ANALYTICAL BIOCHEMISTRY 54, 484-489 (1973)

New Method for Quantitative Determination

of Uranic Acids
NELLY BLUMENKRANTZ AND GUSTAV ASBOE-HANSEN
Univer& of Copenhagen, Departme& of Dermatology (with Connective Tissue
Research Laboratories), Rigshospital, 2100 Copenhngen, Denmark
Received December 26, 1972; accepted March 2, 1973
A new method for determination of uranic acids with meta-hydroxy-
diphenyl is introduced. It is simpler, quicker, more sensitive, and more
specific than other methods, and it needs lesser amounts of fluid. It is
recommended for determination of acid mucopolysaccharides in biological
materials.
Hexosamine and uranic acid are the components of the repeating
unit of all acid mucopolysaccharides (glycosaminoglycans) with the
exception of keratosulfate, in which galactose replaces the uranic acid
moiety. Uranic acid is widely determined as representative of acid
mucopolysaccharides in biological substances. However, the assays
mainly used for quantitative determination of uranic acids are not suf-
ficiently accurate, insofar as hexoses (1,2) or pentoses (3) interfere with
their specificity. The need for a rapid, sensitive, and specific method for
quantitative assay of uranic acids and acid mucopolysaccharides stimu-
lated the elaboration of a new method based upon the appearance of a
chromogen when uranic acid heated to 100C in concentrated sulfuric
acid/tetraborate is treated with meta-hydroxydiphenyl.
MATERIALS AND METHODS
Materials
Glucuronic acid, galacturonic acid, iduronic acid, mannuronolactone,
and chondroitin sulfates A and B were gifts from Dr. Martin B. Mathews
(University of Chicago, Chicago, IL). Hyaluronic acid, chondroitin sul-
fate C, and heparin were kindly donated by Professor Karl Meyer
(Yeshiva University, New York, NY). Glucose and L-arabinose were
obtained from E. Merck A.G., Darmstadt ; glucosamine and galactosamine
were products of Sigma Chemical Co., St. Louis, MO; concentrated
sulfuric acid, specific gravity 1.84 (analytical grade) E. Merck A.G.,
Darmstadt; sodium tetraborate (p.a.) , Riedel-deHa&i A.G., Seelze,
484
Copyright @ 1973 by Academic Press, Inc.
All rights of reproduction in any form reserved.
DETERMINATION OF URONIC ACIDS
485
Hannover ; meta-hydroxydiphenyl (analytical grade) was obtained from
K & K Laboratories Inc., Plainview, NY; para-hydroxydiphenyl (p.a.1,
Koch-Light Laboratories Ltd., Colnbrook, Bucks, England; ortho-
hydroxydiphenyl (analytical grade), Eastman Organic Chemicals,
Rochester, NY; cetyltrimethylammonium bromide (reagent grade), was
obtained from British Drug Houses (BDH).
Reagents
Meta-hydroxydiphenyl solution. A 0.15% solution of meta-hydroxy-
diphenyl in 0.5% NaOH. The reagent solution was kept in the refrig-
erator covered with aluminum foil for more than 1 month.
A solution of ortho-hydroxydiphenyl was prepared in a similar way.
Parahydroxydiphenyl was dissolved in 10% NaOH, and the solution
was diluted with distilled water to a final concentration of 1.5% in 0.5%
NaOH. The solution was then diluted 1: 10 with 0.5% NaOH and used as
reagent.
H,SOJsodiwm tetraborate solution. A 0.0125 M solution of tetraborate
in concentrated sulfuric acid.
Cetyltrimethylammonium bromide solution. A 570 solution of cetyltri-
methylammonium bromide in distilled water.
Method
To 0.2 ml of the sample containing from 0.5 to 20 pg uranic acids,
1.2 ml of sulfuric acid/tetraborate was added. The tubes were refrigerated
in crushed ice. The mixture was shaken in a Vortex mixer and the tubes
heated in a water bath at 100C for 5 min. After cooling in a water-ice
bath, 20 ~1 of the m-hydroxydiphenyl reagent was added. The tubes were
shaken and, within 5 min, absorbance measurements made at 520 nm
in a Beckman DU spectrophotometer.
