| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Endocrinology and Metabolism |
1 Division of Metabolic and Endocrine Diseases and 3
Department of Neuropediatrics, University Children’s Hospital Heidelberg, Heidelberg, Germany.
2 Department of Pediatrics, University Medical Center Nijmegen, Nijmegen, The Netherlands.
4 Nijmegen Center for Mitochondrial Disorders (NCMD), Nijmegen, The Netherlands.
aAddress correspondence to this author at: Division of Metabolic and Endocrine Diseases, University Children’s Hospital Heidelberg, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany. Fax 49-6221-565565; e-mail marina_schwab{at}med.uni-heidelberg.de.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: We report a spectrophotometric method for the analysis of PDHc activity in fibroblasts based on detection of NADH formation via a p-iodonitrotetrazolium violet (INT)-coupled system. We investigated in detail the specific requirements of this assay, such as cofactor requirements and the effects of suggested stimulatory compounds and different cell disruption procedures. The reliability of the optimized assay was studied by investigation of patients previously diagnosed with PDHc deficiency and by comparison with results from the radiochemical method.
Results: Mean (SD) total PDHc activities were 136 (31) and 58 (21) mU/U of citrate synthase in fibroblast homogenates from 10 healthy volunteers and 7 PDHc-deficient patients, respectively, by the spectrophotometric assay. Similar results were obtained in a mitochondrial fraction. Dithiothreitol (DTT) increased the nonspecific inhibitor-insensitive rate with less pronounced effect on the specific rate of PDHc activity. Administration of DTT increased PDHc activity to 193 (3)% of control activity (without DTT), but decreased the inhibitor-sensitive rate from 99 (0.3)% (without DTT) to 69 (2)% (with 0.3 mmol/L DTT).
Conclusion: The simple, optimized spectrophotometric assay for PDHc analysis allows reliable investigation of the enzyme complex in human skin fibroblasts.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PDHc is a multienzyme complex localized in the inner mitochondrial membrane. It consists of six different components: pyruvate decarboxylase (E1; EC 1.2.4.1), dihydrolipoic transacetylase (E2; EC 2.3.1.12), dihydrolipoyl dehydrogenase (E3; EC 1.8.1.4), two regulatory enzymes [PDH kinase (EC 2.7.1.99) and PDH phosphatase (EC 3.1.3.43)], and the E3-binding protein (3).
The first three subunits (E1, E2, and E3) are the major catalytic components and act in sequence. PDHc is regulated mainly by a phosphorylation–dephosphorylation cycle catalyzed by PDH kinase or phosphatase, respectively (4). PDHc activity is also regulated by feedback inhibition by NADH and acetyl-CoA (5). PDHc deficiency is a frequent cause of inherited mitochondrial disorders, mostly affecting the E1 component (6).
The central role of PDHc in mitochondrial energy metabolism and the severe consequences of PDHc deficiency (7) underline the necessity of having a reliable method for PDHc analysis in the diagnostic work-up of mitochondrial disorders. Existing methods for the analysis of PDHc activity are based on measurement of the endproducts of the enzymatic cascade, i.e., NADH, acetyl-CoA, and CO2. The formation of NADH or acetyl-CoA can be detected by spectrophotometric analysis (8)(9), and 14CO2 can be measured by radiochemical methods using [1-14C]pyruvate as substrate (10). For diagnostic purposes, PDHc is determined mainly in fibroblasts and/or muscle tissues obtained from patients with suspected PDHc deficiency. Because muscle biopsies are more invasive than skin biopsies, cultured human skin fibroblasts are used more commonly than muscle tissue for the investigation of PDHc deficiency. Unfortunately, PDHc analysis in human skin fibroblasts is hampered by low specific activity and the largely inactivated (i.e., phosphorylated) multienzyme complex in this tissue compared with muscle (11).
