[65] Assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods

This chapter is based on the assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods based on instruments capable of giving a full-scale deflection of the recorder with 0.25μM NADH, with a noise level less than 2%.

Abstract

This chapter is based on the assays of intermediates of the citric acid cycle and related compounds by fluorometric enzyme methods. An eppendorf fluorometer or a metabolite fluorometer are instruments capable of giving a full-scale deflection of the recorder with 0.25μM NADH, with a noise level less than 2%. At such high sensitivities, the full progress of each enzymatic reaction is recorded. The following accounts for the majority of difficulties and inaccuracies commonly encountered with fluorometrie enzyme methods: All solutions should be dust and particle free; the cuvettes should be temperature equilibrated; Particular care should be taken to avoid contamination of solutions with enzymes, or cross-contamination; Fresh enzyme solution must be made each day; Solutions of pyridine nucleotides are best prepared each day and stored on ice. NAD+ and NADP+ are most stable in a slightly acid solution, and may be diluted with distilled water; and all standard solutions should be neutralized, and assayed spectrophotometrically on the day of use. Metabolic intermediates other than reduced pyridine nueleotides, total CoA, fatty acyl-CoA, and fatty acylearnitine compounds are measured in neutralized perchloric acid extracts of tissues. Perchloric acid is generally more convenient to use than trichloroacetic acid for the extraction, because it may be removed by precipitation as the potassium salt.