ANALYTICAL BIOCHEMISTRY 175,525-530 (1988)

Calorimetric Determination of Neutral Sugars by

a Resorcinol Sulfuric Acid Micromethod

MICHELMONSIGNY, CLAIREPETIT,ANDANNIE-CLAUDEROCHE
Laboratoire de Biochimie Cellulaire et Mol&ulaire des Glycoconjugks, Centre de Biophysique Mokdaire,
Centre National de la Recherche Scientifique, 1, Rue Haute, 45071-Orleans Cedex 02, France
Received May 11, 1988

A microscale calorimetric assay for neutral sugars, in which neutral sugars react with resor-
cinol in the presence of 75% sulfuric acid solution is described. This assay, based on the use
of microtiter plates with 96 U-shaped wells is simple and easy to handle; it allows accurate
determinations with small samples (20 ~1) containing 1 to 100 nmol of neutral sugars and is
quite convenient for detection of glycoconjugates in chromatographic column effluents. 0 1988
Academic Press, Inc.
KEY WORDS: sugar; glycoconjugates; glycoproteins: resorcinol.

The neutral sugar content of glycoconju-
gates is usually determined by specific color
reaction using sulfuric acid and phenol ( 1),
anthrone (2), or orcinol(3). These procedures
require relatively large amounts of sugar (0.1
to 1 pmol) and give accurate results in a nar-
row concentration range (5- to lo-fold). The
present paper describes procedures by which
1 to 100 nmol of sugar gives reliable values.
The method uses a solution of hydrated sul-
furic acid (H2S04, H20) and resorcinol ( 1,3-
dihydroxybenzene). The determination of
glycoconjugate neutral sugar content which
can be conducted by using small tubes or mi-
crotiter plates is suitable for monitoring col-
umn effluents and is quite easy to use. The
presence of protein does not alter the results.
METHODS
Resorcinol, purchased from Merck (Darm-
stad, FRG) was crystallized from a n-butanol
solution by adding chloroform and hexane.
Other phenolic compounds were from Ald-
rich (Milwaukee, WI), sulfuric acid (analyti-
cal grade, 95%; d = 1.834)) was purchased
from Prolabo (Paris). Neutral sugars and bo-
vine serum albumin were from Sigma (Saint
Louis, MO) and pristane was from Janssen
Chimica (Beerse, Belgium). Absorbance
spectra were recorded with an Uvikon double
beam spectrophotometer (Kontron, Plym-
outh, MA); samples were contained in either
a 5 X 5-mm or a 10 X 2-mm Hellma cuvette
(Hellma, Baden-Wurtt, FRG). For the mi-
croassay, 96-well polystyrene crystal plates
(Greiner, Labortechnik, Nurtingen, FRG)
were used and the optical density was read
with a microtiter plate reader (Artek, Farm-
ingdale, NY) using an interference filter ei-
ther at 430 + 10 nm or at 480 f 10 nm (Ea-
ling, South-Natick, MA). Sugars were dried
under vaccum at 50°C for 72 h. Sugars were
dissolved in 0.0 1 M acetic acid. Resorcinol so-
lution was kept at 4°C in the dark for up to 1
month. A 75% sulfuric acid solution was pre-
pared by mixing 100 ml of concentrated sul-
furic acid (d = 1.834) and 24 ml of distilled
water and kept at room temperature in the
dark for up to 3 weeks.
Regular assay. To a sample containing 5 to
100 nmol of neutral sugars in a volume of 200
~1 in a 0.6 X 7-cm assay tube are added 200
~1 of 6 mg/ml resorcinol and 1 ml of 75% sul-
furic acid. The tubes are shaken by vortex
and heated at 90°C in a temperature-regu-

525 0003-2697188 $3.00

Copyright 0 1988 by Academic Press, Inc.
All rights of reproduction in any form reserved.
526 MONSIGNY, PETIT, AND RGCHE

300 350 400 450 500 550 600 75% H2S04 1,~‘)

