2.4- Dinitrophenylhydrazones amino acids, from

Although alcaptonuria is a relatively harmless condition, such is not the ca.se with other errors in amino acid metabolism. In maple syrup urine dis-eim, the oxidative decarboxylation of cy-kctoacids derived from valine, isoleucinc, and leucine is blocked because the branched-chain dehydrogenase is missing or defective. Hence, the levels of these cx-ketoacids and the branched-chain amino acids that give rise to them are markedly elevated in both blood and urine. The urine of patients has the odor of maple syrup-hence the name of the disease (also called branched-chain ketoaciduria). Maple syrup urine disease usually leads to mental and physical retardation ss the patient is placed on a diet low in valine, isoleucinc, and leucine early in life. The disease can be readily detected in newborns by screening urine samples with 2,4-dinitrophenylhydrazine, which reacts with a-ketoacids to form 2,4-dinitrophenylhydrazone derivatives, A definitive sis can be made by mass spectrometry. 

The oxidation product has been isolated as its 2,4-dinitrophenylhydrazone and corresponds with -keto-6-aminovaleric acid. Putrescine, arginine, Manske s 1 (+) acetylornithine (118) and other related amino acids are oxidized much less readily if at all. The same authors have extracted an ornithine dehydrogenase from the young roots of Datura tatula. It requires the cooperation of a coenzyme not yet identified, and appears unable to oxidize putrescine and amino acids other than ornithine and to a lesser extent glutamic. Either of these systems, or the two linked into a H-transfer chain, would seem able to catalyze the oxidation of ornithine in the living tissues. No carbon dioxide was released from ornithine by the poly-phenolase system but on addition of an unwashed belladonna tissue-suspension carbon dioxide was liberated, presumably by decarboxylation of the a-keto-5-aminovaleric acid formed by the oxidation. 

Hydrolysis of the labeled compound with 2 N sulfuric acid furnished 3-amino-4-methyl-2-hexanone which, on treatment with iodine in the presence of sodium hydroxide, furnished isoleucine and iodoform. The iodoform was oxidized to carbon dioxide, while treatment of isoleucine with ninhydrin furnished 2-methylbutanal, isolated as its 2,4-dinitrophenylhydrazone. Kuhn-Roth oxidation of the aldehyde gave acetic acid which was degraded by the Schmidt procedure to carbon dioxide and methylamine. The 2-methylbutanal was also degraded stepwise by the method of Strassman. Determination of the radioactivity of the various degradation products showed that 94% of the activity was equally shared between the lactam C(2) and side-chain C(10) carbonyl atoms. The remaining activity was shared between C(4) of the lactam ring and the a> carbon atom of the ec-butyl side chain. The results are consistent with derivation of tenuazonic acid from isoleucine and two molecules of acetic acid. However, the direct incorporation of isoleucine into tenuazonic acid was not investigated. 

Neubauer B) and Knoop (Ba) demonstrated in the early part of this century the metabolic interconversion of a-amino and a-keto acids. In 1920, Thunberg (S) observed that glutamate was oxidized in the presence of frog muscle, and, in 1936, Weil-Malherbe (4) prepared an extract from brain tissue which catalyzed glutamate oxidation and identified the product, a-ketoglutarate, as its 2,4-dinitrophenylhydrazone. 

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