A SIMPLE AND SENSITIVE FLUOROMETRIC ASSAY METHOD FOR TAURINE USING HIGH-VOLTAGE PAPER ELECTROPHORESIS

A SIMPLE

AND

METHOD

FOR

SENSITIVE TAURINE

PAPER Kazuaki

FLUOROMETRIC USING

ASSAY

HIGH-VOLTAGE

ELECTROPHORESIS

YOSHIKAWA

and Kinya KURIYAMA

Department of Pharmacology, Kyoto Prefectural University of Medicine, Kamikyo-ku, Kyoto 602, Japan Accepted July 6, 1976

Abstract-A simple and sensitive fluorometric assay method for taurine (2-amino ethanesulfonic acid) has been developed. For the separation of taurine, high voltage paper electrophoresis subsequent to column chromatographic procedures was employed. Fluorescent product of taurine was yielded by spraying fluorescamine (4-phenylspiro [furan-2(3H), 1'-phthalan]-3, 3'-dione) and borate buffer on the paper, and the fluo rescence was assayed spectro-fluorometrically after eluting with 50% ethanol. The linear relationship between the concentration of taurine and fluorescence developed was achieved over the concentration ranges of 0.5-10 nmoles, and the recoveries obtained were 90-100%. The specificity of this method for taurine was satisfactory and structural analogues involved in the metabolic pathway of taurine did not interfere with the assay. Examples for tissue levels of taurine in various organs of the rat as de termined by this new method are also presented.

Although taurine is known to be present in almost all organs of animals, physiological or functional role of this compound is not fully understood.

For the assay of taurine,

ninhydrin or o-phthaldehyde reaction (1) has been usually employed after separating this compound by column (2), thin layer (3), paper chromatographic voltage paper electrophoresis (4).

procedures, and/or high

These colorimetric methods, however, lack the sensitivity

capable of determining taurine contents in a small amount

of tissue.

Recently, more

sensitive radiometric (5), fluorometric (6) or enzymatic assay (7) methods have been reported. These methods, however, are troublesome and have disadvantages in terms of rapidity, simplicity and difficulties in selecting the assay conditions.

High-voltage electrophoresis,

used in many laboratories today, is thought to have the advantages of rapidity and efficiency in isolating amino acids.

On the other hand, it is known that fluorescamine reacts with

primary amines and develops fluorescence. This reaction has been reported to retain simplicity as well as rapidity (8, 9). Described herein is a sensitive and rapid assay method for taurine using high-voltage paper electrophoresis and fluorescamine as a fluorescent probe. MATERIALS AND METHODS Column chromatography Ion exchange column (cation exchanger; Dowex 50W x 2, 200-400 mesh, H+ form, 0.3 x 10 cm) was prepared according to the procedure described by Iversen and Kravitz (10).

The column was eluted with distilled water, the initial 0.4 ml of eluent was discarded and the following 0.8 ml was collected as a taurine fraction. to dryness in a dryer chamber at 100'C.

This fraction was then evaporated

The residue was redissolved in an adequate volume

of water and taurine content in the solution was adjusted to 0.5-10 nmoles of taurine per 10 t11(see `Results'). High-voltage paper electrophoresis High-voltage

paper electrophoretic

cooling solvent (Toyo Cool X) was used.

apparatus

(Toyo HPE-406) equipped with the

The paper (Toyo No. 51A, nearly equivalent to

Whatmann No. 1, 2 x 40 cm, weight 85 g/m', thickness 0.16 mm, smooth surface) was used without any pre-treatment.

The 10 /al of sample was applied at the point 17 cm from the

anodal end with a micropipette.

The conditions for electrophoresis based on the method

of Mabry and Todd (11) were as follows; Buffer solution used: formate: acetate: H2O=6: 24:170 (pH 2.0), Potential gradient: 90 V/cm, Duration

of electrophoresis:

25 minutes.