The most commonly used assay for PDHc measurement is a radiochemical method, but this method has the disadvantages of high (up to 50% blank rate) nonspecific decarboxylation of [1-14CO2]pyruvate (12), high costs, and the use of radioactive compounds. The only spectrophotometric assay adapted for fibroblasts in the literature is a coupled enzyme assay using pigeon liver arylamine acetyltransferase for measurement of acetyl-CoA (9). A particular disadvantage of this method is that it does not reliably determine PDHc activity in fibroblast extracts (13). Therefore, the major aim of the present study was to develop a reliable spectrophotometric assay with high sensitivity and specificity for the analysis of completely activated PDHc activity in tissues low in mitochondria, such as human skin fibroblasts.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
controls and patients
Human skin fibroblast cell cultures from seven patients with PDHc deficiency were included in this study. All patients had decreased PDHc activity as measured by radiochemical analysis in different tissues, i.e., muscle and fibroblasts. In two patients, PDHc deficiency was confirmed to result from disease-causing mutations in the E1
gene of the PDH complex. In five patients, mutation analysis of this gene did not reveal any disease-causing mutation, suggesting an involvement of other components of the PDHc. Mutation analysis of the E1 gene was performed according to Brown et al. (14). In parallel with the spectrophotometric analysis, PDHc deficiency was investigated in fibroblasts and/or muscle tissue of these patients by a radiochemical method with [1-14C]pyruvate as substrate, as described by Robinson et al. (15) with the modifications of Sperl et al. (16). Control human skin fibroblasts from forearm skin biopsies were obtained from 11 healthy control individuals. Skin biopsies and subsequent experiments in human skin fibroblast cultures were performed after receipt of informed consent. The study was approved by the ethics committee.
human skin fibroblast cultures
Human skin fibroblasts from forearm skin biopsies were cultivated to confluency under standard conditions at 37 °C in DMEM supplemented with 100 mL/L fetal calf serum, 100 mg/L penicillin, 100 mg/L streptomycin, 2.5 mg/L fungizone, and 200 µmol/L uridine (17)(18). The medium was changed twice a week. Each cell culture was tested for contamination by Mycoplasma spp. before enzyme measurement.
preparation of submitochondrial particles from bovine heart
To establish and optimize the spectrophotometric PDHc analysis, we used submitochondrial particles (SMPs) from bovine heart as a model for PDHc in our study. SMPs were isolated as described previously (19) and were stored in liquid nitrogen until they were used for the experiments.
preparation of crude tissue homogenate
Confluent fibroblast cell cultures (
1–2 mg of total cell protein; one or two 75-cm2 flasks) were harvested with 10 mL of 0.5 g/L trypsin–0.2 g/L EDTA, washed twice with PBS, and subsequently centrifuged (300gmax). Cells were then suspended to a final protein concentration of 1 g/L in PBS. DCA (5 mmol/L) was added to specifically inhibit the inactivating kinase (but not the activating phosphatase) of PDHc, as described previously (20). The suspensions were incubated at 37 °C for 15 min. After activation with DCA, cultured fibroblasts were collected by centrifugation (300gmax). The cells were washed once with PBS, and the cell pellet was resuspended in 3 mL of buffer containing 250 mmol/L sucrose, 1 mmol/L EGTA, and 10 mmol/L HEPES (adjusted to pH 7.5) and homogenized by 20 strokes in a tight-fitting ground-glass Potter-Elvejhelm. The homogenization procedure was carried out at 0–4 °C. After centrifugation at 13 000gmax (4 °C), the supernatant was discarded. The pellet was stored in liquid nitrogen until measurement. The enzyme measurements were performed within 3 months after freezing. Immediately before PDHc measurement, the crude tissue pellet was frozen and thawed three times (–196 and 25 °C) and suspended at a protein concentration of 4 g/L in 1 mL/L Triton X-100, 1 mmol/L CaCl2, 5 mmol/L MgCl2, 50 mmol/L KCl, 250 mmol/L sucrose, and 20 mmol/L Tris-HCl (pH 7.5). For some experiments (Table 1
), the suspended samples were sonicated for 20 s (power output control 20% of maximum value) rather than being subjected to the freezing–thawing procedure.
|
To stimulate the phosphatase, which activates the E1 component of the PDHc, by calcium and magnesium, we preincubated the cell suspension at 37 °C for 10 min before measurement (10). To measure basal PDHc activity in tissue homogenate, we omitted the stimulation with DCA, calcium, and magnesium. The PBS used for our experiments was free of calcium and magnesium. The citrate synthase activity and protein concentration of each sample were determined to normalize PDHc activity (21)(22).
