Rapid determination of tricarboxylic acid cycle enzyme activities in biological samples
© Goncalves et al; licensee BioMed Central Ltd. 2010
Received: 21 August 2009
Accepted: 28 January 2010
Published: 28 January 2010
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.
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 α-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.
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.
Primary deficiencies in tricarboxylic acid cycle enzymes in humans.
1. Progressive encephalopathy
2. Hereditary leiomyomatosis and renal cell cancer
No disease identified so far
No disease identified so far
No disease identified so far
Congenital lactic acidosis
Succinyl CoA ligase
Encephalomyopathy with mtDNA depletion
1. Encephalopathy (Leigh syndrome)
2. Pheochromocytoma and paraganglioma
Residual activities associated with TCAC impairments in humans vary widely and may determine the magnitude of organic acid accumulation . Organic acid accumulation has been proven instrumental in initiating tumor formation related to SDH or fumarase deficiency .
The ratios between TCAC enzymes are consistent for each mammalian tissues presumably reflecting their metabolic demand, as shown three decades ago in the seminal study by Pette and Hofer . This echoes the occurrence of metabolons in the mitochondrial matrix [11–13], allowing for efficient channeling of substrates and co-factors through the Krebs cycle and related enzymes such as transaminase . Consequently, in addition to the determination of residual absolute activities, estimation of ratios between enzyme activities is an effective means of detecting partial but potentially harmful deficiencies. When used to assess respiratory chain activities, this approach enabled the identification of several gene mutations, even in patients with partial respiratory chain deficiencies [15, 16].
At present, TCAC enzyme activities are measured using a series of independent assays that are both laborious and time consuming. We therefore developed a limited set of assays allowing both measurement of all TCAC enzyme activities and detection of abnormalities in enzyme activity ratios. We used these assays successfully to detect severe and partial isolated deficiencies in several TCAC enzymes.
Given that TCAC enzyme activity ratios, because of their consistency , are important in comparing data between samples, we devised a method for measuring the activities of all eight TCAC enzymes using only three assays, which allows rapid determination of enzyme activity ratios. To define appropriate assay conditions, we first used mouse heart samples and assessed various parameters (detergent, pH, ionic force) that are known to independently stimulate each activity, but which might interfere with the measurement of other activities. We found that two media were sufficient for assaying all TCAC activities. The difference between these two media lies in the presence of phosphate required by some of the enzymes and in the use of electron acceptors to cope with the various reduced equivalents.
The second assay starts with measurement of the reduction of pyridine nucleotides (NAD+/NADP+) by KDH. This enzyme, one of the limiting steps of the TCAC, requires the presence of Ca++ ions, thiamine pyrophosphate, and coenzyme A to catalyze the oxidation of α-ketoglutarate. After KDH measurement, cis-aconitate is added for measurement of aconitase activity based on the formation of isocitrate, which, in the presence of IDH, is readily used up to reduce NAD+/NADP+. Finally, the maximal activity rate of IDH is determined after addition of a large isocitrate excess. Citrate synthase, the last TCAC enzyme to be measured, condenses acetyl-CoA and oxaloacetate into citrate while concomitantly releasing coenzyme A, whose thiol residue readily reacts with Ellman's reagent (dithionitrobenzene). It is measured using the standard procedure which, in the case of cultured skin fibroblasts, concomitantly allows the detection of mycoplasma .
The renewed interest in measuring TCAC enzyme activity, shown to be sensitive targets under various pathological conditions, prompted us to devise a rapid assay method for detecting TCAC deficiencies in biological samples. Our previous work on the mitochondrial respiratory chain established that, in addition to absolute residual activities, relative ratios of enzyme activities in a metabolic pathway are effective in detecting even partial deficiencies in a given enzyme. We therefore developed a set of three assays that conveniently estimate all TCAC enzyme activities in tissue homogenates and permeabilized cells. Although the experimental conditions had to be adapted to allow for the measurement of many enzymes using a small number of assays, they were largely based on the pioneer work done in the 1940s by Krebs and colleagues. In particular, the concentrations of substrates and cofactors and the metal requirements for each enzyme were as determined by these authors.
As a first result of this work, we fully confirmed that TCAC enzyme activity ratios in each of the different tissues or cell investigated are consistent under basal conditions, as previously observed by Pette and colleagues as early as 1960[10, 22].
