The lsolationa nd ldentificatioonf Abnormal Metabolites from Urine of Patients with lsovaleric Acidemia and their Metabolic Significance

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Recently, isovaleric acidemia, an inborn error of leucine metabolism, was reported from our laboratory.Main clinical features of this disease are episodic ketoacidosis and coma associated with great elevation of isovaleric acid in blood.Evidence obtained to date suggested that the disease is due to deficiency in the enzyme, isovaleryl CoA dehydrogenase.However, serum isovaleric acid levels, although still 2to5 times above normal, were markedly less in remission than those found during attacks of ketosis. One of the hypotheses on the episodic nature of this disease was that there might be an alternate pathway which can metabolize isovaleryl CoA efficientlys o that serum isovaleric acid does not accumulate during remission.Therefore, urinary metabolites of leucine were sought for by gas-liquid chromatography (GLC) and two unknown compounds were found in large amounts. A huge peak (Compound B) was observed by GLC after methylation in ethyl acetate extracts of acidified urine of patients which were obtained both in remission and ketosis (Fig.1). This was purified through repeated siliciac cid chromatography and recrystallization (Fig.2).Analytical data including mass (Fig.3), nuclear magnetic resonance (Fig.4), and infrared spectra (Fig.5) indicated that compound B was N-isovalerylglycine and this was confirmed by demonstration of isovaleric acid and glycine after hydrolysis of this compound, and also by the identity of ele. mental analysis, melting point and infrared spectrum of Compound B with those of synthesized N-isovalerylglycine. Another unknown compound (Compound D) was found in urine which was obtained when a patient was in ketotic attacks. Methyl ester of Compound D was isolated by preparative GLC. The isolated Compound D was identified as β-hydroxyisovaleric acid by identity of retention time on GLC, and also of mass spectrum with those of the synthesized compound. One of the patients (3 1/2 yr) excreted N-isovalerylglycine in amounts of 1,100 and 1,300 mg per day in two occasions of ketotic attack. Excretion by the same patient ranged from 330 to 1,000 mg per day during remission.The other patient (5 1/4 yr) excreted from 550 to 1,700 mg per day during remission.Although excretion of this compound in ketosis increased 2 to 3 times of those in remission, serum levels of isovaleric acid increased about 100 times of those in remission. β-Hydroxyisovaleric acid is excreted in large amounts (60 to 340 mg per day) only in ketosis where free isovaleric acid was increased large excess, and excretion in remission was below 7 mg per day. On the basis of these and other data, it was concluded as follows: 1) Isovaleryl CoA which could not be oxidized by patients with isovaleric acidemia is conjugated with glycine, protecting the patients from toxity of isovaleric acid; 2) When isovaleryl CoA is produced inexcess and overloads this conjugating system following excessive protein intake or excessive catabolism, isovaleryl CoA not conjugated is hydrolyzed, and free isovaleric acid produced causes toxic effects observed in ketosis; 3) Free isovaleric acid which increased during ketosis may be hydroxylated directly to β-hydroxyisovaleric acid; 4) It may be concluded that episodic nature of this disease is explained by the presence of conjugating system with glycine, and short duration of ketotic attacks is due to the presence of hydroxylating system.
Japan Society of Clinical Chemistryの論文

The lsolationa nd ldentificatioonf Abnormal Metabolites from Urine of Patients with lsovaleric Acidemia and their Metabolic Significance

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