Estimation of Sugars

There are many methods for detecting and estimating sugars and they cannot be considered in individual detail here however, most of them fall into a few, simple categories. 

Two types of reaction yield formyl pyruvic acid these are some P-eliminations (see below) and the periodate oxidation of most sialic acids. In each case the formyl pyruvate produced can be made to react with thio-barbituric acid to give an intensely coloured product, so giving the means of following P-eliminations and estimating very small amounts (1-2 (jig) of sialic acid. 

Periodate oxidation is widely used in carbohydrate chemistry, for example in the detection of carbohydrate of glycoprotein in polyacrylamide gels and in analysing linkages. It is considered more fully below. 

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The third method is of limited application and is used only in special cases, The second is the most accurate and rapid method, and is of considerable technical importance. The chemical method (described below), although less accurate than the polarimetric method, is of great value for the estimation of sugars in biological fluids. In fact, for such purposes, it is often to be preferred to the polarimetric method owing to the probable presence of other substances having high optical rotations. 

In RNA, most residues have Ch -endo sugar puckers (N-conformations) with small 3/hi H2 coupling (<2-3Hz) and with the corresponding Hl -H2 cross-peaks not observed in homonuclear 2D COSY or TOCSY spectra. These cross-peaks are observed only for flexible residues and residues locked in the S-conformations the presence or absence of TOCSY Hl -H2 cross-peaks is often used for qualitative estimation of sugar puckers. 

Gas-liquid chromatography This technique allows the quantitative and qualitative estimation of sugar alcohols. It is limited by the volatility and stability of derivatives and by the acciuacy with which quantitative conversion of polyols to their volatile derivatives can be achieved. The volatile derivatives of sugar alcohols commonly used are trimethylsilyl (TMS) ethers. To prepare TMS derivatives, samples are dried, dissolved in pyridine, and reacted by sequential addition of two volumes of hexamethyldisilazane (HMDS) and one volume of trimethylchlorosilane (TMCS). Other... 

Mount JN and Laker ME (1981) Estimation of sugar alcohols by gas-liquid chromatography using a modified acetylation procedure. Journal of Chromatography 226 191-197. 

Refractometry This allows for the rapid, reasonably accurate estimation of sugar levels based on the measurement of soluble solids. Correction tables exist allowing for other dissolved substances, e.g., acids, and for changes in temperature however, many modern refractometers make automatic allowance for this. 

Of the numerous copper reduction methods for the estimation of sugar in urine that of Shaffer and Hartmann (81) is particularly satisfactory when adapted for the estimation of glucuronic acid (22,23). The method cannot be satisfactorily used if sugar is present and must be used with caution for ether glucuronides (see end of section). 

The estimation of sugar phosphates with enzymes offers many possibilities. For instance, fructose 1,6-diphosphate can be estimated using aldolase, glucose 6-phosphate with the corresponding dehydrogenase, etc. Methods for the analysis of glucose i-phosphate, glucose 6-phosphate, fructose i-phos-phate and fructose 1,6-diphosphate have been described . 

Breslow studied the dimerisation of cyclopentadiene and the reaction between substituted maleimides and 9-(hydroxymethyl)anthracene in alcohol-water mixtures. He successfully correlated the rate constant with the solubility of the starting materials for each Diels-Alder reaction. From these relations he estimated the change in solvent accessible surface between initial state and activated complex " . Again, Breslow completely neglects hydrogen bonding interactions, but since he only studied alcohol-water mixtures, the enforced hydrophobic interactions will dominate the behaviour. Recently, also Diels-Alder reactions in dilute salt solutions in aqueous ethanol have been studied and minor rate increases have been observed Lubineau has demonstrated that addition of sugars can induce an extra acceleration of the aqueous Diels-Alder reaction . Also the effect of surfactants on Diels-Alder reactions has been studied. This topic will be extensively reviewed in Chapter 4. 