FIG. 3. Influence of the volume of 75% sulfuric acid

FIG. 1. Absorbance spectra of ( 1) a mixture of an aque-
ous solution (200 11) of mannose (13.5 &ml), of an
aqueous solution (200 ~1) of resorcinol(6 mg/ml), and of
75% sulfuric acid (1 ml) after heating at PO’C for 30 min
and of (2) a blank in which the mannose solution was
replaced by pure distilled water (regular assay).
added in the microplate wells on the optical density de-
termined at 430 nm after heating at PVC for 30 min: to
20 pl of an aqueous solution of mannose (50 &ml) was
added 20 ~1 of 6 mg/ml resorcinol solution and the indi-
cated volume of 75% sulfuric acid (microplate assay).

of 20 ~1 in a U-shaped well of a 96-well micro-

lated water bath for 30 min and subsequently
titer plate are added 20 ~1 of 6 mg/ml resor-
placed in a cold-water bath for 30 min in the
cinol, 100 ~1 of 75% sulfuric acid, and 50 ~1
dark. The optical density of the solution was
of pristane. The solutions are homogenized
determined at 430 or 480 nm. Alternatively,
by shaking the plates with a vortex apparatus.
the same assay can be conducted by using
The plates are heated at 90°C in an oven for
half the volumes indicated above with similar
30 min and subsequently kept at room tem-
results.
perature for 30 min, in the dark. The optical
Microplate assay. To a sample containing
density of each well is automatically recorded
1 to 100 nmol of neutral sugars in a volume
by using a microtiter plate reader equipped

1.0

E 0.50
R
:

\t
I

&

5 3
z
I

4
.^r
; 0.2

.:0
4
5

8‘g0.0
0.5
LLL
.s
0”
0.(X r
0 075 1.25 2.5 3.75 10 o c 6 12 16 20
Mannose ( pg /ZOO@) Rcsorc~nol (mg /ml,

FIG. 2. Effect of mannose concentration on the optical
density determined at 430 nm ofa mixture ofan aqueous
solution (200 ~1) of mannose at the indicated concentra-
tions, of an aqueous solution of resorcinol (6 mg/ml),
and of 75% sulfuric acid (1 ml) after heating at 90% for
30 min (regular assay).
FIG. 4. Effect of the resorcinol concentration on the
optical density at 430 nm after heating at POT for 30
min: to 20 pl of an aqueous solution of mannose (50 ag/
ml) was added 20 ~1 resorcinol solution at the indicated
concentration and 100 ~175% sulfuric acid (microplate
==Y).
 COLORIMETRIC DETERMINATION OF SUGARS 527

nitrophenol, salicylic acid, a-hydroxy /?-

naphthyl sulfonic acid, Bhydroxy cY-naph-
thy1 sulfonic acid, various dihydroxybenzoic
acids, and resorcinol. Resorcinol appeared to
be the best phenolic compound with regard
to the reproducibility and the sensitivity of

ii,L/ 0 5 10 15 20 30 45
the assay.

FIG. 5. Influence of the heating time on the optical
density at 430 nm. A mixture of an aqueous solution (20
~1) of mannose (50 &ml), of 6 mg/ml resorcinol solu-
tion (20 al), and of 75% sulfuric acid (100 ~1) was heated
at 9o’C for the time indicated (microplate assay).
with an interferential filter at 430 or 480 nm.
For quantitative purposes, blanks, neutral
sugar standards, and samples are assayed in
quadruplicate.
Regular Assay
Subsequently, a resorcinol sulfuric acid as-
say was set up as briefly described by Roche et
al. (4). Under the conditions described under
Methods, the maximum coloration was
found to occur at 430 nm (Fig. 1) and optical
density values for neutral sugar were linear at
least in the range of 3.75 to 25 pglml(4 to 28
nmol) (Fig. 2).

