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Technical Briefs |
1 Department of Paediatrics and Child Health, University of Sydney, Sydney, 2006 Australia;
2
Western Sydney Genetics Program, Children’s Hospital, Sydney, 2145 Australia;
3
Department of Clinical Immunology, Royal Prince Alfred Hospital, Sydney, 2050 Australia;
aaddress correspondence to this author at: Western Sydney Genetics Program, Royal Alexandra Hospital for Children, Locked Bag 4001, Westmead, NSW 2145 Australia; fax 612-9845-1864, e-mail Johnc{at}chw.edu.au
Complex III (ubiquinol:ferricytochrome c reductase; EC 1.10.2.2) catalyzes the reduction of cytochrome c by ubiquinol (1)(2). The assay measures the reduction of cytochrome c as catalyzed by complex III at 550 nm (3) in the presence of reduced decylubiquinone (DBH2) (2).
Despite numerous in vivo methods to screen for disorders of ATP production, biochemical assays for the respiratory chain (RC) remain the best way to identify and characterize this group of disorders (4). Although investigators have documented various methodologies to measure complex III in a variety of tissue types (3)(5)(6)(7), these are based on manual methods that are unsuitable for screening large numbers of samples. We recently automated mitochondrial and RC-specific enzyme (8) and protein (9) assays on a random access analyzer (Roche Mira S). The benefits of automation include enhanced speed and simplicity; savings in costs, including labor and reagents, and in the amount of sample needed; and improvements in precision. We adapted and automated the complex III assay (2)(6) on the Roche Mira S random access automated analyzer and compared methods of isolation, performed stability studies, and examined the precision of this new automated method.
Skin fibroblasts from healthy human controls and patients were grown from skin explants. Fibroblasts were cultured in DMEM (high glucose) supplemented with 100 mL/L fetal bovine serum and 225 µmol/L uridine (10).
Two procedures were used to isolate mitochondria from cultured fibroblasts, with all sample preparation being carried out at 4 °C. The first method used a glass-on-Teflon homogenizer, based on the long method of Pitkänen et al. (11), except that four confluent T75 flasks were used, and the final mitochondrial pellet was resuspended in 400 µL of the sucrose buffer. The comparative method was based on the use of 1 g/L digitonin for 35 s before centrifugation and resuspension of the pellet in MOPS buffer (12).
Samples prepared by either method were subjected to three freeze-thaw cycles and sonication (Branson sonifier 250; six pulses, using 30% duty cycle, output control 3), immediately before being assayed (10). For both extraction procedures, isolated mitochondrial extracts were snap-frozen and stored at -80 °C until ready for biochemical analysis.
Decylubiquinone (Sigma) was reduced to DBH2 according to the method of Trounce et al. (2), with minor modifications. To 250 µL of 10 mmol/L decylubiquinone we added 
5 mg of potassium borohydride and 10 µL of 0.1 mol/L HCl. The DBH2 was stabilized with 10 µL of 1 mol/L HCl.
The reagents used for the assay were as described by Rahman et al. (6), except that decylbenzylquinol was replaced with DBH2 and the final reaction volume was 500 µL. All automated assays (including citrate synthase and protein) were performed at 37 °C on a Mira S automatic analyzer (Roche). For the automated complex III assay, the main reagent vessel contained potassium phosphate buffer, potassium cyanide, rotenone, bovine serum albumin, and maltoside (all obtained from Sigma). The start reagent 1 vessel contained DBH2, and the start reagent 2 vessel contained ferricytochrome c.
Complex III (ubiquinol:ferricytochrome c oxidoreductase) activity was calculated based on the initial quasilinear rate. The reduction of ferricytochrome c was measured by the increase in absorbance at 550 nm after addition of the sample at cycle 13. The main reagent buffer was loaded into the cuvette in cycle 1 (426–431 µL). At cycle 13, sample was added (5–10 µL), followed by start reagent 1 (DBH2; 6 µL) in cycle 14 and start reagent 2 (ferricytochrome c; 7.5 µL) in cycle 15. Absorbance readings were then taken until cycle 40, each cycle being 25 s. Absorbance readings were expressed as µmol · min-1 · mg of protein-1 and also relative to citrate synthase. Both citrate synthase and protein were also measured on the Mira S as reported by Williams et al. (8). Samples were measured in duplicate.
Extracted mitochondrial pellets from healthy controls and from patients with a defect in complex III (all assayed for complex III, citrate synthase, and protein) were kept under different conditions (duration and temperature) to determine the stability of the complex III enzyme and the repeatability of the assay. Digitonin- and mortar-and-Teflon pestle-extracted samples were kept at 4 °C for 1 week before being assayed, and aliquots were also kept at room temperature for 24 h before being assayed. In addition, samples underwent one freeze-thaw cycle for the interrun comparison. The volume used in the complex III stability studies was 10 µL.