The 1995 Canadian and United States sugar alcohol (polyol) production is shown in Table 2. The market share of each is also given. Liquids comprise 48% crystalline product comprises 39% and mannitol comprises 13% of the polyol market. An estimate of total U.S. sorbitol capacity for 1995 on a 70% solution basis was 498,000 t. ADM, Decatur, lU., produced 68,200 t Ethichem, Easton, Pa., 13,600 t Lon2a, Mapleton, lU., 45,400 t Roquette America, Gurnee, lU., 68,200 t and SPI Polyols, New Castle, Del., 75,000 t (204). Hoffman-LaRoche, which produces sorbitol for captive usage in the manufacture of Vitamin C (see Vitamins), produced about 27,300 t in 1995. 

Quantitative Estimation of Deoxy Sugars and Related Compounds with Special Reference to Periodate Oxidation. 

In as far as other analytical methods are concerned, many specific reactions have been elaborated for the quantitative determination of 2-deoxy aldoses. 2-Deoxy-D-ribose (2-deoxy-D-erythro-pentose), a compound which was recognized early as playing an important role in biological systems, has been of particular interest. Overend and Stacey (43) have given a critical review of the methods available until 1952 for the estimation of 2-deoxy pentoses. A recent summary of specific methods for the identification and quantitative estimation of the different classes of deoxy sugars has been prepared by Dische (13). 

To obtain reliable, accurate, and reproducible methods for quantitative estimation of deoxy sugars, certain conditions must be fulfilled. Thus, it is necessary that the chromogen be formed quantitatively from the sugar. The chromogen must then react quantitatively with the compound used for color formation, and lastly, the dye, once formed, should be stable and have a well defined molar extinction coefficient. In methods in which all of these conditions are not or cannot be fulfilled, recourse must be had to simultaneous determinations with suitable standard substances, a requirement not always easy to fulfil. 

Despite the above-mentioned short-comings, this approach to the estimation of those deoxy sugars which yield malonaldehyde when oxidized with periodate, seemed promising, since, as has been seen (58,59), the dye is formed quantitatively in the reaction of malonaldehyde with 2-thiobarbituric acid also, more recently, its constitution (49,57) and molar extinction coefficient (36) have been established. Thus, if conditions could be found in which malonaldehyde, while being formed quantitatively from the deoxy sugars, would be stable, an ideal method, independent of standard compounds, would be available for the quantitative determination of all of these sugars. 

Results of inhibition studies with nojirimycin and its analogs published up to 1988, and additional data from the author s laboratory, are summarized in Table VI. It should be noted that the inhibition constants are given in pM instead of mM (as in Tables II - V). Data for glycosylamines are included, in order to facilitate an estimation of the effects caused by the different positions of the basic group in the two types of basic sugar derivative. Not included are the data of Reese and coworkers and of Grover and Cushley on nojirimycin, because these authors were apparently unaware of the slow and only partial dissociation of the nojirimycin hydrogensulfite adduct which had been used instead of free nojirimycin. 

Basically, there are four major types of measures that are used in taste intensity measurements (a) threshold measures or estimates of the physical level at which the sensation of sweetness begins, (b) equal-sweetness matches between a sugar and other sweeteners, (c) category or rating scales, and (d) ratio scales. Each method has found its adherents and uses, and each possesses specific advantages and defects that indicate its use for one application, but contraindicate its use for another. These methods and their applications have been critically analyzed and reviewed, " " and it is, therefore, superfluous to deal with the topic here. 

The significance of n.m.r. spectroscopy for structural elucidation of carbohydrates can scarcely be underestimated, and the field has become vast with ramifications of specialized techniques. Although chemical shifts and spin couplings of individual nuclei constitute the primary data for most n.m.r.-spectral analyses, other n.m.r. parameters may provide important additional data. P. Dais and A. S. Perlin (Montreal) here discuss the measurement of proton spin-lattice relaxation rates. The authors present the basic theory concerning spin-lattice relaxation, explain how reliable data may be determined, and demonstrate how these rates can be correlated with stereospecific dependencies, especially regarding the estimation of interproton distances and the implications of these values in the interpretation of sugar conformations. 

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