RESULTS AND DISCUSSION

Basis of Resorcinol Surfuric Acid Method
With the aim of adapting the very useful
phenol sulfuric acid method (1) to microscale
assay, preliminary experiments showed that
the results were dependent on the rate of ad-
dition of concentrated sulfuric acid and on
the shape of the test tubes used. Better results
were obtained when sugar samples in 100 ~1
of distilled water or saline were placed in 0.6
x 7-cm tubes followed by addition of 100 ~1
of 50 mg/ml distilled phenol and 500 ~1 of
concentrated sulfuric acid, and the tubes
were then shaked with a vortex and heated at
100°C for 5 min in a boiling water bath. To
circumvent the uncontrolled spontaneous
temperature increase upon addition of con- o.oY, . . . . . . , * .
centrated sulfuric acid to the aqueous solu- 0 12 3 4 5 6 7 8 910 12 15
Mannose (,.1g/20~1,
tion, 75% sulfuric acid solution was prepared
and allowed to cool at room temperature be- FIG. 6. Effect of the sugar concentration on the optical
fore use. Unfortunately, when 75% sulfuric density determined at 430 (0) or 480 nm (W). A mixture
acid solution was used, weak color reactions of an aqueous solution (20 ~1) of mannose at the indi-
were obtained with phenol. A series of other cated concentration, of an aqueous resorcinol(6 mg/ml)
solution (20 pl), and of 75% sulkuic acid (100 al) was
phenolic compounds were tested, including heated at 9o’C for 30 min. Inset: optical density of solu-
orcinol, anthrone, cu-naphthol, P-naphthol, tions containing mannose in a low concentration range,
o-nitrophenol, 1,3dihydroxynaphtalene, p- monitored at 430 nm (microplate assay).
528 MONSIGNY, PETIT, AND RGCHE

A 45 min (Fig. 5). Under optimal conditions,

oso- neutral sugar values were linear in the range
of 1 to 20 nmol upon reading at 430 nm and
in the range of 10 to 100 nmol upon reading
at 480 nm (Fig. 6).

Interferences
5 To determine whether the resorcinol sulfu-
:
ric acid assay is or is not modified by the pres-
0" ODOi .
0 10 20 30 40 ence of proteins, various amounts of bovine
Bovhe Serum Albumin ‘vg /20@, serum albumin were added to a solution of
mannose used as a standard. As shown in Fig.
PIG. 7. Influence ofthe presence of bovine serum albu- 7, no change was found in the optical density
min on the optical density at 430 nm. A mixture of an
aqueous solution (20 ~1) of mannose (50 g/ml) contain- whatever the amount of bovine serum albu-
ing the indicated concentrations of bovine serum albu- min added (0 to 2 mg/ml).
min, of an aqueous solution (20 ~1) of resorcinol(6 mg/ The solvent in which sugars or glycoconju-
ml), and of 75% sulfuric acid (100 ~1) was heated at 9o’C gates are dissolved can be either distilled wa-
for 30 min (microplate assay).

TABLE 1
INTERFERENCE~FS~LVENTINTHERES~RCINOL
Microplate Assay
SULFIJRICACIDMICROPLATEASSAY
With the aim to both scale down and speed
Relative
up the assay, test tubes were replaced by mi- Solvent” absorbance b
crotiter plates with polystyrene crystal 96 U-
shaped wells. The main difficulty was to ob- Water 100
tain a good mixing of sulfuric acid solution Sodium chloride (150 mM) 100
with sugar and resorcinol solutions. Shaking Phosphate-balanced saline
(PBS; pH 7.4) 100
with a vortex led to reproducible results. 100
Tris, HCl(50 mM, pH 7.4)
However, to avoid any projection of sulfuric Sodium acetate buffer (1 M; pH 5) 101
acid solution, a layer of pristane (2,6,10,14- Borate buffer (0.1 M; pH 8.2) 92
tetramethyl pentadecane, boiling point, Acetic acid ( 100 mM) 100
>25o”C; d = 0.785) was added in each well Acetic acid ( 1 M) 87
HCl(O.1 M) 107
before the shaking step. Optimal conditions 104
HCl(1 M)
which are described under Methods were de- HCOzH (0.1 M) 94
fined on the basis of the following experi- HCOzH (1 M) 53
ments. MAC15 (0.1%)’ 104
Triton X- 100 ( 1% in PBS) 82

Determination of Optimum Reaction a To 20 pl of listed solvent are added 20 ~1 of 6 mg/

