Comparison of two methods for the measurement of rat liver methylmalonyl-coenzyme A mutase activity: HPLC and radioisotopic assays

Methylmalonyl-coenzyme A mutase (MCM) is a 5'-deoxyadenosylcobalamin-linked mitochondrial enzyme that catalyzes the isomerization of L-methylmalonyl-c oenzyme A to succinyl-coenzyme A. In vitro assays of total and holo-MCM act ivities are important tools for investigating the cobalamin pathway. Severa l methods have been described for measuring MCM activity. The most commonly -used method is a radioassay based on the permanganate oxidation of DL[CH3- C-14]methylmalonyl-coenzyme A, but radiometric methods are insensitive, lab orious and time-consuming. Therefore, we have compared this method with a n onradiametric assay, potentially most sensitive, based on the separation of methylmalonyl-coenzyme A and succinyl-coenzyme A by high performance liqui d chromatography (HPLC). We determined the optimal assay conditions and the reproducibility and sensitivity of each technique. The results obtained by the two techniques were very different: the specific activities obtained b y the permanganate oxidation method (0.039 +/- 0.013 nmol/min/mg protein fo r the holo-MCM activity and 1.90 +/- 0.69 nmol/min/mg protein for the total -MCM activity) were threefold lower than those obtained with the HPLC metho d (0.124 +/- 0.011 nmol/min/mg protein for the holo-MCM activity and 6.15 /- 0.76 nmol/min/mg protein for the total-MCM activity). The coefficients o f variation for the radiometric method (18.4-40.6%) were three to five time s greater than those for the HPLC assay (3.5-12.2%). This demonstrates the lack of sensibility and reproducibility of the permanganate radioassay. Thu s, the radiometric method is not suitable for measuring low mutase activiti es such as the hole activities in tissues. The intrinsic inconvenience of t he radiometric assay indicates that the HPLC method is a method of choice f or measuring MCM activity. (J. Nutr. Biochem. 10:56-62, 1999) (C) Elsevier Science Inc. 1999. All rights reserved.