A method for the colorimetric determination of urinary methylmalonic acid in pernicious anemia.

Abstract The detection of increased methylmalonic acid in the urine serves as a useful tool in the diagnosis of vitamin B 12 deficiencies, including pernicious anemia. A simple method for determination is described here. The acid is isolated from urine by using Dowex 3 × 4 anion-exchange resin, allowed to react with diazotised p-nitroaniline to form a green color, and determined spectrophotometrically at 620 mμ. A number of urinary components were examined and found not to interfere significantly with the assay. Methylmalonic acid was identified by paper and thin-layer chromatography as the chromogenic agent in the urine of a patient with untreated pernicious anemia. Malonic acid, which also yields a green color with the diazo reagent, was not detectable in this urine. The elevated level of urinary methylmalonic acid in this patient showed a prompt response to therapy, declining from 750 mg. to 92 mg. per day within a 24 hour period following the parenteral administration of vitamin B 12 . Two other patients with pernicious anemia who were studied showed pretreatment levels of 40 and 610 mg. methylmalonic acid per day. The coefficient of variation for the method ranged from 4 to 15 per cent in urine samples containing from 72 to 1,020 mg. methylmalonic acid per liter. The error was considerably greater at the apparent concentration of 5 mg. of methylmalonic acid per liter in normal urine.

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