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Urinary Methylmalonate and Hepatic Methylmalonyl Coenzyme A Mutase Activity in the Vitamin B₁₂-deficient Rat^1,2,

Department of Biochemistry and Biophysics, University of California Medical Center, San Francisco, California 94122

The amount of methylmalonate (MMA) excreted in the urine by rats maintained on vitamin B₁₂-deficient diets was found to be a useful indicator of the severity of B₁₂ deficiency. MMA was determined quantitatively by a new method as its ammonium salt, after separation from an ether extract of urine by one-dimensional paper chromatography. The extent of vitamin B₁₂ deficiency in the liver was estimated by measuring the activity of methylmalonyl coenzyme A (MMCoA) mutase, decreased in the deficient state owing to depletion of the vitamin B₁₂ coenzyme required for its function. In rats fed B₁₂-deficient diets containing propionate, urinary MMA excretion > 1.5 µmoles per gram body weight per 24 hours reflected a decrease in hepatic MMCoA mutase activity to about 50% (or less) of the vitamin B₁₂-supplemented control value. To simplify measurement of MMA, a rapid, semiquantitative screening test, detecting as little as 50 nmoles (about 6 µg) of MMA, was devised. The test can be done directly on filtered rat urine, without prior extraction, and provides a practicable method for evaluating the degree of vitamin B₁₂ deficiency in the intact rat.

² Supported in part by National Institutes of Health Training Grant nos. 2 T1-GM-372, 5 TO 1 GM00372-07 and -08, Fellowship no. 1-F3-AM-24-536-01 to E. B. Reed and by ACS Institutional Grant no. IN-33E.

Manuscript received 13 January 1970.