After electrophoresis, the papers were dryed with a dryer (hanging type) and the next process was the development of the fluorescence. Fluorescence development The papers were set vertically and the 0.015% fluorescamine (Fluram, Hoffmann-La Roche Co.) in acetone (W/V) was sprayed on manually. buffer (pH 9.5) was applied by spraying. dryed with a dryer. 0

A few minutes later 0.5 M borate

The papers were kept for 10 minutes and then

After detecting the fluorescent spots under the U.V. lamp (wave length:

3650 A), the taurine spot was cut out and eluted for 10 minutes or longer with 3 ml of 50 ethanol

in test tubes.

excitation Preparation

capitated,

of the eluent was measured

rats weighing

and immediately at -80°C

150-200

g or male ddY mice weighing

each organ was removed

and frozen.

in a deep freezer until each assay.

10 Vol. of 75'/0' ethanol

type, 1 ml in a total volume).

dryness

in vacuo and the residue was dissolved

and mixing with 0.2 ml of chloroform, al of the upper

aqueous

at

the tube was centrifuged applied

with

into the microtube

was then evaporated

in 0.1 nil of distilled

was then

g were de

These frozen tissues were

was transferred

The homogenate

phase

25-30

Each tissue was homogenized

and 0.2 nil of the homogenate

(conical

Seventy

spectrofluorometrically

wave lengths of 390 and 490 nm, respectively.

of tissue extracts

Male Wistar

stocked

Fluorescence

and emission

water.

to a near

After adding

at 3,000 r.p.m. for 10 minutes. to the

column

as described

previously. The recovery

of taurine was estimated

by using an internal

4-10 nmoles taurine were added to the known amount to the extraction

procedures

as described

above.

m mole) was also added to each homogenate completing

extraction

extract

was transferred

(12).

The radioactivity

with 50'./ ethanol into a counting

standard

of tissue homogenate Trace amount

for estimating

from the fluorescent vial containing

in these vials was determined

method,

before subjection

of "C-taurine

the recovery spot,

(1-5 mCi;

of taurine.

After

1 ml of the fluorescent

10 ml of Bray's scintillation in a Packard

in which

cocktail

3390 liquid scintillation

spectrometer. RESULTS A linear relationship rescence was obtained ranges

between

as shown in Fig. 1.

of 0.5-10 nmoles.

standard

from fluorescent

the column

compounds could

(i.e. primary

amines

In this column

such as amino

in the taurine

fraction.

To exclude

of duplicate measurements. Ten standard solution with varying taurine

was

applied

to the paper and the fluorescence was measured following electrophoresis and fluorescamine treatment (see "Methods"). Fluorescence units.

intensity

acids,

Only

from actual

from the

blank areas on the The extraction

is given in arbitrary

pH.

The elution

was

pattern

procedure,

fluorescamine

reactive

polyamines

and biogenic

amines)

O-phosphoethanolamine,

with a similar migrating

FIG. 1. Fluorescence intensity at varying con centrations of taurine. A typical example is shown. Each point represents the

of

of fluo

within 5 minutes and the stability of fluorescence

in Fig. 2.

reactive compounds

concentrations

by the extrapolation

for over several hours at neutral-alkali

is shown

(11), was eluted closely to taurine.

average tl of

value calculated

well with the value obtained

spots was completed

not be eluted

fluorescamine

and the intensity

usually the former value was used as an assay blank.

found to be maintained from

of taurine

The linearity was achieved over the concentration

Since the blank

curve corresponded

same chromatogram,

the concentration

rate in electrophoretic

this compound

from

one of the procedures

the taurine

fraction,

FiG. 2. Elution pattern of taurine in column procedures. Taurine and 0-phosphoetha nolamine, both 50 nmoles in 10 Id, were applied to the column and every 0.1 ml of the fraction was collected. To each frac tion, 0.2 ml of 0.2 M borate buffer (pH 9.0) and 0.1 ml of 0.015 % (W/V) fluorescamine in acetone were added and mixed. After adjusting the final volume to 3 ml with distilled water, the fluorescence was mea sured. "C-taurine was also applied for identifying the taurine fraction in the pre sence of taurine and 0-phosphoethanol amine. The intensity of fluorescence is given in arbitrary units. The horizontal solid bar in the figure indicates the taurine fraction used (0.8 ml).

