0022-1

554/82/08082404$02.75

The

Journal

of Histochemistry
1982 by The

and
Histochemical

Cytochemistry
Society, Inc.

Vol.

(),

No.

8,

pp.

824-82.

1982

Copyright

Printed in U.S.A.

II

Study
MELVIN

of Serotonin
H. VAN WOERT, lB

in Neuropsychiatric
MAGNUSSEN, YOUNG HEE

Disorders
LOWE,

and
School ofMedicine. Nezi York.

EUNYONG
Departments Neu York 10029 for

CHUNG
ofNeurology and (M.H.V.W.: Department March

HWANG
Y.H.L.; of Neurology 16, 1982; E.C.H.). (1.M.). accepted and Aarhus March 25, Pharmacology Kommunehospital. 1982 (OA (M.H.V.W.). DK-8000. 82-157SA) Mount Sinai Aarhus. C. Denmark

Received

publication

KEY

WORDS:

Senotonin, Cenebnospinal

Humans; fluid
;

High-pressure

liquid

chno-

matognaphy;

Plasma;

Myoclonus.

Disturbances of serotonin tected in numerous human and understood caused by the originating testine.
5-HT

(S-HT) metabolism diseases. Perhaps

have been dethe best known

2. HIAA),

Cerebrospinal

fluid

(CSF)

5-hydroxyindoleacetic

acid

(5-

serotonin disorder rapid synthesis of the entenochnomaffin specific possess

is the carcinoid syndrome, S-HT by a carcinoid tumor cells subcellular in the storage small sites infor

and
possible

the turnover
to

major metabolite of 5-HT in

increase the

of 5-HT, the central

of this

reflects nervous
index

the synthesis system. It is

of S-HT turn-

sensitivity

from Platelets

over
blocks

by

pretreatment
the secretion

of
of

the
S-HIAA

patient
out

with
of

probenecid,
the CSF and

which
brain,

gut. and also

and preferentially accumulate 5-HT Abnormalities of platelet accumulation

hemotological disorders such

synthesized in the of 5-HT are found

pool disease

thus over some types

3.

giving a given

a measure period

of of time. depression before

and

the

accumulation CSF 5-HIAA and also and after

of CSF levels are in patients with the administration

also have

5-HIAA lower certain of

been 5-HT reis

in

in several

as storage

Hermansky-Pudlak There is considerable may be present

syndrome. evidence in a number of 5-HT disorders

diseases.

patients with of myoclonus (3,18,19).

S-HT abnormal Platelet to be

that

5-HT

abnormalities diseases. of
have

probenecid
ported

of neuropsychiatnic to the is less

Several

plasma in some

S-HIAA neurological

However, the relationship these central nervous system

compared to peripheral S-HT

pathogenesis well understood

techniques

diseases.

usually measured is concentrated in plasma

5-HT

in platelets about 25,000

instead times

of in plasma because it more in platelets than attacks platelet with headacheS-HT

uptake

been used to uncover abnormalities neunopsychiatric disorders: I . Evaluation of the effect of S-HT on
and

of

5-HT

metabolism and with

brain

in man (9). levels decrease intervals

in Platelets

During migraine headache significantly as compared On the

with a

agonists
that

antagonists neurological
S-HT de-

free
4.

(15).
patients

other
infantile high

hand,
autism affinity

platelet
(6). S-HT

is often
system

clinical
psychiatric

signs

and
diseases.

symptoms
The

in patients
hypothesis

increased

contain

ficiencies play a role in the pathogenesis of a subcategory mental depression and certain types ofmyoclonus is supported
by studies with 5-HT precursors. Both tryptophan and

of
L-5-

which is similar to that present in serotonergic neurons. thermore, the 5-HT uptake system in both platelets and contains pressant
of platelet

Furbrain antide-

specific drug
5-HT

binding that blocks

uptake

sites S-HT
and

for

5H-imipramine, uptake (5,16).