preparation of a mitochondria-enriched fraction
Confluent fibroblasts (4–5 mg of total protein; five to six 75-cm2 flasks) were harvested with trypsin and washed twice with PBS. To activate PDHc, the pellet was incubated at 37 °C with DCA as described above. The cells were then washed once more with PBS. Isolation of mitochondria from the cell pellet was performed at 4 °C according to Krähenbühl et al. (23) with some modifications. Briefly, the cell pellet was frozen at –80 °C for at least 15 min. After thawing, the pellet was suspended in 2 mL of isolation buffer containing 250 mmol/L sucrose, 1 mmol/L EGTA, 10 mmol/L HEPES, and 5 g/L BSA (pH 7.5) and then was centrifuged at 500gmax for 2 min. The supernatant was discarded, and the remaining pellet was suspended in 3 mL of isolation buffer. The cell suspension was homogenized in a tight-fitting ground-glass Potter-Elvejhelm. After centrifugation at 1500gmax for 10 min, the supernatant was kept on ice. The pellet was homogenized and centrifuged as described above. The two supernatants were pooled and centrifuged at 10 000gmax for 10 min. The resulting mitochondrial pellet was washed twice with BSA-free isolation buffer and stored in liquid nitrogen until measurement. For the analysis of PDHc activity, the mitochondrial pellet was thawed and suspended at a protein concentration of 4 g/L in 1 mL/L Triton X-100, 1 mmol/L CaCl2, 5 mmol/L MgCl2, 50 mmol/L KCl, 250 mmol/L sucrose, and 20 mmol/L Tris-HCl (pH 7.5). Similar to the preparation of tissue homogenates, the mitochondrial suspension was preincubated for 10 min at 37 °C before PDHc measurement, and an aliquot of the mitochondrial suspension was used for measurement of citrate synthase activity and protein content (21)(22).
spectrophotometric assay for measurement of PDHC activity
The reaction mixture contained 5 mmol/L L-carnitine (10), 2.5 mmol/L NAD, 0.2 mmol/L TPP, 0.1 mmol/L CoA, 5 mmol/L pyruvate, 1 mL/L Triton X-100, 1 mmol/L MgCl2, 1 g/L BSA, 0.6 mmol/L INT, and 6.5 µmol/L PMS in 0.05 mol/L potassium phosphate buffer (pH 7.5) according to Hinman and Blass (24) with some modifications. For some experiments, 25 mmol/L oxamate (Fig. 1
and Table 1
) (8) was added to the test buffer.
|
Experiments in the presence of DTT (Fig. 2
) (24) were performed by adding 0.3 mmol/L DTT just before enzyme measurement. Assays were performed at 25 °C in a dual-wavelength spectrophotometer; we monitored the increase in absorbance at 500–750 nm, which was attributable to reduction of INT, the final electron acceptor (12.4 mmol · L–1 · cm–1). Stock solutions of INT were prepared at a saturating concentration of 3 g/L in 0.05 mmol/L potassium phosphate, stirred at 4 °C for 1 h to aid solubilization, and then filtered (Whatman no. 1 filter paper; 0.22 µm pore size). PMS stocks were prepared at a concentration of 0.2 g/L. Both solutions were prepared fresh daily. A stock solution of 3-fluoropyruvate at a concentration of 600 mmol/L in 0.05 mol/L potassium phosphate (pH 7.5 at 25 °C) was prepared. Aliquots of 3-fluoropyruvate were stored in liquid nitrogen until measurement.
|
The enzyme activity measurements were performed in thermostated 96-well microtiter plates in at least three independent experiments. The PDHc activity was measured in two patient samples, with each experiment consisting of at least six to eight single measurements: three to four measurements without inhibition, and three to four measurements with inhibition by fluoropyruvate. Briefly, 50 µL of cell suspension [mitochondria-enriched fraction (MEF) of fibroblasts (200 µg of protein), fibroblast homogenate (200 µg of protein), or SMPs (60 µg of protein)] was added to the wells of the microtiter plate. To start the enzyme reaction, we added 250 µL of the reaction mixture to each well. To examine the specificity of the determined enzyme activity, we inhibited PDHc activity with 3-fluoropyruvate (5 mmol/L), a potent inhibitor of the E1 component (25); for some experiments with arsenite (1 mmol/L), we added a specific inhibitor of the E2 component (26). These two components were added separately to the well (2.5 µL of a 600 mmol/L stock solution). Fluoropyruvate revealed a good inhibitory response (95–100% of control activity). The total volume of each well was 300 µL.
