Nutrition and Metabolite Kits

Sigma manufactures several unique enzymatic-based kits for the quantitation of nutrients and metabolites. These kits utilize spectrophotometric and gravimetric detection making them easy to use, yielding high sensitivity results.

For the quantitative, enzymatic determination of ammonia in food and biological samples. Ammonia reacts with α-ketoglutaric acid and NADPH in the presence of L-glutamate dehydrogenase to form L-glutamate and NADP. The decrease in absorbance at 340 nm, due to the oxidation of NADPH, is proportional to the ammonia concentration. L-glutamate dehydrogenase reacts specifically with ammonia. The Ammonia Assay Kit may be used to determine ammonia concentrations in the range of 0.02-15 µg/ml.

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For the determination of total dietary fiber. Uses a combination of enzymatic and gravimetric methods. This procedure is based on the method published in Official Methods of Analysis, 15th ed., AOAC, Arlington, VA, Vol. II, Sec. 985.29, 1105 (1990). Samples of dried, fat-free foods are gelatinized with heat stable a-amylase and then enzymatically digested with protease and amyloglucosidase to remove the protein and starch present in the sample. Ethanol is added to precipitate the soluble dietary fiber. The residue is then filtered and washed with ethanol and acetone. After drying, the residue is weighed. Half of the samples are analyzed for protein and the others are ashed. Total dietary fiber is the weight of the residue less the weight of the protein and ash.

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Standards for use as internal controls in conjunction with the Total Dietary Fiber Assay Kit (TDF100A).

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For the quantitative, enzymatic determination of fructose in food and other materials. Fructose is phosphorylated by ATP using hexokinase. Fructose 6-phosphate is then converted to glucose 6-phosphate by phosphoglucose isomerase. Glucose-6-phosphate is then oxidized to 6-phosphogluconate in the presence of NAD by glucose-6-phosphate dehydrogenase. During this oxidation, an equimolar amount of NAD is reduced to NADH. The consequent increase in absorbance at 340 nm is directly proportional to fructose concentration.

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For the quantitative, enzymatic determination of glucose in food and other materials. Glucose is oxidized to gluconic acid and hydrogen peroxide by glucose oxidase. Hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to form a colored product. Oxidized o-dianisidine reacts with sulfuric acid to form a more stable colored product. The intensity of the pink color measured at 540 nm is proportional to the original glucose concentration.

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For the quantitative, enzymatic determination of glucose in food and other material. Glucose is phosphorylated by ATP using hexokinase. Glucose-6-phosphate is then oxidized to 6-phospho-gluconate in the presence of NAD by glucose-6-phosphate dehydrogenase. During this oxidation, an equimolar amount of NAD is reduced to NADH. The consequent increase in absorbance at 340 nm is directly proportional to glucose concentration.

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For the quantitative, enzymatic determination of starch in food and other materials. The hydrolysis of starch to glucose is catalyzed by a-amylase and amyloglucosidase. Glucose is oxidized to gluconic acid and hydrogen peroxide by glucose oxidase. Hydrogen peroxide reacts with o-dianisidine in the presence of peroxidase to form a colored product. Oxidized o-dianisidine reacts with sulfuric acid to form a more stable colored product. The intensity of the pink color measured at 540 nm is proportional to the original glucose concentration.

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For the quantitative, enzymatic determination of native starch in food and other materials. The hydrolysis of starch to glucose is catalyzed by amyloglucosidase. Glucose is phosphorylated by hexokinase. Glucose-6-phosphate is then oxidized to 6-phosphogluconate in the presence of NAD in a reaction catalyzed by glucose-6.phosphate dehydrogenase. The increase in absorbance at 340 nm is directly proportional to the glucose concentration.

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For the quantitative, enzymatic determination of sucrose in food and other materials. Sucrose is hydrolyzed to glucose and fructose by invertase. Glucose and fructose are phosphorylated hexokinase. Glucose-6-phosphate is then oxidized to 6-phosphogluconate in the presence of NAD in a reaction catalyzed by glucose-6-phosphate dehydrogenase . The increase in absorbance at 340 nm is directly proportional to sucrose concentration.

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For the measurement of glycerol, true triglycerides, or total triglycerides in serum or plasma. Triglycerides are first hydrolyzed by lipoprotein lipase to glycerol and free fatty acids. Glycerol is then phosphorylated by ATP forming glycerol-1-phosphate and using glycerol kinase. Glycerol-1-phosphate is then oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate and hydrogen peroxide. Peroxidase catalyzes the coupling of hydrogen peroxide with 4-aminoantipyrine and sodium N-ethyl-N-(3.sulfopropyl) m-anisidine (ESPA) to produce a quinoneimine dye that shows an absorbance maximum at 540 nm. The increase in absorbance at 540 nm is directly proportional to triglyceride concentration of the sample. Many of the triglyceride reagents which are commercially available, do not differentiate between endogenous glycerol and glycerol derived by hydrolytic action of lipase on glycerides. The kit also includes sufficient reagent for an additional 250 free glyceride tests for true triglyceride determination.

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Measures free, endogenous glycerol using coupled enzyme reactions and does not include initial lipase hydrolysis.

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