Conditions
The maximal optical density was obtained
when 120 + 30 ~1 of 75% sulfuric acid was
added to 40 ~1 of neutral sugar and resorcinol
mixture (Fig. 3). The resorcinol concentra-
tion was not very critical as long as it was
above 4 mg/ml (Fig. 4). The optimal heating
time at 90°C was found to be between 20 and
ml resorcinol, 100 ~1 of 75% sulfuric acid, and 50 pl of
pristane. Plates were shaken, placed in an oven at 9o’C
for 30 min, and subsequently left for 30 min at 25’C and
optical density was determined with a microtiter plate
reader eqiupped with a 430-nm interference filter.
b The absorbance obtained with water is taken as 100;
the absorbance with other solvents is related to that of
water.
c MAC stands for monosaccharide alkylamine conju-
gate; MAC1 5 is a gluconoylamidoheptane (4).
 COLORIMETRIC DETERMINATION OF SUGARS 529

TABLE 2 TABLE 4
SPECIFICITYOFTHERESORCINOL SULFURICACID NEUTRAL SUGAR CONTENT OF SOME GLYCOCONJU-
METHOD IN MICROPLATE ASSAY GATES (GLYCOPROTEINS AND NEOGLYCOPROTEINS)
DETERMINEDBYUSINGTHEMICROPLATEASSAY"
Sugar’ Relative absorbance b
Neutralsugar
Man 100
Rib 90 Glycoconjugate Found Expected
Fuc 74
Gal 72 Ovalbumin 2.OkO.l 26
Gal UA 24 Ovomucoid 8.7 + 0.3 8-9’
GalNAc <2 Orosomucoid 14.7 f 0.3 14.5d
Glc 58 ManIs-BSA 4.1 kO.2 4.0e
Glc UA 30 Man2,-BSA 4.4 -t 0.2 4.4’
GlcNAc <I Ma%-BSA 8.0 + 0.3 7.9’
Glucuronolactone 40
NeuSAc 0 ’ Glycoconjugates were dissolved either in phosphate-
balanced saline, pH 7.4, or in distilled water, at a concen-
’ To 20 pl of distilled water, containing 10 nmol of the tration of 1 mg/ml. Neutral sugar content was deter-
listed sugar, are added 20 ~1 of 6 mg/ml resorcinol, 100 mined by using the microplate assay in quadruplicate
~1 of 75% sulfuric acid, and 50 pl of pristane. Plates were and calculated from the values of the optical density at
shaken, placed in an oven at 90°C for 30 min, and subse- 430 nm.
quently left for 30 min at 25°C and optical density deter- b According to Johansen et al. (5); mannose was used
mined with a microtiter plate reader equipped with a as the standard.
430-nm interference filter. ‘According to Monsigny et al. (6); mannose was used
b The absorbance obtained with mannose is taken as as the standard.
100; the absorbance with other sugars is related to that of d According to Jeanloz (7); a 1: 1 mixture of galactose
mannose. and mannose was used as the standard.
e Neoglycoproteins were prepared according to Mon-
signy et al. (8). Mannose was used as the standard. The
ter or a buffer such as phosphate-balanced sa- expected content was calculated on the basis of the num-
line which does not interfere in the microas- ber of mannopyranosylphenylthiocarbamyl residues
say (Table I). bound to one serum albumin molecule determined from
SpeciJicity the yield ofthe coupling reaction. The sugar content (A)
is related to the number (n) of sugar residues bound to
The optical density is dependent on the na- the protein according to .x% = n.M,,/(M, + n .I%&)
ture of the neutral sugars as in the case of the where M, is the molecular mass of the sugar residue, Mp
is the molecular mass of the unsubstituted protein, and
MgpTc is the molecular mass of the glycosylphenylthio-
TABLE 3
carbamyl residue.
Emcr OF THE SUGAR SUBSTITUANT IN THE
DETERMINATIONOFMAIWOSEINMICROPLATEASSAY