FIG. 3. High voltage paper electiophoretic profiles of taurine, its structural anal ogues, and profile of extract from mouse cerebral cortex. Standard solutions, containing 5 nmoles of each compound in 10,al, were applied and treated in the same manner as described in Methods. The fluorescence was measured directly on the paper with a fluorodensitometer (Shimazu CS-910) at excitation and emission wave lengths of 365 and 450 nm, respectively. The shaded arrow indicates the origin of sample application.

FIG. 4.

Relationship

Mouse cerebral were transferred

between

the

amount

of tissue

used

genate which corresponded to 0.5, 1, 2, 4 mg of tissues. average of duplicate measurements.

the column chromatographic

the

taurine

content.

procedure

Each point represents

the

procedures were applied and the separation of O-phosphoe

thanolamine from taurine was achieved (Fig. 2). chromatographic

and

cortex was homogenized and various amounts of the homogenate respectively to microtubes so that each tube contained the homo

was approximately

The recovery of taurine during this column 100%.

To examine the specificity for

taurine, structural analogues involved in the metabolic pathway of taurine and possibly to be eluted out from the column (hypotaurine, cysteic acid, cysteinesulfinic acid) were also tested with the extract from mouse cerebral cortex (Fig. 3).

The migration pattern of the extract

showed a single peak identical with that of authentic taurine and no overlapping of peaks of the structural analogues and that of taurine was observed, indicating that the taurine fraction was not contaminated by these compounds.

The linear relationship between the

amount of cerebral cortical tissues used and the amount of taurine obtained is shown in Fig. 4. The concentration of taurine in mouse cerebral cortex was 11 amoles/g wet weight,

TABLL 1.

Taurine

Each

value

three

separate

concentration

represents

the

in various

mean

_ __ S.D.

organs

obtained

of rat

from

determinations.

as calculated from the curve shown in Fig. 4. Tissue levels of taurine in various organs of the rat measured by this method are shown in Table 1. The values obtained were essentially in agreement with those in previous reports (12, 13, 14, 15, 16); the pituitary gland had the highest level of taurine, and relatively high levels in heart, spleen and adrenal gland were also noted among the various organs tested. The recovery of tissue taurine through the entire assay procedures was 90-100 % as determined by either an internal standard "Methods") .

procedure

or the recovery of added '4C-taurine (see

DISCUSSION There is a considerable volume of literature on the fluorescamine reaction in the aqueous phase (9, 17), but quantitative fluorometric procedures using paper electrophoresis have not been documented. In our preliminary experiments, it was found that the fluorescence of taurine yielded by fluorescamine was 2-3 times higher than fluorescence of other amino acids and excellent linearity was retained.

On the contrary, color development of taurine

by ninhydrin reaction was extremely poor as compared with other amino acids (18). For the assay of taurine using electrophoresis,

there is no doubt that the use of fluo

rescamine as a fluorescent probe is superior to that of ninhydrin. disadvantages of this method which should also be noted;

There are, however,

1) Absolute fluorescence intensity

is variable, so that standards are always necessary in each group of assays when fluorescence is developed.

Usually, standards with two different concentrations

the paper before electrophoresis were satisfactory for routine assays.

of taurine applied to 2) Although it is a

somewhat time consuming processes, the use of column procedures before applying electro phoresis is advisable to exclude other interfering substances including O-phosphoethanol amine and also to obtain constant migration patterns.

This method

is also considered

useful to separate

compounds,

since no interference

was counted

directly in a liquid scincillation

Acknowledgernent: and 048254 (1975))

was detected

when the radioactivity

taurine

or related

of fluorescent

spots

system using Bray's Solution (12).

This work was supported

from the Ministry

the radioactive

of Education,

in part by research

grants (Nos. 087119

Japan.

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