and

Magnussen

(4).

 

Recoveries

of

5-HT

and

5-HIAA

were

70-77Y

and

74-84%,

respectively.

Normal

5-HT

levels

range

from

26-185

ng

pen

2.5

x

i0

platelets

(approximately

 

1.0

ml

ofblood).

Normal

5-HIAA

levels

 

range

from

19-133

ng

per

ml

of

plasma.

 

Quantitative

 

Determination

of

The

advantage

ofisolating

platelets

 

(PRP)

for

5-HT

determination

.5-Hydroxytryptophan

 

(5-HTP),

 

5-HT

and

 

is

to

avoid

the

breakdown

 

of

5-HT

by

the

penoxidase-like

 

activity

of

.5-HJAA

 

in

Blood

of

Patients

Treated

with

oxyhemoglobin.

Furthermore,

 

the

percentage

 

release

of

5-HT

from

 

clotting

 

blood

(approximately

50Y

)

is

quite

variable.

On

the

other

L-5-HTP

and

Carbidopa

hand,

it

must

be

recognized

that

circulating

platelets

are

heteroge-

The

serotonin

precursor

therapy,

L-5-HTP

with

a peripheral

decar-

neous

in

size,

density,

and

sedimentation

 

characteristics

and

only

a

bylase

inhibitor,

canbidopa,

has

been

used

in

clinical

trials

of

several

fraction

 

of

the

platelets

 

in

the

blood

sample

are

obtained

by

centnif-

neurological

 

and

psychiatric

disorders.

It

is

important

to

measure

the

ugation.

 

phanmacokinetics

of

any

new

drug

therapy

so

that

one

can

determine

 

factors

 

such

as

absorption

of

drug,

dosage,

optimal

time

intervals

L-5-HTP

 

Pharmacokinetics

 

in

Myoclonic

 

between

 

doses,

catabolism,

etc.

In

our

laboratory

we

have

used

the

Patients

 

following

method

to

study

the

pharmacokinetics

 

of

L-5-HTP

in

pa-

tients.

 

Myoclonus

 

consists

of

involuntary

 

muscle

jerking

movements

 

that

may

be

associated

 

with

many

 

diseases

of

the

central

ner-

 

L-5-HTP.

Plasma

L-5-HTP

is

determined

 

by

HPLC

separation

vous

system.

Myoclonus

due

to

certain

disease

processes,

pan-

and

fluonometnic

detection

after

o-phthaldialdehyde

(OPT)

as

de-

ticulanly

 

post-anoxic

 

intention

 

myoclonus

and

progressive

my-

scnibed

 

by

Engbaek

HCIO.,

and

Magnussen

(10).

Add

4

ml

ofacidified

 

butanol

oclonus

 

epilepsy,

may

 

result

from

a

brain

S-HT

deficiency.

(4

ml

of

70’ /

per

liter

of

1-butanol)

 

to

750

jal

plasma,

mix,

These

patients

 

have

low

levels

of

CSF

S-HIAA

(post-anoxic

826

intention

myoclonus,

 

61.5

±

7.0

ng/ml;

progressive

 

myo-

clonus

epilepsy

4

1.9

±

6.5

ng/ml;

controls

90.3

±

1 1.0

ng/

ml)

after

probenecid

pretreatment

and

they

respond

 

to

therapy

with

L-5-HTP

in

combination

with

carbidopa

 

(2

1).

The

pe-

ripheral

L-aromatic

 

amino

acid

decarboxylase

inhibitor,

car-

bidopa,

prevents

the

conversion

of

L-5-HTP

to

5-HT

outside

the

brain

and

thereby

increases

S-HT

synthesis

from

L-S-HTP

 

in

the

central

nervous

system

while,

at

the

same

time,

de-

creasing

the

peripheral

side

effects

of

the

precursor

 

amino

acid.

Addition

of

fluoxetine,

a specific

5-HT

uptake

inhibitor,

 

to

the

drug

combination,

reduces

the

dose

of

L-5-HTP

to

about

#{188}with

the

same

improvement

in

myoclonus

 

and

de-

creased

side

effects

(20).

Since

fluoxetine

 

potentiates

sero-

tonin

only

at

the

serotonergic

 

synapse

this

clinical

 

study

pro-

vided

further

evidence

for

brain

serotonin

deficiency

in

certain

types

ofmyoclonus.

Finally,

the

S-HT

antagonist

methysergide

 

has

been

reported

to

counteract

the

anti-myoclonic

 

action

of

L-5-HTP

and

carbidopa

( I

1).

We

have

investigated

the

pharmacokinetics

 

of

L-5-HTP

administered

with

and

without

peripheral

decarboxylase

in-

hibitors

using

the

methods

 

described

above.

Venous

blood

samples

were

drawn

(for

L-S-HTP,

plasma

 

5-HIAA,

 

and

plate-

let

5-HT)

every

half

hour

for

2

hr

and

thereafter

 

hourly,

from

1 1 :00

to

1 7:00

hr.

We

observed

that

a

single

oral

dose

of

carbidopa

(50

mg

1

hr

prior

to

L-5-HTP)

is

equipotent

to

14

days

pretreatment

(50

mg

4

times

a

day)

in

inhibiting

the

peripheral

decarboxylation

of

L-5-HTP,

 

indicating

 

that

pre-

loading

with

carbidopa

is

unnecessary

 

(13).

Carbidopa

 

en-

hances

the

rise

in

plasma

concentration

 

of

L-5-HTP

 

5-

to

15-

fold

after

administration

of

a

single

 

oral

dose

of

L-5-HTP

as

well

as

counteracting

the

increase

in

plasma

5-HIAA

 

levels

induced

by

L-S-HTP

alone

(12,13).

 

Platelet

5-HT

remained

 

normal

after

a single

oral

dose

ofL-5-HTP

 

(2

mg/kg

body

wt),

with

on

without

carbidopa,

over

a

6

hr

period

of

examination.

The

reason

for

the

lack

of

change

in

platelet

5-HT

levels

in

patients