data analysis
Data are expressed as the mean (SD). Experiments were performed at least in triplicate. pH dependence of PDHc activity and Michaelis–Menten parameters were analyzed by use of PsiPlot software package 5.02a. Statistical analysis was performed by ANOVA followed by the post hoc Bonferroni multiple comparison test or the Student t-test (for two groups). Statistics were calculated using SPSS for Windows 10.0 software. P <0.05 (Student t-test) and P <0.001 (ANOVA) were considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
; n = 3–8). Omission of TPP led to significantly decreased enzyme activity. In addition, carnitine and magnesium/calcium also increased PDHc activity (Fig. 1
). The specificity of PDHc activity was confirmed by complete inhibition with the specific E1 inhibitor 3-fluoropyruvate (Fig. 1
) (25). NADH is oxidized to NAD by mitochondrial complex I and lactate dehydrogenase (LD). We investigated whether inhibition of these enzymes influenced PDHc activity in our assay. However, 2-n-decylquinazolin-4-yl-amine (DQA), a potent inhibitor of complex I, and oxamate, an inhibitor of LD, did not significantly influence PDHc activity (Fig. 1
). Furthermore, DCA increased PDHc activity in fibroblasts, as described previously (Table 1
) (20).
We observed similar effects in MEFs and homogenates from human skin fibroblasts (Table 1
), except for oxamate (25 mmol/L), which had no effect in MEFs but mildly decreased PDHc activity in homogenates (Table 1
) and in SMPs (Fig. 1
). In addition to tissues with low mitochondrial content, we also measured PDHc activity in tissues with a high mitochondrial content, such as liver, with the optimized spectrophotometric assay (data not shown).
influence of PH on PDHC activity
We determined the pH optimum (pHopt) for the spectrophotometric assay in SMPs, varying the pH from 6.5 to 8.2 (n = 9). The pHopt for PDHc activity was 7.5. We calculated the pHopt according to the method of Brandt and Okun (28) and obtained a pKa of 6.85 and a pKb of 8.14 (see Figs. 1 and 2 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol51/issue1/). In a previous study using spectrophotometric analysis of PDHc, pH 7.8 was used (24). Notably, we demonstrated that even a small shift from pH 7.5 (i.e., pHopt) to pH 7.8 decreased the mean (SD) Vmax to 72 (10)% of pHopt. Because this effect was more pronounced in control cells than in PDHc-deficient cells, measurement at pH 7.8 also decreased the diagnostically relevant difference between both groups (see Fig. 4 in the online Data Supplement).
michaelis–menten parameters
The Michaelis–Menten constant (Km) was calculated at 27 (3) µmol/L pyruvate [Vmax = 347 (2) mU/U of citrate synthase; n = 8]. A Lineweaver–Burk plot for pyruvate indicated nonlinearity (see Fig. 3 in the online Data Supplement).
correlation between PDHC activity and protein concentration
In fibroblasts and SMPs, absorbance correlated with the protein concentration (up to 320 µg of protein; r2 = 0.99). Cell homogenates showed similar results (see Figs. 7, 8, and 9 in the online Data Supplement).
influence of different preparation procedures on PDHC activity
To allow reliable PDHc measurements, sufficient disruption of cells before PDHc activity measurement is necessary, particularly when crude tissue homogenates are used. For this purpose, we compared standard methods, i.e., the freezing–thawing procedure, sonication of samples, and the use of a detergent (Triton X-100). The freezing–thawing procedure increased PDHc activity in MEFs and fibroblast homogenates (Table 1
; n = 3). In contrast, sonication of samples decreased PDHc activity in all investigated tissues (Table 1
; n = 3), most likely because of partial disintegration of PDHc. Omission of the polyoxyethylene detergent Triton X-100 significantly decreased PDHc activity in all investigated tissues (n = 3; Fig. 1
and Table 1
; see also Fig. 14 in the online Data Supplement). On the basis of the results of these experiments, we chose the following standard conditions for spectrophotometric PDHc analysis: freezing–thawing; addition of CaCl2, MgCl2, DCA, Triton X-100, and carnitine; and adjustment to pH 7.5 (Table 1
).
dtt increases nonspecific, inhibitor-insensitive PDHC activity.
