Pyruvic dinitrophenylhydrazone

This enzyme is a pyridoxal protein which is present in excessive amounts in blood serum during diseases of the liver or cardiac muscle. It may be assayed by an ultra-violet method using malate dehydrogenase in an indicator reaction [274]. Alternately, the oxalacetate formed is decomposed to pyruvate which is treated with DNP to form pyruvate-dinitrophenylhydrazone. In the presence of sodium hydroxide, an intense brown colour is produced with an absorption maximum at 505 nm [275]. 

An improved method for the preparation of A" -3-ketones from 4-bromo compounds was described by Mattox and Kendall. This procedure involves dehydrobromination of the 2,4-dinitrophenylhydrazone and subsequent cleavage of the hydrazone with pyruvic acid ... 

The scope of this reaction was investigated by Djerassi, °° who showed that 4-bromo ketones in the series and 2-bromo ketones in the 5a series give unsaturated 2,4-dinitrophenylhydrazones in 80-90% yield on warming under nitrogen with 1.1 moles of 2,4-dinitrophenylhydrazine in acetic acid. Cleavage with pyruvic acid affords the pure unsaturated ketones in 60-70 % yield. 

Pyridine base eliminations of a-bromo ketones cannot be recommended for general use because of the side reactions already discussed. The semi-carbazone-pyruvic acid method should be employed if strict absence of isomerization is required in the dehydrobromination of 2- or 4-bromo-3-ke-tones. This procedure is not applicable for the preparation of A1,4-3-ketones, due to rearrangement. The use of semicarbazide for the dehydrobromination of 6-bromo-A4-3-ketones is reported to be satisfactory for the preparation of A4,6-3-ketones65 but appears to be a neglected approach. (The corresponding 2,4-dinitrophenylhydrazones cannot be cleaved.)... 

Timmen and Dimick (1972) characterized the major hydroxy compounds in milk lipids by first isolating the compounds as their pyruvic ester-2,.6-dinitrophenylhydrazones. Concentrations as weight percent of the compounds from bovine herd milk lipids were 1,2-DGs 1.43, hydroxyacylglycerols 0.61, and sterols 0.35. Lipolysis tripled the DG content. The usual milk fatty acids were observed, except that the DGs lacked 4 0 and 6 00, again indicating that these lipids were in part intermediates in milk lipid biosynthesis. With the large hydrazone group... 

Esters of pyruvic acid 2,6-dinitrophenylhydrazone were described by Bassette et al. 

Pyruvic acid was first found in agar194 and then in several carrageenans.113,195,196 It may be determined quantitatively in the form of its 2,4-dinitrophenylhydrazone,197 but an enzymatic procedure with the available lactate dehydrogenase seems to be more convenient.198... 

In addition, the DNPH (2,4-dinitrophenylhydrazine) [38] and NADH (dihydronicotinamide adenine dinucleotide) [36] studies with purified Iso-4 provided the evidence that the 70 Da moiety was a pyruvate derivative (C3H2O2). In the DNPH study, treatment of Iso-4 with acid and 2,4-dinitrophenylhydrazine produced the 2,4-dinitrophenylhydrazone of the pyruvic acid liberated from Iso-4. In the NADH study, the amount of NAD+... 

A procedure developed by Mattox and Kendall for the liberation of cortisone acetate from the C3 2,4-dinitrophenylhydrazone utilizes a mixture of pyruvic acid, acetic acid, chloroform, and hydrogen bromide and affords the parent compound in 80% yield. For comparison. DePuy° prepared a mixture of 1 g. of A -cholestene-S-one 2,4-DNP with 100 ml. of chloroform and 100 ml. of the usual levulinic-hydrochloric acid and heated it under reflux for 3 hrs. Chromatography afforded pure A -cholestene-3-one in high yield. 

Detection systems for GC are chosen for their sensitivity and selectivity for a particular class of VOCs. Detectors for GC include FID, the BCD, the photoionization detector (PID), the pulsed discharge detector (PDD), and the reduction gas detector (RGD). A variety of mass spectrometers can also be interfaced with a GC for confirmation of molecular structure and quantitation. Singlewavelength ultraviolet-visible detectors (190 to 600 nm) and diode array detectors are used to detect carbonyls as their 2,4-dinitrophenylhydrazone derivatives. The absorption maxima for aliphatic carbonyls, aromatic carbonyls, and dicarbonyls are near 360 nm, 385 to 390 nm, and 415 to 430 nm, respectively. Formic, acetic, and pyruvic acid are detected by ion conductivity. 

Carbonyl products were identified by paper chromatography of the 2,4-dinitrophenylhydrazones (20). The irradiated N-acetylalanine showed only pyruvic acid and acetaldehyde. These were determined quantitatively by the method of Johnson and Scholes (13) with minor modifications. Chloride ion was determined by the method of Luce et al. (15) after Hayon and Allen (12). 

The absorption spectra of equal concentrations of the 2.4-dinitrophenylhydrazones of pyruvic acid and a-oxogtutaric acids, showing an isosbestic point at 431 nm. 

Oxopropionaldehyde (MethylgJyoxdl, Pyruvic aldehyde) di 254 52 " 6/5-2,4-Dinitrophenylhydrazone 299 300, red, PhNO. Dioxime... 

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. 

For the determination of keto-acids, e.g. pyruvic, a-ketoglutaric and oxalacetic acids in mixtures and in biological materials, a method has been proposed employing paper electrophoretic separations of the 2,4-dinitrophenylhydrazones of these acids.< ) After elution, the polarographic wave corresponding to the reduction of the nitro-groups (in the 2,4-dinitrophenylhydrazone formed) was followed in 0-1 N HCl and the first wave at —0T4 V measured. 

In the colorimetric, end-point, Reitman-Frankel method, the 2,4-dinitrophenylhydrazone derivative of pyruvate is measured. (The 2,4-dinitrophenylhydrazone derivative of 2-oxoglutarate is also present but this is not as chromogenic.) This method is now little used. 

The oxidation of NADH to NAD can be followed spectro-photometrically at 340 nm. A variation of this is to measure the pyruvate formed by pyruvate kinase as its 2,4-dinitrophenylhydrazone derivative. 

---