Agreement was assessed graphically by Bland–Altman plots (13). CVs and SDs for intra- and interrun precision comparisons were estimated from duplicate assays performed on samples.
For a given sample volume, the intra- and interrun variability was very similar for the comparisons of both isolation methods, with the intrarun difference in CV being <1% for 5 and 10 µL of test sample (Table 1
). These results suggest that either method was suitable for isolating mitochondria-containing extracts from primary fibroblasts in culture.
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When we compared 5- and 10-µL digitonin-extracted pellets, there was a threefold difference in the intrarun CV (Table 1
). Bland–Altman plots (values not shown) of the difference between the duplicates vs the mean of complex III activity showed that for the 5-µL sample, the majority of measurements were scattered in the 10–20% range. Doubling the volume to 10 µL for the same digitonin-extracted samples gave improved reproducibility: the Bland–Altman plot showed that most samples had complex III activity measurements clustered between 5% and 10%. Mortar-and-pestle-isolated 5-µL (CV, 9.1%) and 10-µL (CV, 3.9%) mitochondrial extracts showed a comparable trend in reproducibility and precision (Table 1
).
The effects of storage temperature and duration on complex III activity in isolated mitochondrial extracts are shown in Table 1
. Loss of enzyme activity varied, depending on the extraction method, with digitonin-extracted samples showing greater losses of enzyme activity than mortar-and-pestle-isolated samples. As expected, samples stored at -80 °C for 7 days showed the least enzymatic deterioration.
Isolated complex III deficiency is less common than isolated defects in complexes I and IV of the RC (14), but it occurs more frequently as part of a multicomplex defect (4). Although automated enzyme assays are commonplace in many diagnostic laboratories, laboratories are unlikely to invest time or resources in automating biochemical assays of RC enzymes because it is perceived that these disorders are relatively rare. However, recent studies suggest that mitochondrial RC disorders may be as common as 1 per 8500 individuals (15).
Automation of the complex III assay may seem unnecessary at first. We have been using cybrid studies to evaluate inheritance patterns in RC disorders (10), which requires isolation and evaluation of many clones (>20). Because complex III has subunits encoded by both nuclear and mtDNA gene(s) (16), incorporation of automated assay methods into cybrid studies saves both time and resources.
A comparison between the longer, traditional method (mortar and pestle) as described by Pitkänen et al. (11) and the quicker, crude (digitonin) whole-cell extract method used by Williams et al. (8) is shown in Table 1
. Although the differences in within- and between-run imprecision are minimal, as evidenced by the differences in the CV being <1%, the crude (digitonin) method has the advantages of being faster and requiring less starting material.
A comparison of the intrarun CVs at the different sample volumes showed that 10-µL samples had a lower CV (one-third lower) than 5-µL samples; therefore, in subsequent studies we used only a 10-µL sample volume.
When compared with published methods for complex III assays (7), our method requires a much smaller volume (10 µL vs 500 µL) and proportion of test sample (2.5% vs 50%); in addition, reagent costs for our assay are one-half the costs for the published assays. With the crude (digitonin) extraction method, we were able to use less starting material (2 x 10-cm Petri dishes), thus saving time in both the culture of cells and in the preparation of the mitochondrial pellet.
The stability of the enzyme assay was demonstrated by the minimal loss of activity, especially when samples were stored at -80 °C for 7 days, which would allow batching of large numbers of samples in a single run.
In summary, we have developed a method that may permit faster, less expensive screening of test samples and be well suited to research programs where large numbers of assays must be performed rapidly without compromising precision.
Acknowledgments
We thank Dr. John Coakley (Clinical Chemistry Department, Children’s Hospital at Westmead) for granting us access to the Mira S automated analyzer and Dr. Zhanhe Wu (Cytogenetics Department, Western Sydney Genetics Program, Children’s Hospital at Westmead) for the initial establishment of skin fibroblast lines. We also thank Dr. David Thorburn and Denise Kirby (Murdoch Children’s Research Institute, Melbourne) and Dr. Ian Trounce (Genomic Disorders Research Center, Melbourne, Australia) for helpful advice regarding some of the methods used.
References
The following articles in journals at HighWire Press have cited this article:
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  K. A. Kramer, D. Oglesbee, S. J. Hartman, J. Huey, B. Anderson, M. J. Magera, D. Matern, P. Rinaldo, B. H. Robinson, J. M. Cameron, et al. Automated Spectrophotometric Analysis of Mitochondrial Respiratory Chain Complex Enzyme Activities in Cultured Skin Fibroblasts Clin. Chem., November 1, 2005; 51(11): 2110 - 2116. [Abstract] [Full Text] [PDF]
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