It has been suggested in a previous study (24) that the addition of sulfhydryl group-protecting agents, such as DTT, is necessary for INT-coupled spectrophotometric measurement of PDHc activity. We demonstrated that the addition of DTT to the test buffer increased PDHc activity in SMPs and decreased the inhibitory response to 3-fluoropyruvate (Fig. 2A
; n = 4), i.e., DTT mostly increased nonspecific PDHc activity, whereas the increase in the specific activity was less pronounced. This notion was further supported by the finding that reduction of INT occurred even in the absence of SMPs after addition of DTT to the test mixture (Fig. 2A
); we therefore suspected that this reduction is artificial. In contrast, the rate for nonspecific binding in a blank containing test buffer was <1% of control activity when DTT was omitted (Fig. 2A
). Finally, we demonstrated that the DTT-induced increase in absorbance at 500–750 nm was concentration-dependent: absorbance correlated to the amount of DTT in the test buffer up to a DTT concentration of 0.3 mmol/L (r2 = 0.99; Fig. 2B
). It can be suggested from these results that the nonspecific, inhibitor-insensitive enzyme activity in the presence of DTT is induced by direct reduction of INT by DTT. This effect was amplified by administration of the electron mediator PMS (see Figs. 11 and 12 in the online Data Supplement).
PDHC activity in human skin fibroblasts
We next investigated PDHc activity in fibroblasts from seven patients with PDHc deficiency. The results from the spectrophotometric analysis were subsequently compared with those obtained by the radiochemical method in the same patients (Tables 2
and 3
; also see Fig. 10 in the online Data Supplement). The comparative investigations were not performed with the same fibroblast homogenates, but all experiments were performed at least in triplicate, and our assay showed a low interday variability (<5%). In all patients, mutation analyses and/or enzymatic PDHc measurements in muscle and/or fibroblasts were performed in parallel. The results of the radiochemical assay in fibroblasts (Table 2
) indicated a significant decrease in PDHc activity in patients 1–3 (Table 3
). Patient 4 had a mutation in the thiamine-binding site of the E1 gene (640T>C), which led to an amino acid substitution (W214R) (29); spectrophotometric, but not radiochemical, analysis demonstrated a significant decrease in total PDHc activity. Whereas basal PDHc activity in fibroblasts was decreased in both methods (Tables 2
and 3
), radiochemical analysis of fresh muscle tissue showed normal PDHc activity in this patient (data not shown). Patient 1 had a polymorphism on the cDNA level (291G>A) in the E1 gene that was present in the control population and did not lead to an amino acid substitution (G97G). It is therefore unlikely that this polymorphism is pathogenic. Notably, the patient had decreased total PDHc activity in fibroblasts and in fresh muscle tissue (data not shown) by radiochemical analysis. Spectrophotometric analysis gave results similar to the results of the radiochemical investigations in fibroblasts (Table 2
). We found no mutation in the E1 gene in patient 2, but radiochemical and spectrophotometric analysis in fibroblasts from the same patient showed clearly decreased PDHc activity (Tables 2
and 3
). No muscle tissue was available for PDHc measurement in patient 2. Patient 3 had a disease-causing mutation in the E1 gene of the PDHc complex in fibroblasts (1133 G>A), which led to an amino acid substitution (R378H) (30). This finding was confirmed by spectrophotometric and radiochemical analysis in fibroblasts (Tables 2
and 3
) as well as in muscle (radiochemical analysis; data not shown).
|
|
Patients 5–7 had significantly deceased fibroblast PDHc activity, as measured by the spectrophotometric method (Table 3
). Radiochemical analysis confirmed the decreased PDHc activity in muscle samples from these patients, whereas the PDHc activity in fibroblasts was normal (Table 2
). However, mutation analysis in the E1 component could not confirm PDHc deficiency in these patients.
