Dinitrophenylhydrazine assay

F9. Floor, E., and Wetzel, M. G., Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay. J. Neurochem. 70, 268-275 (1998). 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

The dinitrophenylhydrazine (DNPH) assay detects a-keto acids in the urine [1, 4]. 

Assay Transfer 2.0 ml of camphor spirit to a suitable pressure bottle containing 50 ml of freshly prepared dinitrophenylhydrazine TS. Close the pressure bottle, immerse it in a water bath, and maintain at about 75° for l6 hours. Cool to room temperature, and transfer the contents to a beaker with the aid of 100 ml of dilute sulfuric acid (1 in 12). Allow to stand not less than 12 hours at room temperature, transfer the precipitate to a tared filter crucible, and wash with 100 ml of dilute sulfuric acid (1 in 12) followed by 75 ml of cold water in divided portions. Continue the suction until the excess water is removed,dry the crucible and precipitate at 80° for 2 hours, cool, and weigh. The weight of the precipitate so obtained, multiplied by O.U58I, represents the weight of 10 l6 n sample taken. 

Dinitrophenylhydrazine Reaction. This assay is based on a method developed by Wittenberg et al. (1956). It takes advantage of the interaction of p-dinitrophenylhydrazine with a free aldehyde to yield a yellow-colored hy-drazone derivative. The absorbance of the reaction mixture can be read on a spectrophotometer at 395 nm, and aldehyde can be determined quantitatively by this procedure. The reader is urged to consult this very informative article. 

In the widely used colorimetric assay of SGOT and SGPT (R2), serum is incubated at 37 °G with phosphate-buffered L-aspartate/a-oxoglutarate or DL-alanine/a-oxoglutarate, respectively, the reaction terminated after a definite interval by addition of 2,4-dinitrophenylhydrazine in dilute HCl, and after a period at room temperature the hydrazones of oxalace-tate or pyruvate so formed are treated with dilute alkali and the optical density measured against water at 505 mp the results are read from standard curves of optical density against transaminase activity. 

The fact that DHA possesses vitamin C activity was recognized early in studies of AA and was studied by several groups (13,14), Enzymes that catalyze the reduction of DHA to AA have been demonstrated in many systems and are discussed later in this chapter. Dimeric DHA, or bisdehydroascorbic acid (BDHA), and DHA both are important in nutrition this importance has been taken for granted in that the common assay of ascorbic acid, the dinitrophenylhydrazine (DNPH) method, does not distinguish between these forms. 

Reduction of Aldehydes and Hydroperoxides. Stock solutions of approximately 100-200 ppm of 2-ethylhexanal in 95% ethanol, hexane, ana 2-ethylhexanol were prepared. Accurate carbonyl assays were conducted (ASTM method E411-70, "Trace Quantities of Carbonyl Compounds with 2,4-Dinitrophenylhydrazine ) initial carbonyl levels were... 

The dinitrophenylhydrazine methods are very sensitive and remarkably specific and are the methods of choice for most analyses. Micromodifications are available for assays in microgram quantities of tissues. 

Traditionally, the extent of oxidation is determined by the peroxide value and some of the carbonyl compound assays. Most involve the formation of color products that result from the reaction of the carbonyl group with the amine group of a cyclic leuco-compound. The most popular old method involves determination of carbonyl compounds in reaction with 2,4-dinitrophenylhydrazine (carbonyl value). The reaction with benzidine (benzidine value) was used for the first time by Wode in 1957 Soviet authors referred to it as the Lyubavina technique. This technique was abandoned due to carcinogenicity of benzidine. 

A number of other assay approaches based on the reactivity of the intact C-17 side chain described for related steroids are presumably applicable to dexamethasone These include a) 2,4-dinitrophenylhydrazine reagent - this... 

Some enzyme reactions can be studied colorimetrically when either the substrate or product can be converted chemically to a coloured product suitable for measurement in a u.v. or visible light spectrophotometer. In the case of alanine aminotransferase, the pyruvate formed in the reaction can be converted to pyruvate-2,4-dinitrophenylhydrazone by the addition of 2,4-dinitrophenylhydrazine (DNP). Addition of sodium hydroxide yields a product with an absorption maximum at 505 nm. Other examples of colorimetric procedures will be found in the last section. Colorimetric procedures are used for enzyme assays in the sampling mode, whereby samples of the reaction mixture are analysed at certain fixed times after starting the reaction. Graphs depicting the reaction rate must then be constructed by plotting amount of substrate transformed against time. 

The semicontinuous surfactant-free synthesis of a core/shell styrene/acrolein latex and its analysis for surface aldehyde functions are described. A calorimetric assay based on reaction with dinitrophenylhydrazine is compared with reduction by tritiated sodium borohydride and integration of aldehyde peaks in the proton NMR spectrum of the dissolved polymer. X-ray photoelectron spectroscopy confirmed the surface location of the aldehyde functions and the analytical reaction products. The three assay procedures were in reasonable agreement, suggesting all the aldehyde functions were accessible to aqueous reagents. Relevance to protein carriers is indicated. 21 refs. CANADA... 

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