1973_Blumenkrantz-New Method for Uronic Acids

8/12/2019 1973_Blumenkrantz-New Method for Uronic Acids

1/6

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 Tiss ue

Research Laboratories), Rigsh osp ital, 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 morespecific than other methods, and it needs lesser amounts of fluid. It isrecommended for determination of acid mucopolysaccharides in biologicalmaterials.

Hexosamine and uranic acid are the components of the repeatingunit of all acid mucopolysaccharides (glycosaminoglycans) with theexception of keratosulfate, in which galactose replaces the uranic acidmoiety. Uranic acid is widely determined as representative of acidmucopolysaccharides in biological substances. However, the assaysmainly used for quantitative determination of uranic acids are not suf-ficiently accurate, insofar as hexoses (1,2) or pentoses (3) interfere withtheir specificity. The need for a rapid, sensitive, and specific method forquantitative assay of uranic acids and acid mucopolysaccharides stimu-lated the elaboration of a new method based upon the appearance of achromogen when uranic acid heated to 100C in concentrated sulfuricacid/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 wereobtained from E. Merck A.G., Darmstadt ; glucosamine and galactosaminewere products of Sigma Chemical Co., St. Louis, MO; concentratedsulfuric acid, specific gravity 1.84 (analytical grade) E. Merck A.G.,Darmstadt; sodium tetraborate (p.a.) , Riedel-deHa i A.G., Seelze,

484Copyright @ 1973 by Academic Press, Inc.All rights of reproduction in any form reserved.


2/6

DETERMINATION OF URONIC ACIDS 485

Hannover ; meta-hydroxydiphenyl (analytical grade) was obtained fromK & 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), wasobtained 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 solutionwas diluted with distilled water to a final concentration of 1.5 in 0.5NaOH. The solution was then diluted 1: 10 with 0.5 NaOH and used asreagent.

H,SOJsodiwm tetraborate solution. A 0.0125 M solution of tetraboratein 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 refrigeratedin crushed ice. The mixture was shaken in a Vortex mixer and the tubesheated in a water bath at 100C for 5 min. After cooling in a water-icebath, 20 ~1 of the m-hydroxydiphenyl reagent was added. The tubes wereshaken and, within 5 min, absorbance measurements made at 520 nmin a Beckman DU spectrophotometer.

As carbohydrates produce a pinkish chromogen with sulfuric acid/tetraborate at lOOC, a blank sample was run without addition of thereagent, which was replaced by 20 ,J of 0.5 NaOH. The absorbanceof the blank sample was subtracted from the total absorbance.

Determination of Acid Mucopolysaccharides in Urine ujith m-Hydroxy-diphenyl. Urinary acid mucopolysaccharides were precipitated withcetyltrimethylammonium bromide according to Teller et al. (41. How-ever, a modification of this method was introduced by using 3 or 6 mlof urine instead of 15 or 30 ml as stated by Teller et al. When the specificgravity of the urine was 1.020 or higher, 3 ml of urine and an equalamount of water were added and then precipitated with 0.2 ml of thequaternary ammonium compound. In the case of lower specific gravity,6 ml of urine was used. Before the addition of the precipitating agent,


3/6

486 BLUMENKRANTZ AND ASBOE-HANSEN

the urine was brought to pH 5 by 2 N HCI with the use of indicatorpaper (4). After the addition of the cetyltrimethylammoniun~ bromidesolution (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 finalprecipitate was dissolved in 1 ml of distilled water. One-hundred to two-hundred microliters of this solution was used for determination of uranicacids with the method described above.

RESULTS

Assays of pure standards of glucuronic, galacturonic, iduronic, andmannuronic acids with the new method indicated that the absorbancesobtained 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 methodpresented by Dische (1)) the modification proposed by Bitter and Muir(2), and the orcinol reaction of Brown (3). The sensitivities of the fourmethods are presented in Fig. 2. The specificity of our reaction is takenfrom Table 1. The assay method presented in this paper showed highersensitivity and specificity than the other methods. Since hexosamines didnot produce any chromogen with nL-hydroxydiphenyl, the absorptionshown for chondroitin sulfates in Fig. 3 must be due solely to their uranicacid moiety.