binding

an

hydroxytryptophan blockers, leviate tocols ulated

(FDA)

(L-5-HTP), been reported (18,19). The

as well

as specific and investigation Drug

S-HT specific

uptake and are alproreg-

Investigation
may in-

have myoclonus used and (see

to improve methodology Food and

depression

H-imipramine

in human monitored Appendix).

pharmacological by the

dicate orders.
binding

abnormalities of these In fact, platelet 5-HT

to platelet membranes

systems uptake
(2)

in neuropsychiatnic ( 1 7) and 5H-imipramine from levels depressed and 5-HT syndrome

for brain

dis-

Administration

patients uptake as well

S-HT ab-

were have
5.

lower been
The

than shown
most

normal.

Platelet

5-HT

to be decreased
direct method of

in Downs
looking

(7,14). Supported by U.S. Public Health Senvicegnants NS 12341, Clinical Center for Research on Parkinsons and Allied Diseases NS I 1 63 1, RR 0007 1 from the Division of Research Resources, General Clinical

normalities centrations

is the and

measurement H-lysergic receptor particularly

of

5-HT acid

and

S-HIAA

con(LSD), This new

3H-5-HT,

diethylamide

Research
Clinical 824

Center
Pharmacology

Branch,
(to

and
I.M.).

a Merck

International

Fellowship

in

and 5H-imipramine approach has been

binding in autopsy tissue. productive in providing

SEROTONIN

IN

NEUROPSYCHIATRIC

DISORDERS

825

information depression.

regarding

defects

in S-HT

metabolism

in mental

centrifuge,

and the Richmond,

add

6 ml

of n-hexane resin, column acetate mI/mm with

to the Aminex buffer (adjusted and the

supennatant

and

vortex.

After
tories,

centnifugation,

apply
CA). rate of The

25 jal ofextnacted

plasma
A-S consists temperature fluorogen

to HPLC
(Bio-Rad 5.0 of reagent of9 with

column
Labonaacetic

containing

cation-exchange

of 0. 1 M sodium 70#{176}C. The (3 mmol and

penchlonate

and 0. 1 M sodium
a flow of the 0.3 column is reacted

to pH

Materials
Quantitative Homovanillic
In order
mine The and and model LC-4A to

and

Methods
of 5-HIAA in CSF
CSF
liquid 6000 Waters coupled (W. uBondapak x 30 cm). phosphate tetnaacetic mI/mm of 2 nA/V. and electrochemical buffer acid the detector (0. 1 M, (EDTA) column is is set of potassium ethylenediamine flow rate The a sensitivity is 2.0

acid) effluent

with

Determination Acid
measure

and

(HVA)
simultaneously

ofOPT, 20 mg FC-134 the fluorescence detected

length: 360 nm; emission

and 150 mg Bnij in 1 liter with a fluoronephalometen

measured at 480 nm).

N HCI),

(excitation

wave-

5-HIAA

and HVA

(dopa(HPLC), lected system MA) carbon chnocol5-HT

metabolite) LC system U6K from (3.9 The 3.7) 7Yc mm mobile containing acetonitnile. at ambient

by high-performance

chromatography A solvent Associates to a TL-5 Lafayette, C-18 delivery (Milford, glassy IN). reverse-phase

and

5-HIAA.

Thirty-six
tube containing

milliliters

of venous

blood
saline with

is col1/

we developed

the following
consists loop injector

method.
of a model from detector Systems

in a 50 ml plastic

4 ml of0.7%

Ampenometnic Bioanalytical column inner plase

electrode matographic umn pH and

The

is a Waters diameter consists 1 mM The

EDTA as anticoagulant, and centrifuged at 1300 rpm for 10 mm at 26#{176}C an International in 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 electronic 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}C until
assayed 1 week for after 0.4 ml of at 5000 5-HIAA. collection N 5f x Both 5-HT blood and 5-HIAA sample. are measured within 7.5 EDTA is cenml of the

operated at a potential

temperature. V and

Three
of ice-cold and 0.07

milliliters

of PRP
penchlonic sodium g for

or PFP
acid metasulfite

is thoroughly
0.15 solution.