As carbohydrates produce a pinkish chromogen with sulfuric acid/
tetraborate at lOOC, a blank sample was run without addition of the
reagent, which was replaced by 20 ,J of 0.5% NaOH. The absorbance
of the blank sample was subtracted from the total absorbance.
Determination of Acid Mucopolysaccharides in Urine ujith m-Hydroxy-
diphenyl. Urinary acid mucopolysaccharides were precipitated with
cetyltrimethylammonium bromide according to Teller et al. (41. How-
ever, a modification of this method was introduced by using 3 or 6 ml
of urine instead of 15 or 30 ml as stated by Teller et al. When the specific
gravity of the urine was 1.020 or higher, 3 ml of urine and an equal
amount of water were added and then precipitated with 0.2 ml of the
quaternary ammonium compound. In the case of lower specific gravity,
6 ml of urine was used. Before the addition of the precipitating agent,
486 BLUMENKRANTZ AND ASBOE-HANSEN
the urine was brought to pH 5 by 2 N HCI with the use of indicator
paper (4). After the addition of the cetyltrimethylammoniun~ bromide
solution (0.2 ml) the samples were cooled in a water-ice bath for 30 min.
Washing was performed as indicated by Teller et al. (4). The final
precipitate was dissolved in 1 ml of distilled water. One-hundred to two-
hundred microliters of this solution was used for determination of uranic
acids with the method described above.
RESULTS
Assays of pure standards of glucuronic, galacturonic, iduronic, and
mannuronic acids with the new method indicated that the absorbances
obtained were proportional to the uranic acid content (Fig. 1).
Sensitivity and Specificity of the Reaction. The sensitivity and specific-
ity of the new method were compared with those of the carbazole method
presented by Dische (1)) the modification proposed by Bitter and Muir
(2), and the orcinol reaction of Brown (3). The sensitivities of the four
methods are presented in Fig. 2. The specificity of our reaction is taken
from Table 1. The assay method presented in this paper showed higher
sensitivity and specificity than the other methods. Since hexosamines did
not produce any chromogen with nL-hydroxydiphenyl, the absorption
shown for chondroitin sulfates in Fig. 3 must be due solely to their uranic
acid moiety.
Comparison of meta-, ortho-, and para-hydroxydiphenyl as reagents
showed decreasing sensitivity in the mentioned order.
I 200
I
x- GLCURONlC ac,o
fig URONIC ACID
FIG. 1. Assay of different uranic acids with meta-hydroxydiphenyl.
DETERMINATION OF URONIC ACIDS
487
/q GLUCURONIC ACID.
FIG. 2. Comparison of the sensitivity of the m-hydroxydiphenyl reaction with
other reactions used for the determination of uranic acids.
Stability of Color Reaction. The color produced by uranic acids and
nz-hydroxydiphenyl was stable for at least 12 hr. Absorbance measure-
ments followed Lambert and Beers law within the range 0.5-15 pg for
glucuronic, galacturonic, and iduronic acid, and 0.5-30 pg for man-
nuronic acid. The absorbances obtained for mannuronic acid and m-
hydroxydiphenyl were lower than those obtained for the other uranic
acids.
pg CHONDROITIN SULFATE (CS)
FIG. 3. Determination of the uranic acid content in chondroitin sulfates with the
m-hydroxydiphenyl method.
