Japan. J. Pharntacol. 25, 303-310 (1975)

EFFECTS

OF

VARIOUS

TOTAL

DRUGS

TRYPTOPHAN

TRYPTOPHAN Heitaroh

303

ON

SERUM

LEVELS

AND

METABOLISM

IWATA,

Hiroshi

FREE

IN

OKAMOTO

AND

BRAIN RATS

and Setsuko

KOH

Department of Pharmacolo y, Faculty of Pharmaceutical Sciences, Osaka University, Toncyama, Toyonaka, Osaka, Japan Accepted April 2, 1975

Abstract Various drugs known to bind to serum albumin were examined to deter mine whether or not they influenced the level of free tryptophan in serum in vitro and in vivo. Possible relationships between the serum free tryptophan level and scrotonin (5-1-IT) synthesis in the brain and the hypothermic effects of these drugs were investigated. Of the drugs examined, sodium salicylate, sodium benzoate and indomethacin caused a significant increase in the concentration of serum free trypto phan and stimulated the synthesis of 5-HT in the brain. Hypothermia induced by salicylate and indomethacin was potentiated by pretreatment with pargyline, a mono atnine oxidase inhibitor. Administration of benzoate did not cause any change in body temperature, but after pargyline a hypothermia did occur. However, pretreat ment with parachlorophenylalanine, an inhibitor of 5-f-IT synthesis, did not influence the hypothermia induced by salicylate and indomethacin. Relationship between the hypothermic effect and the increase of 5-HT synthesis in the brain after a large dose of salicylate and indomethacin is discussed. The rate of synthesis availability changes

of 5-HT in the brain is known

of the precursor

amino

acid, L-tryptophan

in the level of free, but not total

with change

in the brain

serum albumin, to compete

tryptophan

and a wide variety

with L-tryptophan

sible that certain

by increasing

the synthesis

in vivo.

of compounds,

increasing

tryptophan

centrations

drugs

and

15:00 hr.

of tryptophan,

stored

at

-20'C

it was shown

that

are well correlated

is the only amino

including

by the

salicylate, albumin

acid bound

to

have been found (3, 4).

may also compete

It is pos

with tryptophan

in the serum and there

drugs which bind to serum albumin

in rat serum in vitro and on 5-HT synthesis the synthesis

AND METHODS

rats,

weighing

The brain was quickly

160 to 220 g, were decapitated removed,

5-1-IT and 5-hydroxyindoleacetic

tryptophan

in

of 5-HT in the brain and the

was also examined.

Male Sprague-Dawley

Serum for analyzing then

in the plasma

the level of tryptophan

between

MATERIALS

tween 13:00

Recently,

site on serum

the effects of various

The relationship

effect of some

Animals:

tryptophan, L-Tryptophan

principally

of 5-HT in the brain.

we examined

on the ratio of free to bound

hypotherric

(I).

drugs which bind to serum albumin

In this work,

the brain

(2).

for this binding

for its binding site; consequently,

to be regulated

was obtained

until analyzed.

homogenized

acid (5-H1AA)

by the centrifugation

be

and the con were measured.

of blood after clotting

304

H. IWATA, H. OKAMOTO & S. KOH Measurement of tryptophan in the serum and brain:

Tryptophan

in the serum and

brain was assayed fluorometrically by the method of Denkla and Dewey (5).

Serum free

tryptophan was determined using 0.1 ml samples of ultrafiltrates, prepared by centrifuging 1 ml of serum in dialysis tubing (Size 8/32, Visking Co.) at 3,000 g for 2 hr at 0-4°C. Studies on rat serum in vitro: aspirin, atropine sulfate, and pento NaCI.

Indomethacin,

Sodium salicylate, sodium benzoate, sulfathiazole, and phenobarbital sodium were dissolved in 0.9

phenylbutazone and aminopyrine were dissolved in a NaOH solu

tion and diluted with 0.9% NaCI.

Next, 0.1 ml of the test drug solution was added to

0.9 ml of normal rat serum and the mixture was analyzed for free tryptophan.

As con

trols, 0.1 ml of saline or dilute alkali was added in place of the drug solution.

The final

pH range of these mixture was 6.5 to 8.0, in which only a negligible change of free trypto phan level was observed. Measurements of 5-HT and 5-HIAA:

Brain 5-HT was determined by the method

of Bogdanski et al. (6) and brain 5-H[AA by the method of Curzon and Green (7) and Udenfriend et al. (8). Measurements of body temperature:

Body temperature

a thermistor probe about 5 cm into the rectum.

was measured by inserting

All experiments were conducted at a

room temperature of between 20 and 22°C. RESULTS Effects of various drugs on the ratio of free to total tryptophan in rat serum in vitro Fig. 1 shows the in vitro effects of various drugs, known to bind to serum albumin, on the ratio of free to total tryptophan in rat serum.

The drugs were tested at final con

centrations of 2 mM; and of those examined indomethacin,

salicylate, benzoate, sulfa

thiazole and phenylbutazone significantly increased the serum concentration of free trypto

FIG. 1. Effects of various drugs on the ratio of free to total tryptophan serum in Vitro. Drugs were added to serum at a concentration of 2 mM. Values arc means of results in 4-8 experiments. * Significantly different from control (P