Rapid determination of tricarboxylic acid cycle enzyme activities in biological samples

Abstract

Background: In the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays.

Results: We developed three assays that measure the full set of TCAC enzymes. One assay relies on the sequential addition of reagents to measure succinyl-CoA ligase activity, followed by succinate dehydrogenase, fumarase and, finally, malate dehydrogenase. Another assay measures the activity of alpha-ketoglutarate dehydrogenase followed by aconitase and isocitrate dehydrogenase. The remaining assay measures citrate synthase activity using a standard procedure. We used these assays successfully on extracts of small numbers of human cells displaying various severe or partial TCAC deficiencies and on frozen heart homogenates from heterozygous mice harboring an SDHB gene deletion.

Conclusion: This set of assays is rapid and simple to use and can immediately detect even partial defects, as the activity of each enzyme can be readily compared with one or more other activities measured in the same sample.

Figure 1

Tricarboxylic acid cycle enzyme assays. A, The first segment of the tricarboxylic acid cycle can be conveniently evaluated using a single assay to measure five enzymes by spectrophotometrically recording the reduction of DCPIP. The sequential assay begins by measurement of succinyl-CoA ligase activity based on oxidation of the produced succinate by succinate dehydrogenase, which forwards the electrons to the electron acceptors (DQ, DCPIP, and PMS). Coupling of these two activities to estimate succinyl-CoA ligase activity is permitted by the much higher activity of SDH than of succinyl-CoA ligase. After SDH inhibition by malonate, simultaneous addition of glutamate and aspartate aminotransferase ensures elimination of any oxaloacetate in the assay medium, thereby allowing further measurement of MDH activity. Incidentally, the required presence of NAD+ permits the measurement of glutamate dehydrogenase activity. Adding more DCPIP allows subsequent measurement of fumarase and MDH activity. Again, the coupling assay to estimate fumarase activity using MDH activity is permitted by the much higher activity of MDH. B, A second spectrophotometric assay subsequently measures pyridine dinucleotide reduction by three additional enzymes starting with α-ketoglutarate dehydrogenase. The next enzyme to be measured is aconitase, whose product, isocitrate, is readily oxidized by isocitrate dehydrogenase, producing NADPH. A saturating isocitrate concentration is finally added to enable measurement of isocitrate dehydrogenase activity. C, respective proportions of TCAC enzyme activities in mouse heart. The inset shows the TCAC depicted as two interacting enzyme cycles, A and B. Cycle A: α-ketoglutarate dehydrogenase (6), succinyl CoA ligase (1), succinate dehydrogenase (2), fumarase (4), and malate dehydrogenase (5); Cycle B: citrate synthase (9), aconitase (7), and isocitrate dehydrogenase (8). The two cycles interact via the activity of aspartate aminotransferase (10).

Figure 2

Proportionality between TCAC enzyme activities and protein concentration in mouse heart homogenate. 1-2, 4-8: Enzyme activities plotted as a function of protein concentration: succinyl-CoA ligase (1), succinate dehydrogenase (2), fumarase (4), malate dehydrogenase (5), α-ketoglutarate dehydrogenase (6), aconitase (7), and NADP⁺/NAD⁺-isocitrate dehydrogenase (8). Experimental conditions were as described in Figure 1. Enzyme numbering according to Figure 1.

Figure 3

Detection of severe and partial TCAC enzyme deficiencies in various biological samples. A, severe enzyme deficiencies. (a) control fibroblasts; (b) SDHA-mutant fibroblasts homozygous for a deleterious R554W mutation, and (c) fumarase-mutant fibroblasts homozygous for a deleterious E319Q mutation. Note that only the addition of organic acids and inhibitors is indicated, although the experiments also involved additions of cations, cofactors, etc, similar to those in Figure 1. B, partial enzyme deficiencies. (a) fumarase-mutant lymphoblasts heterozygous for an N64T mutation; and (b) heart homogenate from a mutant mouse heterozygous for a deleterious SDHB deletion of exon 2. Numbers along the traces are nmol/min/mg protein. The shaded areas show the decreases compared to control values. C, Graphical representation of the values obtained for the various samples investigated. Dark symbols indicated statistically significant deficiencies. Values are means ± 1 SD. A minimal number of three independent assays (up to ten) were performed to calculate the mean values. Experimental conditions were as in Figure 1. Abbreviations: P1, patient harboring a homozygous SDHA mutation; P2, patient harboring a homozygous fumarase mutation; P3 patient harboring a heterozygous mutation; succ, succinate; fum, fumarate.