488 BLUMENKRANTZ AND ASBOE-HANSEN
TABLE 1
Specificity of the nl-Hydroxydiphenyl Reaction with Different Sugars as
Compared with Other Reactions
Recovered as glucuronic acid
Carbohydrates added Orcinol
(d
(Brown)
Carbazole
(Dische)
m-Hydroxy-
diphenyl
1 GAa 0.92* 1.0s 0.92
2 GA
2.11 2.13 1.99
4GA
4.00
4.00 4.00
10 G
0 6
1.58 0.12
20 G 1.51 3.81
0.33
40 G 2.98 7.73 0.66
10 A 23.82 0.56 0.06
20 A 45.53 1.21 0.12
40 A 68.85
1.77 0.25
1 GA+ 10G 1.93 2.60
0.92
lGA+20G
2.80
4.09 1.08
lGA+40G
4.68
8.83 1.26
2GA+ 10G 2.81 3.72 1.86
2GA+20G 4.18
6.39
2.00
2 GA + 40 G 6.06 9.021 2.10
4GA+ 10G
5.19
5.95 3.84
4GA+20G 6.20 7.90 3.96
4GAf40G 8.63 12.09 4.01
lGA+lOA 24.37 1.209 0.92
lGAf20A 47.74 2.98 1.0
lGAf40A 73.44 3.44 1.06
ZGA+ 10A 25.34 3.55 1.88
2GA+20A 48.19 3.81 1.96
2GAf40A 73.44
4.46
2.01
4GA+ 10A 27.63
6.7
3.90
4GA+20A
59.67
6.97 3.96
4GAf40A 73.44 7.53 4.12
a Abbreviations: GA, glucuronic acid; G, glucose; A, Larabinose.
h Resulk are expressed as Mg glucuronic acid.
Effect of Heating Time on the Reaction. After 5 min of boiling, the
reaction showed no change in sensitivity.
Reagent Concentration. A 0.15% concentration of the reagent in 0.5%
NaOH is optimal.
Acid Mucopolysaccharides in Urine. Urinary acid mucopolysaccharides
expressed as glucuronic acid are presented in Table 2. Similar values
were obtained for the uranic acid content of the acid mucopolysac-
charides precipitated with cetyltrimethylammonium bromide, whether
Teller et als procedure (4) or our modification with reduction of urinary
DETERMINATION OF URONIC ACIDS
489
TABLE 2
Twenty-four-Hour Urinary Excretion of Acid Mucopolysaccharides
Uranic acid (mg)a
Carbazole
Orcinol (Brown)
Carbazole (Dische) (Bitter and Muir) m-Hydroxydiphenyl
5.50 + 0.26b 4.83 * 0.21 4.87 f 0.21 4.57 * 0.19
a Twenty determinations were made by each method. Uranic acid was measured as
glucuronic acid.
6 Figures represent mean values and standard error of the mean.
volume was followed. The values were also similar whether the method
of Dische (1)) Bitter and Muir (2)) Brown (3)) or the m-hydroxydi-
phenyl method was used.
DISCUSSION
The methods most commonly used for quantitative determination of
uranic acids have the disadvantage that neutral sugars interfere with
their specificity. The orcinol reaction of Brown (3) is highly sensitive
to pentoses. Therefore, their interference must be taken into consideration.
The carbazole reaction originally described by Dische (I) and subse-
quently modified by other authors (2,5) allows no possibility of dif-
ferentiation between uranic acids and hexoses. The modification intro-
duced by Galambos (5) reduced significantly the interference of hexoses,
but, in our experience, the sulfamate added in this modification pre-
cipitates when the tubes are cooled. Although this precipitate can be
redissolved by reheating, this step is time consuming. The method pre-
sented in this paper has the advantages of increased sensitivity and
specificity, together with the practical aspect of being simpler and quicker
than the other methods available. The need for smaller volumes of urine
makes it even more practical. Although the mechanism of the proposed
method is unexplained, its evident advantages render it recommendable
for quantitative assay of uranic acids.
REFERENCES
1. DISCHE, Z. (1947) J. Biol. Chem. 167, 189.
2. BITTER, T., AND MUIR, H. M. (1962) Anal. Biochem. 4, 339.
3. BROWN, A. H. (1946) Arch. Biochem. 11, 269.
4. TELLEB, W. M.. BURKE, E. C.. ROSEVF.AR. J. W.. AND MCKENZIE. B. F. (1962)
J. Lab. Clin. Med. 59, 95.
5. G.AL.AMROS. J. (1967) And. Biochem. 19, 119.