Shown in Fig. 3
is an overview of total PDHc activities in MEFs (Fig. 3A
) and homogenates (Fig. 3B
) from human skin fibroblasts of healthy controls (n = 10–11) and patients with PDHc deficiency (n = 7) as measured by the spectrophotometric method. The measured PDHc activities in fibroblast cell lines from controls and PDHc-deficient patients are summarized in Table 3
. In all fibroblast cultures from PDHc-deficient patients, citrate synthase activity was within the range of values obtained for the controls and was used as the mitochondrial reference enzyme (data not shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we describe a sensitive and specific spectrophotometric assay for measurement of completely activated PDHc that can be used to measure PDHc activity in tissues with low activity, such as fibroblasts. Measurement of PDHc activity in tissues rich in mitochondria, such as liver, can also be performed with this optimized assay.
To investigate the sensitivity and specificity of our assay, we omitted the known cofactors of the PDHc (TPP, NAD, and CoA) and inhibited the enzyme with 3-fluoropyruvate, a specific inhibitor of the E1 component (25), and with arsenite, a specific inhibitor of the E2 component (26). We demonstrated that this spectrophotometric PDHc assay revealed an appropriate cofactor requirement and a good inhibitory response to 3-fluoropyruvate (inhibition >95%), a well-described inhibitor of the E1 component, confirming the specificity of the assay (Fig. 1
). The assay had a negligible blank rate.
To determine maximum PDHc activity, many different variables, such as pH, incubation period, disruption procedure, and phosphorylation/dephosphorylation of the PDHc, dramatically influence enzyme activity and must be considered carefully. Hinman and Blass (24) used pH 7.8 for the INT-coupled assay adapted for mouse brain homogenate and rat kidney mitochondria without exact calculation of the pHopt of their assay. We demonstrated that even a slight pH shift from pH 7.5 (i.e., pHopt) to 7.8 significantly decreased the PDHc activity to 72% of Vmax (Fig. 4 in the online Data Supplement) and thus would impair the diagnostic work-up of PDHc deficiency in fibroblasts. Furthermore, a prolonged incubation period up to 60 min, as is often required for the radiochemical method, decreased PDHc activity (Figs. 5 and 6 in the online Data Supplement). In addition to pH- and time-dependent effects, the method used to disrupt the mitochondrial membrane is of utmost importance. As described previously for the measurement of E1 activity (33), a freezing–thawing procedure significantly increased PDHc activity, whereas the addition of Triton X-100 was less effective (Table 1
and Fig. 1
). In the present study, we demonstrated that a combination of freezing–thawing and Triton X-100 further increased the PDHc activity compared with use of either of these alone, whereas the addition of a sonication step decreased the PDHc activity.
PDHc activity is regulated mainly by phosphorylation and dephosphorylation and is strongly inhibited by NADH and acetyl-CoA via a feedback loop. Considering these regulation mechanisms, we tested the influence of different compounds (magnesium, calcium, DCA, carnitine, and oxamate) suggested to activate PDHc activity. In accordance with other reports, magnesium (5 mmol/L) and calcium (1 mmol/L) increased PDHc activity (Table 1
and Fig. 1
) (10), most likely through stimulation of the endogenous PDH phosphatase and subsequent dephosphorylation of the E1 component (34). In line with the report by Sheu et al. (20), activation of PDHc could also be induced by the addition of DCA, an inhibitor of PDH kinase (Table 1
). We added L-carnitine to the test mixture to prevent feedback inhibition by acetyl-CoA. In fact, L-carnitine increased the enzyme activity in our assay, whereas this effect was lower (64% vs 29% in our assay) than that reported previously in another study (10). In contrast to the results obtained by Chretien et al. (8), who found increased PDHc activity in the presence of oxamate, an LD inhibitor that prevents LD-induced NADH reoxidation, we could not confirm a significant effect of this compound in our assay (Table 1
and Fig. 1
). To exclude oxidation of NADH by mitochondrial complex I, we inhibited this multiprotein complex with the specific inhibitor DQA. However, this compound also had no significant influence on PDHc activity (Fig. 1
).
