ANALYTICAL

4, 330-334

BIOCHEMISTRY

A Modified

(1962)

Uranic

Acid

T. BITTER
From

the

Medical

Unit,

AND

Carbazole
H. M. MUIR1

St. Marys

Received

Reaction

March

Hospital,

London,

England

12, 1962

The reaction of uranic acids with carbazole (l-2) is the most

satisfactory method of estimating uranic acids in chromatographic
fractions but requires 2 hr for the full development of color and, with
certain compounds, the color is partially
suppressed by salts (3-5).
The color is unstable and sensitive to overheating or dilution by water
(9), and impurities in the reagents or the sample interfere with it (6-8).
In keeping with the finding of Aminoff, Morgan, and Watkins (10)
for the determination
of hexosamines it was established by Gregory
(11) that borate ion similarly increases the color yield of the uranic
acid-carbazole reaction while in the orcinol-sulfuric
acid reaction for
hexoses it produces an immediate appearance of color (12).
The present modification
using borate in concentrated sulfuric acid is
both rapid and quantitative,
and results in the following advantages:
(a) further increase of sensitivity beyond that recorded by Gregory
(11)) (b) immediate
appearance of color, (c) marked increase of
stability of the color, (d) greater reproducibility,
and (e) reduction of
interference by chlorides and oxidants. A preliminary
report of some of
these results has been given (13).
EXPERIMENTAL

Reagents

(a) 0.025 M Sodium tetraborate. lOH,O (analytical grade) in sulfuric

acid, sp.gr. 1.84 (analytical grade).
(b) 0.12570 Carbazole? in absolute ethanol or methanol (analytical
grade). Stable for 12 weeks at 4C in the dark.
(c) Glucuronolactone
standards of 440 ,ug/ml were prepared by dilution with either glass distilled or deionized water saturated wit,h benzoic
Supported
by grants
from
the Empire
Rheumatism
Council
and the Pearl
Insurance
Company.
*The
carbazole
obtained
from
B. D. H. (Poole,
Ds., England)
did not need
recrystallizing
as recommended
by Dische,
and can be stored
in the dark
at 4C
for 6 months.
330

A MODIFIED

acid from a stock standard

for 6 months at 4C.

CARBAZOLE

in water

REACTION

saturated

with

331
benzoic acid. Stable

Procedure
5 ml of sulfuric acid reagent is placed in tubes fixed in a rack and
cooled to 4C; 1 ml of the sample or standard is carefully layered on to
the acid. The tubes are closed with ground glass or Teflon stoppers and
the rack shaken at first gently, then vigorously
with constant cooling.
At no time should the temperature of the mixture exceed room temperature. If these precautions are omitted the temperature can rise to 135C
at the interface.
The tubes are then heated for 10 min in a vigorously boiling distilled
water bath and cooled to room temperature.
(For extreme accuracy it is
recommended that the tubes be cooled to -70C
before the sample is
layered and then allowed to warm up to room temperature
while being
shaken.)
Carbazole reagent (0.2 ml) is then added; the tubes are shaken again,
heated in the boiling bath for a further 15 min, and cooled to room
temperature. The optical density (OD) is then read at 530 rnp in a l-cm
cell. The OD of the blank against sulfuric acid should be below 0.025.
For economy of sample or reagent when assaying chromatographic
fractions, the following proportions
can be used: 0.5 ml sample, 3.0 ml
sulfuric acid reagent., and 0.1 ml carbazole reagent.
RESULTS

AND DISCUSSION

The color is stable for at least 16 hr. The sensitivity of the reaction
is approximately double that of the original procedure of Dische (2)
for glucuronolactone and most connective tissue heteropolysaccharides
tested except heparin. The low color yield of dermatan sulfate (chondroitin sulfate B) due to iduronic acid is 41% of that of chondroitin
4- and 6-sulfates in the Dische method (14). In the present modification
the color yield increased to 83% of that of chondroitin 4-sulfate (see
Table 1).
Solutions containing 1 pg/ml uranic acid will give a definite color but
reproducibility below 4 pg/ml is poor. The optical density is a linear
function of concentration between 4 and 40 kg/ml uranic acid. Using
glucuronolactone (aDZ3+ 19.4)) the final solution containing all reagents had E iFrn 530 mp = 1160 t 3%.
No difference was observed if the acid reagent was added t.o the
sample or vice versa. Borate ion concentration can be varied between
0.025 and 0.1 M and carbazole concentration between 0.125 and 0.5~~.
Pentoses and tryptophan gave no color at concentrations of 100 pg,iml,

332

BITTER

AND

MUIR

TABLE
COMPARATIVE
Polyuronides

Hyaluronate
(L. Light & Co.)
Chondroitin
4sulfate
Dermatan
sulfate
Heparan
sulfate
Aortic
heparan
sulfate (15)
Heparin
(Hoffmann-La
Roche)
Pectin
Alginate
(L. Light & Co.)