Compound” MMb Relative absorbance’ other methods (Table 2). This assay is suit-
able to quantitatively determine pentoses,
Man 180.2 100 hexoses, and 6-deoxy hexoses. Therefore the
Man-PITC 312 100 sugar used as the standard must be selected in
Man-PNOI 301.5 100
Man-MUF 338.4 100 relation to the type of sugar contained in the
glycoconjugate under experiments. Uranic
a Aliquots (20 pl) of sample containing 10 nmol of the acids give a weak coloration less than one-
listed compounds are assayed as described under Meth- third that of the corresponding neutral sugar.
ods. Amino sugars and IV-acetyl neuraminic acid
b Molecular mass.
’ The absorbance obtained with mannose is taken as do not interfere (Table 2).
100; the absorbance with other sugars is related to that of The nature of the aglycone moiety in the
mannose. case of current glycosides usually does not in-
530 MONSIGNY, PETIT, AND RGCHE
terfere (Table 3). It may therefore be conve-
nient to usep-nitrophenyl or 4-methylumbel-
liferyl glycosides as standards. The concen-
tration of the arylglxcoside solutions may be
determined by measuring the absorbance at
300 nm in the case of nitrophenyl glycosides
(EM = 10,800) or at 320 nm in the case of 4-
methylumbelliferyl glycosides (Q,, = 12,400).
The absorbance of these arylglycosides is not
affected by the nature of the sugar.
The microplate assay is also suitable for de-
termination of the neutral sugar content of
glycoconjugates (Table 4). Using this assay,
accurate results are obtained by using less
than 100 pg of the glycoconjugates in experi-
ments done in quadruplicate.
Precision
Reproducibility was estimated over a 2-
month period by replicate assays of samples
containing 1,2, and 10 pg of mannose kept
at 4°C. The mean and standard deviations of
10 measurements in triplicate were 1.Of 0.05
pg (range, 0.95-1.05 pg; CV = 7%) and 2.0
+ 0.07 pg (range, 1.92-2.1 pg; CV = 4%)
upon reading at 430 nm, and 10.0 + 0.1 pg
(range, 9.90-10.1 pg; CV = 1%) upon reading
at 480 nm.
CONCLUDING REMARKS
The microplate assay, described in this pa-
per, has been used for several years in our lab-
oratory and has been found to be suitable to
quantitatively determine the sugar content of
glycoproteins and neoglycoproteins and to
monitor the sugar content of effluents of
chromatography columns as well as of high-
performance liquid chromatography col-
umns. Peak detection from chromatography
fractions can be achieved just by looking at
the coloration of the wells, within 30 min. Ac-
curate results can be obtained within 1 h. The
microplate assay appears to be more conve-
nient than test tube assays with regard to the
small volume of samples required and to the
limited amount of sulfuric acid solution used;
furthermore, the monitoring of microplates
does not require a transfer of solutions in
spectrophotometer cuvettes, and the addition
of pristane prevents any deleterious effect of
casual liquid projections. Finally, the use of
microtiter plates allows sugar determination
of several tens of samples quickly and easily.
ACKNOWLEDGMENTS
This work was supported partly by grants from the In-
stitut National de la Sante et de la Recherche Medicale
(INSERM, 8 114 84 20 16) and from the Association
pour le Developpement de la Recherche sur le Cancer
(ARC). ACR is Directeur de Recherche INSERM.
REFERENCES
1. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers,
P. A., and Smith, F. (1956) Anal. Chem.28,350-
356.
2. Dreywood, R. (1946) Ind. Eng.Chem.Anal.Ed. 18,
499-503.
3. Rimington, C. (1940)B&hem. J. 34,93l-940.
4. Roche, A. C., Barzilay, M., Midoux, P., Junqua, S.,
Sharon, N., and Monsigny, M. (1983) J. Cell. Bio-
them.22,131-140.
5. Johansen, P. G., Marshall, R. D., and Neuberger, A.
(1960) Biochem. J. 77,239-247.
6. Monsigny, M., Adam-Chosson, A., and Montreuil,
J. (1968) Bull Sot. Chim.Biol.50,857-886.
7. Jeanloz, R. W. (1972) in Glycoproteins (Gottschalk,
A., Ed.), pp. 565-611, Elsevier, Amsterdam.
8. Monsigny, M., Roche, A. C., and Midoux, P. (1984)
Biol.Ceil.51, 187-196.