In a previous study (24) in which PDHc activity was measured by an INT-coupled spectrophotometric assay, the sulfhydryl group-protecting agent DTT was suggested as a necessary ingredient in the test buffer for PDHc measurements; there was a complete loss of PDHc activity in the absence of DTT. Unfortunately, the specificity of this finding has not been confirmed by analysis of the inhibitor-sensitive rate of this assay. In our study, DTT mostly increased the inhibitor-insensitive, i.e., nonspecific, rate of PDHc activity from 1% (without DTT) to 31% (with DTT), whereas it had a less pronounced effect on the inhibitor-sensitive rate. We demonstrated that DTT increased the absorbance at 500–750 nm in a concentration-dependent way even in the absence of PDHc protein in the assay system, thereby mimicking PDHc activity (Fig. 2B
). To further unravel the underlying mechanism of this effect, we investigated changes in absorbance at 500–750 nm by combining DTT with different components in the absence of SMPs according to the previously published protocol of Hinman and Blass (24). Notably, we could demonstrate that DTT directly reduces INT in the test system, explaining the inhibitor-insensitive enzyme activity (Fig. 11 in the online Data Supplement). Because these results highlight a direct reaction between DTT and INT, an indicator of specific PDHc activity in the presence of DTT, we concluded that addition of DTT is not suitable for this assay although it increases the absolute PDHc activity.
Using fibroblasts from patients with PDHc deficiency, we demonstrated that all of these patients had significantly decreased PDHc activity, as measured by our spectrophotometric assay (Table 3
). Radiochemical analysis revealed decreased PDHc activity in muscle tissue from five of the seven patients (patients 1, 3, and 5–7). In four patients, basal and/or total PDHc activity was decreased in fibroblasts, as measured by the same method (patients 1–4). Notably, in one patient, who had a mutation in the thiamine-binding site of the E1 subunit of the PDHc, spectrophotometric but not radiochemical analysis revealed a significant decrease in total PDHc activity in fibroblasts. In contrast, in both assays basal PDHc activity was decreased in fibroblasts from this patient (Tables 2
and 3
). The two patients with pathogenic mutations in the E1 gene (patients 3 and 4) and the patient with the polymorphism in the E1 gene (patient 1) had decreased PDHc activities in fibroblasts, as measured by both methods (Tables 2
and 3
). Radiochemical analysis of muscle samples from patients 1 and 3 confirmed the PDHc deficiency (data not shown). Although patients 5–7 had decreased PDHc activity in muscle tissue, as measured by the radiochemical assay, and in fibroblasts, as measured by the spectrophotometric assay, mutation analysis revealed no mutation in the E1 gene. This phenomenon is possibly attributable to pathogenic mutations in the E1 gene (e.g., mutations in the promoter site of the E1 gene) or in the localization of the disease-causing mutation in the remaining subunits (E2, E3, or E3-binding protein) or regulatory enzymes (PDH phosphatase and PDH kinase) of the PDHc. It can be concluded from these results that the optimized spectrophotometric assay has at least the same sensitivity and specificity as the radiochemical method. However, because the present investigations have been performed in a relatively small group of PDHc-deficient patients in two different laboratories, our comparisons regarding the reliability and validity of these methods has some limitations. This should be investigated in future studies in greater detail.
PDH E1 subunit (pyruvate decarboxylase; EC 4.1.1.1) deficiency (McKusick 312170) is an X-linked error of metabolism, and diagnosis is usually based on measurement of enzyme activity. This may present difficulties in females because of random X inactivation patterns (35). The X inactivation occurs rather frequently in fibroblast cultures (25% of cases), making exclusion of the diagnosis in females rather impossible (35). Patient 2, the sole female patient in our study, had dramatically decreased PDHc activity in fibroblasts, as measured in both the radiochemical and spectrophotometric assays. Mutation analysis revealed no mutation in the E1 gene. The X inactivation pattern and the variable tissue expression of PDHc activity can limit usefulness of enzyme measurements in human skin fibroblasts to exclude the diagnosis of PDH E1 subunit deficiency. Because fibroblasts are easier to obtain than muscle tissue and can be expanded in cell culture, diagnostic work-up is often initially performed in this tissue. If clinical and biochemical features strongly suggest PDHc deficiency and the fibroblast enzyme activity is within the reference interval, screening for mutations in the PDH E1 gene or other investigations, such as immunochemical analysis or enzyme measurement in muscle tissue, are necessary because normal PDHc activity in human skin fibroblasts does not exclude a PDHc deficiency.