mixed
ml of The

with 10f

of 0.85 ml of stoned 6 N at

containing

CSF
and the there 0.05 sample

is obtained

by lumbar
HC1
-

tap in the
promptly assayed for spinal and metabolite acetate buffer 50 acid

lateral
(within

decubitus
ml 2 weeks). metabolites

position,
of CSF and Since in the

mixture

is added 20#{176}C until gradient

to each

tnifuged is adjusted

10 mm

at 4#{176}C. The

pH

of the supennatant

is a concentration

monoamine

CSF,
be after diet 0.5 ascorbic

the same

fraction

of CSF removed
The nest of bed

during
tap fasting,

lumbar
is performed since

puncture
at 9:00 exercise containing standard added shaken acetate acetate taken centrifuge both

should
AM.

assayed can alter ml

in each CSF 0.1 and ofCSF of for to The under a test

patient. monoamine M sodium

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 ified eluted from with the 2.5 OPT method ml of2 in N HCI and (1). 0.2 ml aliquots 0.17 (8). The are cysteine mixture mixed with 1.2 ml 0.004 10 N HCI containing Green as modis heated

a 12 hr period of

and 1% (4to each tube. on layer a is is to 86A 12-

levels. (pH ng) 1.6) are mixture ethyl ethyl are of internal

ofCurzon

acid,

10 jal (containing (duplicate) ethyl 2 mm, tube and into 467c. from the Normal 18-105 acetate and and ethyl the

hydnoxy-3-methoxyphenylpropnionic 1 ml sample Four Vortex transferred repeated. dryness and and

38

(HMPPA) the The with fractions dissolved

to 70#{176}C 20 mm and cooled for to room temperature. An AmincoBowman spectrofluonometer is used for detection of the indole-OPT
fluorescence product (activation: mesh, in for 6 ml of 360 0.5 60c cm nm; emission 470 and of which by nm).

in a 13 ml stoppered is added, centrifuged. extraction acetate residue HPLC. CSF The ng/ml.

milliliters mixer

5-HIAA
AG column) SOW-X4, and

is isolated
200-400 eluted

using a strong

cation-exchange
in diameter methanol as described
70-77Y

column
5.5 0.4 Curzon and 5-HIAA

(Bionad
cm long ml is and

(4 ml) down was from

combined nitrogen was

in 200 ofHVA levels

jal methanol,

processed with OPT Green (8) and Atack

Recoveries respectively. i0 platelets of Normal

fluonometry and 5-HIAA levels range

and Magnussen
5-HT

(4).
were from 74-84%, 2.5 levels x 26-185 Normal ng pen

5-10 5-HIAA ng/ml

jal injected and HVA

recovery

5-HT

5-HIAA

range

(approximately

1.0 ml ofblood).

Quantitative .5-Hydroxytryptophan .5-HJAA in

Determination

of

range from 19-133 ng per ml of plasma. The advantage ofisolating platelets (PRP)

for 5-HT
penoxidase-like release variable. platelets

determination
activity of On are 5-HT the other of from

(5-HTP),
of Patients Carbidopa
therapy,
has new of etc. the disorders. drug drug, In our

5-HT
Treated

and
with

is to avoid oxyhemoglobin. clotting hand,

it

the blood must of the

breakdown Furthermore, (approximately be recognized

of 5-HT the 50Y that blood

by the percentage ) is quite circulating sample

Blood

L-5-HTP
The
bylase

and

heteroge-

serotonin
inhibitor, and such doses, method as

precursor
canbidopa, psychiatric of any absorption catabolism, to study

L-5-HTP
been used

with

a peripheral
trials one we can time have to measure

decarof several the determine intervals used the in pa-

neous
fraction ugation.

