Glucose hexokinase methods

Effect of hemolysis on the assay of glucose by the fluorescence-hexokinase method without protein precipitation. 

Williams, D.C., Huff, G.F., and Seitz, W.R. (1976) Glucose oxidase chemiluminescence measurement of glucose in urine compared with the hexokinase method. Clin. Chem. 22, 372. 

Bondar, R. J. L., and Mead, D. C. (1974). Evaluation of Glucose-6-Phosphate Dehydrogenase from Leu-conostoc mesenteroides in the Hexokinase Method for Determining Glucose in Seram. Clin Chem 20 586. 

Note First results for the determination of glucose in urine and cerebrospinal fluid (CSF) using this hexokinase method have been published (SI99, S224). In this case the samples should be diluted with 1% bovine albumin solution. Good correspondence to a comparative method was described for CSF (S224). A 30% deviation was known for urine samples (SI99). 

S175 Karmen, A. and Lent, R.W. (1987). Measurement of glucose in serum using the hexokinase method with the Ames Seralyzer. Clin. Chem. 33, 1011, Abstr. 637. 

S188 Osuch, M.A.V., Lee, J.K. and Prusinski, E.A. (1987). Evaluation of the accuracy of Seralyzer glucose (hexokinase) and five glucose methods compared to NORM. Clin. Chem. 33, 952, Abstr. 350. 

S222 Stott, A., Clark, B. and Barwise, I. (1988). Measuring serum and plasma glucose by the hexokinase method with a reflectance photometer in a high-risk environment. Clin. Chem. 34, 424. 

Neese JW. Glucose, direct hexokinase method Selected methods. Clin Chem 1982 9 241-8. 

Neese JW, Duncan P, Bayse D. Development and Evaluation of a Hexokinase/Glucose-6-Phosphate Dehydrogenase Procedure for Use as a National Glucose Reference Method. HEW PubHcation No. (CDC) 77-8330, Atlanta Centers for Disease Control, 1976. 

Analytical procedures Protein was determined with the Biorad-method. Glucose was determined by the hexokinase-method (Boehringer), amylase activity by the method of Manning and Campbell (1961) at 60 C. One unit of amylase is defined as the amount of protein which will hydrolyse 10 mg of starch per minute. 

Excellent correlation was found when results at 660 nm and 749 nm were compared using a reference hexokinase glucose method (27). The dose response was excellent up to 300 mg/dL glucose. In general, water-borne coatings do not lend themselves to ranging by antioxidants (qv). 

If the fluorometric method is used after protein precipitation then the glucose can be readily assayed on 1 pi of plasma with the hexokinase procedure. 

In view of the above discussion, it should be clear why the author recommends that the laboratory of Neonatology precipitate proteins with a Somogyi filtrate and then perform the hexokinase procedure on the filtrate. Neither the glucose oxidase i rocedure nor the toluidine method have adequate sensitivity thout the use of special micro-equipment and speciaT techniques. 

A kinetic procedure employing the reverse reaction is coupled to the enzymes hexokinase and glucose-6-phosphate dehydrogenase, as used by Nielson and Ludvigson after the method of Oliver (J ). This procedure was later modified and optimized by Rosalki (38). 

Two other commonly occurring hexoses which are usually found as components of polysaccharides or combined with other molecules in complex structures are galactose and fructose and, in a similar manner to other monosaccharides, enzymic methods are available for their measurement. An enzymic method for the measurement of fructose using hexokinase was described earlier, together with the method for mannose and glucose (Figure 9.22). 

The physiological usefulness of this method for identifying the fuels that are used and their rates of utilisation in different cells is discussed in other chapters (Chapter 3). For example, measurement of the activity of the enzymes hexokinase and glutaminase in immune cells showed, for the first time, that glucose and glutamine are the major fuels utilised by these cells. This finding has had clinical significance (Chapter 17). 

Creatine phosphokinase activity has been reported to be minimally inhibited by hemolysis. Hemoglobin concentrations of 1.25 g/100 ml inhibit 5% and 2.5 g/100 ml, 12% (N5). However, in methods utilizing adenosine diphosphate in the reaction mixture, hemolysates containing 100 mg of hemoglobin per 100 ml may have apparent activities of 5-100 units/liter. The activity is presumably related to adenylate kinase in the erythrocyte (S33). In methods utilizing adenosine diphosphate in a coupled enzyme reaction with hexokinase and glucose-6-phosphatase, the inhibitory effect can be eliminated by adding sufficient adenosine mono-... 

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

Schulze et al. [135] developed fused-silica chips dynamically coated with hydroxypropylmethyl cellulose and utilized them for the separation of aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan. The authors used deep UV laser-induced fluorescence detection for these compounds. Schuchert-Shi et al. [136] identified ethanol, glucose, ethyl acetate, and ethyl butyrate, byproducts obtained in enzymatic conversions using hexokinase, glucose oxidase, alcohol dehydrogenase, and esterase. The authors reported that the quantification for ethyl acetate was possible using contactless conductivity detection. Hu et al. [137] described the separation of reaction products of (3-thalassemia in a multiplex primer-extension reaction using NCE. The method developed was used for patient samples and the results coincided with those of a detection kit. 

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