Uronic acids assay

CAUTION The uronic acid assay involves the use of concentrated sulfuric acid. Wear goggles, gloves, and protective clothing. 

Tomato fruits (Lycopersicon esculentum Mill. var. Castlemart ) were collected from vines grown in the field at the University of California, Davis. Pericarp discs were cut from surface sterilized MG stage fruit (10). Droplets (10 n ) of test solutions (see below) were applied to the cut surface of discs and disc ethylene production was measured as described previously (11). The amounts of test materials used were based on colorimetric assay (6) of uronic acid content. 

Table II. Carbohydrate compositions (weight percentage) of individual oligomer peaks purified (QAE-Sephadex or HPLC ion-exchange separation, respectively) from mixtures of citrus pectin oligomers or B fruit extracts Compositions shown are for peaks whose biological activity is described in Figure 4. Uronic acid values are based on colorimetric assay. Proportions of neutral sugars were determined by GC and adjusted so that totals equal 100%. In fact, some oligomers (G7 peaks 8, 9 and 10. B extract peak 10) produced small (less than 1 % of the total integrated area), unknown peaks in the GC chromatograms.

Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HCl buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.

ChSS was fractionated on a column (550 x 15 mm) of DEAE Sepharo e Fast Flow using a Hiload System (Pharmacia), which was initially equilibrated in 0.005 M NaAc-bufFer pH 5.0. The sample was dissolved in water, the insoluble residue was removed by centrifugation and the supernatant was applied onto the column. After applying the gradient shown in Figure 1, the residual polysaccharides were washed from the column using 0.5 M NaOH. Fractions (23 ml) were collected and assayed by automated methods [2,3] for total neutral sugars and uronic acids. 

Uronic acid content was determined as anhydro-uronic acid (AUA) by the automated colorimetric /n-hydroxydiphenyl assay [2,3,6] using an auto-analyser (Skalar Analytical BV, Breda, The Netherlands). Corrections were made for interference by neutral sugars present in the sample. 

Purity was confirmed by gel-filtration using a HPLC column packed with Asahipak GS-520HQ and elution with 100 mM sodium phosphate buffer containing 300 mM sodium chloride (pH 6.7). The content of total protein, total sugars, uronic acids, sulfates, nucleic acids, phosphate or fatty acids was assayed by the BCA [32] and Lowry method [33], the phenol-sulfuric acid method [34], the Blumenkrantz method [35], nephelometry [36], absorption at 260 nm, the Bartlett method [37] and the GLC method after methyl-esterification [38], respectively. 

E3.3 Determination of the Uronic Acid Content of Plant Cell Walls Using a Colorimetric Assay... 

Colorimetric Assay of Uronic Acid Content of Cell Walls... 

PGase activity assay, 335-348 uronic acid content, colorimetric assay, 735-738... 

One of the most important reactions of hexuronic acids and glycuronans is the decarboxylation caused by treating with strong acids (usually 12% hydrochloric acid). The rapid and stoichiometric loss of 1 mol of carbon dioxide per mole has been developed as an analytical method by many workers.227 Methods based on the formation of colored phenolic compounds in strongly acidic media are widely used to assay total uronic acid.228,229 The method of Blumenkrantz and Asboe-Hausen228 has been adapted for microtiter plates.230... 

B. M. van den Hoogen, P. R. van Weeren, and M. Lopes-Cardozo, A microtiter plate assay for the determination of uronic acids, Anal. Biochem., 257 (1998) 107-111. 

In most cases, uronic acids are liberated from acidic polysaccharides by hydrolysis leading to irreproducible concomitant formation of lactones. Several methods to circumvent this problem have been published describing conversion of the uronic acid into methyl esters followed by reduction with borohydride or borodeuteride reagents and subsequent hydrolysis and GC-MS detection [129]. Other techniques are based on the liberation and quantification of carbon dioxide. Direct determination of uronic acid residues in hydrolyzates has frequently been performed according to colorimetric assays, which are rather insensitive and have thus mostly been replaced by high-performance anion exchange chromatography (HP-AEC) methods [130-132]. 

In the first assay, the sample is mixed with orcinol, FeCls and concentrated HCl and heated at 96°C. The formed purple to red-violet chromophore is extracted with isoamyl alcohol and its absorbance measured at 572 nm. Because of the use of HCl, the method can be used to quantitate the total amount of both free and glycosidically bound sialic acids. Due to the strongly acidic conditions, ester groups are released. As the assay does not discriminate between bound and free sialic acids, it is widely used to monitor the presence of sialic acids in either form during fractionation of biological material. It should be noted that other monosaccharides, especially pentoses, hexoses and uronic acids interfere with the assay, which is of importance when small amounts of sialic acid are present. 

In all the colorimetric methods, galacturonic acid is liberated during the assay by hydrolysis of polymeric pectin. An application of the carbazole method, after extraction of pectin from various fruits and vegetables, has been described (6), as has been an automated carbazole method for monitoring uronic acid levels in pectin fractions (7). 

Analysis of dietary fiber. Total dietary fiber including both water-soluble and water-insoluble components was analysed with an enzymic method as described by Asp et al. (18). The dietary fiber was characterized by gas-liquid chromatographic assay of monosaccharides after acid-hydrolysis and gravimetric determination of acid insoluble lignin. Uronic acids were assayed with a decarboxylation method. These analyses were performed as described by Theander and Aman (19). 

Uronic acid compositions is determined colorimetrically using the m-hydroxydiphenyl assay described by Bramenkrantz and Asboe-Hansen (1973). 

This methodology was used to extract bioactive polysaccharides (PNSPs) from seeds of Pharbitis nil, an annual climbing herb belonging to the family Convolvulaceae. The chemical analysis of PNSPs showed contents of sugar, uronic acid and proteins of 83.6 1.61, 21.8 1.25 and 16.4 0.88% (w/w), respectively. The antioxidant activity of PNSPs was evaluated in-vitro by using ABTS and DPPH radical scavenging assays. At a concentration of 5 mg/mL, PNSPs exhibited values of radical inhibitions of 100% and 89.6% in ABTS and DPPH assays, respectively [123]. 

In addition to the total carbohydrate assay for xanthan using the Dubois method, there are specific analytical methods that may be applied to determine how much of a specific group is present. For example, specific micromethods were applied by Nisbet et al (1982) to assay for 0-acetyl groups, pyruvate ketals and uronic acids. Further discussion of such methods is beyond the scope of this work. 

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