in size,

density,
platelets

and

sedimentation

characteristics
are obtained

and only
by centnif-

in clinical so that

in the

neurological phanmacokinetics factors between following tients. L-5-HTP. and scnibed (4 ml of fluonometnic

It is important therapy dosage, laboratory optimal

L-5-HTP Patients
Myoclonus that may

Pharmacokinetics
consists of involuntary be associated with many Myoclonus due intention post-anoxic muscle diseases

in Myoclonic
jerking of the and movements central nerpanmy-

pharmacokinetics

of L-5-HTP

Plasma and

L-5-HTP after liter of Magnussen per

is determined o-phthaldialdehyde (10). Add 1-butanol) to

by

HPLC (OPT)

separation as butanol mix, de-

vous ticulanly oclonus These

system.

to certain myoclonus

disease

processes, progressive

detection HCIO.,

by Engbaek 70/

4 ml ofacidified 750 jal plasma,

epilepsy, may result from a brain S-HT deficiency. patients have low levels of CSF S-HIAA (post-anoxic

826

VAN

WOERT,

MAGNUSSEN,

LOWE,

HWANG

intention

myoclonus,

61.5

7.0

ng/ml;

progressive

myo

clonus epilepsy 4 1.9 6.5 ng/ml; controls ml) after probenecid pretreatment and they with L-5-HTP in combination ripheral L-aromatic amino bidopa, prevents the the brain and thereby in acid. to about creased tonin vided types
has

90.3 respond

1 1.0 ng/ to therapy pecar-

QII)

30

with carbidopa acid decarboxylase of L-5-HTP S-HT synthesis while, effects a specific reduces the fluoxetine synapse serotonin S-HT the this of at 5-HT dose the the

(2 1). The inhibitor,

conversion increases system side

to 5-HT outside from L-S-HTP same precursor uptake of L-5-HTP and study seroprotime, inhibitor, to dedeamino

the

central the Addition drug #{188} with side only further

nervous peripheral of fluoxetine, combination, the effects same (20).

creasing the

U) 4 -J

10 0
U, 4) 4 0

improvement Since

in myoclonus potentiates clinical deficiency

at the

serotonergic for brain Finally, the to counteract ( I 1). the

evidence

in certain of

ofmyoclonus. been reported

antagonist methysergide anti-myoclonic action of L-5-HTP

2.0-

a.

L-5-HTP

We

and carbidopa have investigated

pharmacokinetics

,c
U,

administered with hibitors using the

and without peripheral decarboxylase methods described above. Venous

inblood platefrom of 14 the preen4 -J to

4, 0

samples were drawn (for L-S-HTP, plasma 5-HIAA, and let 5-HT) every half hour for 2 hr and thereafter hourly, 1 1 :00 to 1 7:00 hr. We observed that a single oral carbidopa (50 mg days pretreatment peripheral loading hances fold well induced with the 1 hr prior (50 mg to L-5-HTP) is equipotent 4 times a day) in inhibiting indicating that (13). Carbidopa of L-5-HTP oral dose in plasma Platelet

0 3 2 0

dose

4
4

decarboxylation carbidopa rise in plasma

of L-5-HTP, is unnecessary concentration of a single the increase alone (12,13).

5- to 15-

after administration as counteracting by L-S-HTP

of L-5-HTP as 5-HIAA levels 5-HT remained

Figure blood

0123456
HOURS
curve of 5-HTP in plasma, 5-HT in in plasma in a 45-year-old woman with

normal after a single oral with on without carbidopa, The reason for the lack

dose ofL-5-HTP (2 mg/kg body wt), over a 6 hr period of examination. of change in platelet 5-HT levels due in to a also chronic of L-5-HTP capacity for 5-HT patients nine might be the amine. and during