In conclusion, our results confirmed that the spectrophotometric assay adapted for the analysis of PDHc activity in human skin fibroblasts reliably measured PDHc activity in tissues with low PDHc activity. Because spectrophotometric measurement in fibroblast homogenates is less time-consuming and gives enzyme activity results identical to those for a MEF (Fig. 13 in the online Data Supplement), we propose to use fibroblast homogenates for spectrophotometric analysis of PDHc complex activity. PDHc activities under different assay conditions reflect both tissue-specific differences (e.g., higher activity in muscle than in fibroblasts) and loss of function or incomplete activation of maximum PDHc activity as a result of methodologic differences (e.g., PDHc leaks from the mitochondria during the isolation of MEFs). Thus, the measured PDHc activity is the result of these two different dimensions. Van Laack et al. (33) observed that the activity of the E1 component of PDHc, as measured by radiochemical analysis of fresh human skeletal muscle, was considerably higher in homogenates than in the mitochondria-enriched supernatants. They also proposed the use of homogenates for E1 measurement in human skeletal muscle. Compared with previously described methods using radioactive-labeled pyruvate or the spectrophotometric arylamine acetyltransferase reaction, our assay has several advantages, i.e., no need for radioactive compounds, a low blank rate, a high specific activity, use of commercially available reagents of standardized quality, and low costs. It is rapid and sensitive and could be easily implemented as a valid and reliable powerful tool in the diagnostic work-up of patients suspected of having a mitochondrial disorder.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
1 Nonstandard abbreviations: PDHc, pyruvate dehydrogenase complex; E1, pyruvate decarboxylase; E2, dihydrolipoic transacetylase; E3, dihydrolipoyl dehydrogenase; TPP, thiamine pyrophosphate; PMS, phenazine methosulfate; INT, p-iodonitrotetrazolium violet; BSA, bovine serum albumin; DCA, dichloroacetic acid; DTT, dithiothreitol; PBS, phosphate-buffered saline; SMP, submitochondrial particle from bovine heart; MEF, mitochondria-enriched fraction; DQA, 2-n-decylquinazolin-4-yl-amine; and LD, lactate dehydrogenase.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subunit gene: identification of further patients and in vitro demonstration of pathogenicity. Hum Mutat 1998;12:114-121.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
-Keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc Natl Acad Sci U S A 1969;62:234-241. subunit of the human pyruvate dehydrogenase complex. Genomics 1989;4:174-181.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
and ß subunits of pyruvate dehydrogenase in a patient with lactic acidosis and unexpected sudden death. Eur J Pediatr 1990;149:487-492.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency. Hum Mutat 2000;15:209-219.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
deficiency. J Inherit Metab Dis 1991;14:140-151.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  D. Feng, A. Witkowski, and S. Smith Down-regulation of Mitochondrial Acyl Carrier Protein in Mammalian Cells Compromises Protein Lipoylation and Respiratory Complex I and Results in Cell Death J. Biol. Chem., April 24, 2009; 284(17): 11436 - 11445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Kumar, V. Kota, and S. Shivaji Hamster Sperm Capacitation: Role of Pyruvate Dehydrogenase A and Dihydrolipoamide Dehydrogenase Biol Reprod, August 1, 2008; 79(2): 190 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Le, M. K. Hailer, S. Buhrow, Q. Wang, K. Flatten, P. Pediaditakis, K. C. Bible, L. D. Lewis, E. A. Sausville, Y.-P. Pang, et al. Inhibition of Mitochondrial Respiration as a Source of Adaphostin-induced Reactive Oxygen Species and Cytotoxicity J. Biol. Chem., March 23, 2007; 282(12): 8860 - 8872. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Kumar, N. Rangaraj, and S. Shivaji Activity of Pyruvate Dehydrogenase A (PDHA) in Hamster Spermatozoa Correlates Positively with Hyperactivation and Is Associated with Sperm Capacitation Biol Reprod, November 1, 2006; 75(5): 767 - 777. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. W. Sauer, J. G. Okun, M. A. Schwab, L. R. Crnic, G. F. Hoffmann, S. I. Goodman, D. M. Koeller, and S. Kolker Bioenergetics in Glutaryl-Coenzyme A Dehydrogenase Deficiency: A ROLE FOR GLUTARYL-COENZYME A J. Biol. Chem., June 10, 2005; 280(23): 21830 - 21836. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and PDHc-deficient fibroblasts (n = 7; 
). (B), PDHc activity in human skin fibroblast homogenate of control cell (n = 10; 
) and PDHc-deficient fibroblasts (n = 7;