To date there has been a lot of efforts to provide convenient assay procedures for respiratory chain enzymes [16, 23–25]. In contrast, to our knowledge, there is no report on any convenient enzymatic procedure to measure the overall activity of TCAC enzymes in the context of screening procedures. Although our assays are rapid and sensitive, they have intrinsic limitations. First, three of the enzymes (succinyl-CoA ligase, fumarase, and aconitase) are measured via coupled assays involving the next enzyme in the cycle (SDH, MDH, and IDH, respectively). Obviously, a severe deficiency in the next enzyme would impair the ability of the assay to measure the first enzyme. Therefore, deficiencies in two consecutive enzymes should be evaluated by assaying each enzyme activity separately via standard methods. Second, although our assays are sufficiently sensitive to detect even partial deficiencies in one TCAC enzyme, measuring the slower enzymes via coupled assays (e.g., aconitase) requires a sample that is large enough to avoid problems with product dilution (e.g., isocitrate in the case of aconitase), which would impair the activity of the coupled enzyme (e.g., IDH in the case of aconitase). Despite these limitations, our set of assays enabled us to detect all TCAC enzyme deficiencies. Even a 40% decrease in fumarase activity in lymphoblastoid cell lines was readily detected.
So far there has been only a limited number of diseases which have been associated with primary isolated or multiple defect of the TCAC [7, 19, 20, 26–28]. Beside primary defects of the TCAC genes, as some of the TCAC proteins harbor oxygen-sensitive iron-sulfur cluster, i.e. aconitase, or require a full set of co-factors, i.e. α-ketoglutarate dehydrogenase, a loss of activity - secondary but yet possibly instrumental in the pathophysiological process - might well be observed in a number of conditions such as aging, Parkinson's disease or heart failure.
Fibroblasts derived from forearm biopsies taken with informed consent from healthy controls and patients with TCAC enzyme deficiencies were grown under standard conditions as described elsewhere  and frozen (-80°C). Before use, cells were resuspended in 1 ml of medium (A) composed of 0.25 M sucrose, 20 mM Tris (pH 7.2), 40 mM KCl, 2 mM ethylene glycol tetra acetic acid (EGTA), 1 mg/ml bovine serum albumin (BSA), 0.01% digitonin (w/v), and 10% Percoll (v/v). After 10 min incubation at ice-melting temperature, the cells were centrifuged (5 min × 2,300 g), the supernatant discarded, and the pellet washed (5 min × 6000 g) with 1 ml of medium A devoid of digitonin and Percoll . Lymphoblasts from patients harboring a deleterious heterozygous fumarate hydratase gene mutation (N64T) were processed similarly to the cultured fibroblasts. Mouse colony was maintained in accordance with national and institutional guidelines. Animal procedures were approved by the ethical review panel of the Robert Debré Institut, Paris, France. Hearts were obtained from mice, snap frozen in liquid nitrogen and stored at -80°C. Frozen tissues were homogenized at ice-melting temperature by hand using a glass-glass potter in medium (1/10-1/20 v/v) composed of 20 mM Tris (pH 7.2), 0.8 M sucrose, 40 mM KCl, 2 mM EGTA, and 1 mg/ml BSA. Large cell debris was removed by low-speed centrifugation (1,500 g for 5 min).
The first assay measures succinyl-CoA ligase, SDH, glutamate dehydrogenase (GDH), fumarase, and malate dehydrogenase (MDH) (see below; Fig. 1). This assay is performed in 400 μl of medium A containing 50 mM KH2PO4 (pH 7.2) and 1 mg/ml BSA. The reduction of dichlorophenol indophenol (DCPIP) is measured using two wavelengths (600 nm and 750 nm) with various substrates and the electron acceptors decylubiquinone and phenazine methosulfate. The second assay measures α-ketoglutarate dehydrogenase (KDH), aconitase, and isocitrate dehydrogenase (IDG) activities. The same volume of the same medium is used, and pyridine nucleotide (NAD+/NADP+) reduction is measured with various substrates using wavelengths of 340 nm and 380 nm. In the third assay, citrate synthase is measured by monitoring dithionitrobenzene (DTNB; Ellman's reagent) reduction at wavelengths of 412 nm and 600 nm as previously described. For this study, all measurements were carried out using a Cary 50 spectrophotometer (Varian Inc., Palo Alto, CA) equipped with an 18-cell holder maintained at 37°C. Protein was measured according to Bradford . All chemicals were of the highest grade from Sigma Chemical Company (St Louis, MO).
List of abbreviations
Bovine Serum Albumin
Ethylene Diamine Tetraacetic Acid
Tricarboxylic Acid Cycle (Krebs cycle)
This work was supported by grants from the AFM (Association Française contre les Myopathies), the AMMi (Association contre les Maladies Mitochondriales), the Leducq foundation, the ANR Genopath MitOxy, and European Fp6 integrated project Eumitocombat
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