1. Time-concentration platelets, and 5-HIAA

patients given a single dose large gastrointestinal storage

L-S-HTP

post-anoxic intention myoclonus treated for 33 months with L-5-HTP and canbidopa. For 2 days prior to the phanmacokinetic study L-5HTP (350 mg) and canbidopa (50 mg) were taken at precisely 05:00,
1 1 :00, 1 7:00 and 23:00 hr. rather than the usual

and

its major in blood

metabolites from

5-HIAA

were

measured

steady state treatment with the the results of this study in one that mg/kg 6 hr),
L-S-HTP

amino acid. Figure 1 shows of these patients. We found of L-5-HTP (16-65

schedule. Venous blood samples were drawn hr. Hatched bans represent normal range.

between

four times 1 1 :00 and

a day 17:00

during

chronic

oral

administration

ylation effects initial appear

of L-S-HTP

may

account

for

the

gastrointestinal

side during the It would

every 6 hr) in combination the time (tmax) elapsing plus carbidopa

with carbidopa (SO mg every after the first morning dose of measured jaM) The L-5-HTP plasma was 1-2 hr (Magnussen concentration of 5-HT

of diarrhea, anorexia, weeks of therapy with that the best way serotonin which

and nausea that occur L-5-HTP and carbidopa. these blocker reduces in the side uptake greatly its action

to maximum

to minimize

effects

is the L-

concentration (Cmax - 1 3.8-77.8 and Van Woert, in preparation).

addition of a specific to L-S-HTP-carbidopa,

5-HTP

like fluoxetine the optimum central nervous

in platelets (mean values from I 1:00-17:00 hr) from the patients treated with L-S-HTP and carbidopa (0.61-5.03 moL/ 10i platelets) were significantly higher (P <0.001, I-test) than found trols tions patients t-test)
study of

dose (20).

by potentiating

system

in twenty (0.06-0.41 of S-HIAA

age

and

sex

matched

untreated The plasma 11:00-17:00

normal

con-

mol/lOr (mean

platelets). values from

concentrahr) in these

Appendix
Regulation
The
metic 5(FDA) tenmine

( 1. 19-3. 16 jaM)were than found in normal

revealed that carbidopa decarboxylation

also significantly higher(p <0.001, controls (0.10-0.70 jaM). This

produces of only L-5-HTP a partial to 5-HT inhibition and

of Clinical
Kefauver-Hannis
of both 1938, mandates

Studies
amendments
that the when

of New
to the Food they and are

Drugs
Food, Drug Drug and CosAdministration

1962
Act

extracerebral

regulate

the
safety

research
and

and

development

of new

drugs

to deto hu-

HIAA.

This

incomplete

inhibition

of extracerebral

decarbox-

efficacy

administered

SEROTONIN

IN

NEUROPSYCHIATRIC

DISORDERS

827

mans.
pass etc. toxicity

Prior
acute Based

to administration
and on chronic whether evidence toxicity

of any new chemical

studies in at least to the company liver, two

to humans, it must species of animals

bone and these marrow, animal

5. Bniley
affinity
1979

MS, Raisman
binding sites

R, Langen
for

SZ: Human
EurJ

platelets
Pharmacol

possess

high
7,

5H-imipramine.

S8:34

to determine studies,

it is injurious

kidney,

of pharmacological

activity

the pharmaceutical

on individual

investigator

6. Boullin DJ, Coleman 5-hydroxytnyptamine

tune 226:371, to 7. 1970

M, OBrien RA: Abnormalities efflux in patients with infantile

in platelet

autism.

Na-

files

with

the

FDA
Exemption

an application
for a New

known
Drug

as a Notice
(IND) for

of Claimed
permission

Investigation

test the chemical A physician who

mission), institutions proposed tional drug. or Human study

(drug) in humans according to a proposed uses the pharmaceutical companys IND

IND, Subjects any each studies must human person with also Research subject obtain permission Review can participating receive in the from the Committee

protocol. (FDA perhis own for investigatrial dinout in clinical by the the 8.