Comparison of meta-, ortho-, and para-hydroxydiphenyl as reagentsshowed decreasing sensitivity in the mentioned order.

I 200I

x- GLCURONlC ac,o

fig URONIC ACID

FIG. 1. Assay of different uranic acids with meta-hydroxydiphenyl.


4/6


/q GLUCURONIC ACID.

FIG. 2. Comparison of the sensitivity of the m-hydroxydiphenyl reaction withother reactions used for the determination of uranic acids.

Stability of Color Reaction. The color produced by uranic acids andnz-hydroxydiphenyl was stable for at least 12 hr. Absorbance measure-ments followed Lambert and Beers law within the range 0.5-15 pg forglucuronic, 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 uranicacids.

pg CHONDROITIN SULFATE (CS)

FIG. 3. Determination of the uranic acid content in chondroitin sulfates with them-hydroxydiphenyl method.


5/6

488 BLUMENKRANTZ AND ASBOE-HANSEN

TABLE 1Spe cificity of the nl-Hydroxydiphenyl Reaction with Different Sugars as

Compared with Other Rea ctions

Recovered as glucuronic acid

Carbohydrates added Orcinol

(d (Brown)Carbazole

(Dische)

m-Hydroxy-

diphenyl

1 GAa 0.92* 1.0s 0.922 GA 2.11 2.13 1.99

4GA 4.00 4.00 4.0010 G 0 6 1.58 0.1220 G 1.51 3.81 0.3340 G 2.98 7.73 0.6610 A 23.82 0.56 0.06

20 A 45.53 1.21 0.1240 A 68.85 1.77 0.25

1 GA+ 10G 1.93 2.60 0.92lGA+20G 2.80 4.09 1.08

lGA+40G 4.68 8.83 1.262GA+ 10G 2.81 3.72 1.862GA+20G 4.18 6.39 2.00

2 GA + 40 G 6.06 9.021 2.10

4GA+ 10G 5.19 5.95 3.844GA+20G 6.20 7.90 3.964GAf40G 8.63 12.09 4.01

lGA+lOA 24.37 1.209 0.92lGAf20A 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 1.962GAf40A 73.44 4.46 2.01

4GA+ 10A 27.63 6.7 3.90

4GA+20A 59.67 6.97 3.964GAf40A 73.44 7.53 4.12

a Abbreviations: GA, glucuro nic acid; G, gluco se; A, Larabinose.

h Resulk are expressed as Mg glucuro nic acid.

Effect of Heating Time on the Reaction. After 5 min of boiling, thereaction showed no change in sensitivity.

Reagent Concentration. A 0.15 concentration of the reagent in 0.5NaOH is optimal.

Acid Mucopolysaccharides in Urine. Urinary acid mucopolysaccharidesexpressed as glucuronic acid are presented in Table 2. Similar valueswere obtained for the uranic acid content of the acid mucopolysac-charides precipitated with cetyltrimethylammonium bromide, whetherTeller et als procedure (4) or our modification with reduction of urinary


6/6


TABLE 2Twenty-four-Hour Urinary Excretion of Acid Mucopolysaccharides

Uranic acid (mg)a

CarbazoleOrcinol (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 sglucuronic acid.

6 Figures represent mean values and standard error of the mean.

volume was followed. The values were also similar whether the methodof 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 ofuranic acids have the disadvantage that neutral sugars interfere withtheir specificity. The orcinol reaction of Brown (3) is highly sensitiveto 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 beredissolved by reheating, this step is time consuming. The method pre-sented in this paper has the advantages of increased sensitivity andspecificity, together with the practical aspect of being simpler and quickerthan the other methods available. The need for smaller volumes of urinemakes it even more practical. Although the mechanism of the proposedmethod is unexplained, its evident advantages render it recommendablefor quantitative assay of uranic acids.

REFERENCES1. DISCHE , Z. (1947) J. Biol. Chem. 167, 189.2. BITTER, T., AND MUIR, H. M. (1962) Ana l. 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.

1973_Blumenkrantz-New Method for Uronic Acids

Documents