Amt.

1
COLOR

Gg/m1)

YIELDS

OD

100
100
100
100
100
100
50
100

(Dische)

OD

0.274
0.237
0.106
0.352
0.370
0.335
0.316
0.178

(borate)

0.554
0.476
0.384
0.614
0.612
0.533
0.632
0.869

Borate/Disc&e
ratio

2.02
2.04
3.65
1.74
1.65
1.59
2.00
4.88

but 60 pg/ml of glucose give an intensity equivalent to 8 pg of uranic

acid using 0.125% carbazole. Heating for 10 min prior to the addition of
carbazole is necessary only for the polymers, as glucuronolactone
gives
similar color yields by the above procedure and after a single heating of
25 min In the presence of carbazole.
Contamination
of chromatographic
samples by dust or chlorinated tap
water gave a green color found to be due to oxidants. Nitrite (5 ~44)
or hydrogen peroxide (0.01 PM) gives a green color with OD 0.030 at
530 mp. In Disches procedure the OD for these concentrations was
0.15 at 530 rnp. It is possible to account for oxidant interference by
subtracting the absorption of the contaminant
at 920 rn,u from the
absorption at 530 mp., at which points the optical densities are almost
equivalent (see spectra, Fig. 1). The present modification
also suffered

Carbmok

rpsctm

--~~}h?icodd
Mm
f f

-lapffi.AFIG.

1.

Equiwlsnt

i,&
points

A MODIFIED

CARRAZOLE

333

REACTION

less than the original procedure

from interference
by impurities
in
reagents. At concentrations
above 0.4 N, potassium,
sodium, and ammonium chloride lower the color yield of heparin and its derivatives
by approximately
33% in the original procedure. In the present modification, only the color yield of heparin itself is depressed. Sodium and
potassium
carbonate, acetate, or phosphate have no effect. It is thus
possible using less than 300 pg of polysaccharide
to distinguish between
heparin, heparin derivatives,
and the other connective tissue polyuronidee by adding chloride ion above 0.4 N and comparing the color yields
in both procedures (see Table 2).
TABLE
SALT

DEPRESSION

Polysaccharides

Chondroitin
Chondroitin
Dermatan
Hyaluronate

4-sulfate
6-sulfat,e
sulfate

Heparan

sulfate
_
Aortic
heparan
sulfate
Heparin

(15)

OF

CARBAZOLE

COLOR

YIELDS

Dische

Borate

Nil
Nil
Nil
Nil
33%
33%
33%

Nil
Nil
Nil
Nil
Nil
Nil
33%

SUMMARY

A modification
of Disches carbazole reaction for uranic acid in the
presence of borate is described. The advantages of the procedure are:
(I) There is an approximately
twofold
increase of sensitivity.
The
OD is a linear function of concentration
between 4 and 40 pg/ml.
(2) Maximum color develops immediately,
(3) The color is stable for at least 16 hr.
(4) There is greater reproducibility
and reduction of interference by
chloride ion and oxidants.
It has been found possible to distinguish
between heparin, heparin
derivatives,
and other polyuronides
of connective tissue by comparing
the effect of chlorides on the color yield in both procedures.
REFERENCES
1.
2.
3.
4.
5.
6.

DISCHE,
Z., Biochem.
Z. 189, 77 (1927).
DISCHE,
Z., J. Biol. Chem. 167, 189 (1947).
RINGERTZ,
N. R., Acta Chem. Stand. 14, 303 (1960).
SCHILLER,
S., Biochim. et Biophys. Acta 32, 315 (1959).
BUDDECKE,
E, Z. physiol. Chem. (Home Seylers) 318, 33 (1960).
GURIN,
S., AND HOOD, D. B., J. Biol. Chem.
131, 211 (1939).
7. SEIBERT, F. B., AND ATNA. J.. J. Biol. Chem. 163, 511 (1946).

334

BITTER

8. HOLZMANN,

G.,

MCALLISTER,

AND

MUIR

R. V., AND NIEMANN,

G.,

J. Biol.

Chem.

171,

27

(1947).
9. DISCHE,
Z., Methods
of Biochem.
Anal. 2, 313 (1955).
10. AMINOFF,
D., MORGAN, W. T. J., AND WATKINS, W. M., Biochem.
J. 51, 376
(1952).
11. GREGORY, J. D., Arch. Biochem.
Biophys.
89, 157 (1960).
12. FRANCOIS, C., MARSHALL,
R. D., AND NEUBERGER, A., Biochem. J. 83, 335 (1962).
13. BITTER, T., AND EWINS, R., Biochem.
J. 81, 43~ (1961).
14. HOFFMANN,
P., LINKER, A., AND MEYER, K., Science 124, 1252 (1956).
15. MUIR, H. M., Biochem.
J. 81, 8p (1961).