Boullin uptake
syndrome.J Cunzon gions

DJ, OBrien RA: Abnormalities and binding by blood platelets

Physiol G, ofnat Green brain. AR: (Lond) Rapid 212:287, method

of 5-hydroxytryptamine from children with Downs

1971 for the determination of 5-

his own before

hydnoxytnyptamine 9. Da Prada
catecholamines

and
BnJ

5-hydnoxyindoleacetic
Phanmacol 39:653, 1970

acid

in small localization
and

neof and

In addition,

must
posed ical

sign a consent
investigational investigator. Clinical studies

form,

following

thorough
answering drugs

description
of questions are usually

of the pro-

M, Picotti
and

GB:

Content

and subcellulan
in human

S-hydnoxytnyptamine

animal

blood platelets: monoamine distribution plasma. BrJ Pharmacol 65:653, 1979

10. Engbaek in plasma detection 1978

between

platelets

of investigational

carried

three

stages:
I . Phase

I consists data) and

of administration to determine excretion.

to normal safety, side

volunteers effects,

(guided absorption,

F, Magnussen I: Determination of 5-hydnoxytnyptophan by high-performance chromatography and fluonometnic after phthaldialdehyde reaction. Clin Chem 24:376,

by

animal

catabolism, 2 . Phase 3. Phase fully patients to determine III designed may nor the the

II involves
requires drug

a therapeutic
safety trial in which this a larger,

trial in a small group

and efficacy. well-organized multicentered, approximately trial is the an usually study) inactive

of patients
careto 3000

1 1. Magnussen I, Dupont E, Engbaek F, de Fine Olivanius B: Posthypoxic intention myoclonus treated with 5-hydroxytryptophan
and an extracerebral 57:289, 1978 12. decarboxylase inhibitor. Acta Neunol Scand

short-term

1000

participate; patient knows

a placebo-con-

trolled,
cian using

double-blind
investigational

investigation
(during drug or

in which

neither
whether compound

the physithey are (pla-

Magnussen canboxylase tryptophan

I, Enbaek F: The effects of aromatic amino acid deinhibitors on plasma concentrations of 5-hydroxyin man. Acta Pharmacol Toxicol 43:36, 1978 MH: Plasma acsingle dose LToxicol 49:184,
concentration of

1 3. Magnussen

cebo.
All New ceutical of the Drug data Application to market from animal and clinical studies (Phases by the I, II, and as pant 14.

I, Jensen TS, Rand JH, Van Woert cumulation and metabolism of orally administered 5-hydroxytnyptophan in man. Acta Phanmacol
1981 McCoy EE, 1968 Rostafinsky MJ, Fishbunn C: The

III) are statistically

company

analyzed
(NDA),

and
the

submitted
which drug. the The entire NDA million

to the FDA
granting

of a

serotonin
12:18,

by platelets

in Downs

syndrome.

J Ment

Defic

Res

is a request

pharmaby the

of a NDA

FDA
for use a drug

allows
until

the pharmaceutical
Currently ofa from successful $20-SO approval to cost

company

to make
process takes dollars. from

the drug
from 7-10

available
years and

15.

by all physicians.

discovering

Muck-Seler D, Deanovic Z, Dupelj and S-HT releasing factor in plasma ache 19:14, 1979

M: Platelet serotonin of mignainous patients.

(5-HT) Head-

is estimated

16.

Paul SM, Rehavi M, Skolnick P. Goodwin specific high affinity binding sites for(5H) platelets. Life Sci 26:953, 1980

FK: Demonstration of imipramine on human

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Tuomisto J, Tukiainen E, Ahlors UG: Decreased uptake of 5hydroxytnyptamine in blood. Platelets from patients with endogenous depression. Psychopharmacology 65:14 1, 1979
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TBB,

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comor

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D, Chung

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2 1 . Van Woent in myoclonus.

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