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Letters to the Editor |
Department of Pediatrics and Laboratory of Pediatrics and Neurology Nijmegen Centre for Mitochondrial Disorders (NC) at the Radboud University Medical Centre, Nijmegen, the Netherlands
aAddress correspondence to this author at:, Laboratory of Pediatrics and Neurology UMC St Radboud, Geert Grooteplein zuid 10, 6525 GA Nijmegen, the Netherlands, Fax +31 24 3618900, E-mail R.Rodenburg{at}cukz.umcn.nl
To the Editor:
Of the mitochondrial respiratory chain enzymes, complex I is often regarded as the most difficult to analyze, particularly in tissues with relatively low mitochondrial content, such as fibroblasts. In 2007 we developed a new spectrophotometric assay for complex I of the respiratory chain, in which 2,6-dichloroindophenol (DCIP)1 is used as terminal electron acceptor for complex I (1). The use of DCIP in a complex I assay has 2 advantages. DCIP helps to keep the quinone used in the assay in the oxidized form, which is desirable because the reduced form (quinol) has been shown to inhibit complex I activity(2)(3). Another advantage is that DCIP has a relatively high molar absorptivity; therefore complex I assays with DCIP produce higher signals than complex I assays that measure NADH oxidation. Using a large panel of fibroblasts and muscle samples from genetically characterized complex I–deficient patients, we showed that the assay can be applied successfully to diagnose complex I deficiencies. Bénit et al. showed that a similar assay enabled detection of partial complex I deficiency in samples from Harlequin mice(2).
Recently, we started the implementation of the decylubiquinone (DQ)-DCIP based complex I assay on an automated spectrophotometric platform. During the assay implementation, we noticed that blank activity, which is the reaction mixture + rotenone, when measured in a separate cuvette, results in an apparent lower rotenone sensitivity compared to a sequential determination of the blank activity, as in the manual assay described previously (1). This situation is mentioned as a main point in the letter by de Wit et al. We found that measurement of the rotenone sensitivity in a separate cuvette in parallel with the assay cuvette results in a rotenone sensitivity that is around 50% for fibroblast-derived mitochondria (Table 1
) and between 70% and 95% for muscle-derived mitochondria (data not shown). These values are similar to recently published observations(2).
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To demonstrate that the DCIP-based complex I assay has no intrinsic reproducibility problem, we tested the reproducibility in an automated spectrophotometer. In this way, possible variation due to manual pipetting is eliminated. In addition, we tested 2 coenzyme Q (CoQ) analogs, DQ and CoQ1. The percentage of rotenone inhibition observed with CoQ1 was slightly higher than with DQ, as were the absolute complex I activities measured in isolated mitochondria from fibroblasts (Table 1
). The intraassay CV was 4.8% (Table 1
). The interassay variation was 7.9% with DQ and 2.5% with CoQ1, compared to 2%–11% for the manual DQ-DCIP method (1), and 41.1% for a recently described CoQ1-based assay(4). The automated assay in which DQ was replaced by CoQ1 gave a further improvement of the %CV (Table 1
). From the results presented here we conclude that(1) incubations with rotenone should be performed in parallel with the incubations lacking rotenone, (2) the reproducibility of the DCIP-based assay is below 8%, and (3) CoQ1 appears to give slightly better results than DQ, although this finding should be tested more extensively in a large panel of samples.
On the basis of these findings, we have adapted the protocol for the measurement of complex I in fibroblast-derived mitochondria as follows. A reaction mixture containing 25 mmol/L potassium phosphate buffer (pH 7.6), 3.5 g/L BSA, 0.06 mmol/L DCIP, 70 µmol/L CoQ1 or 70 µmol/L DQ, and 2% (vol/vol) of mitochondrial preparation (1) is preincubated with or without 1 µmol/L of rotenone for 4 min at 37 °C. Subsequently, 0.2 mmol/L NADH is added, and the reaction is followed at 600 nm for 5 min. Complex I activities are calculated essentially as described previously(1). As with the previously published manual protocol, when a nonlinear and rapid DCIP reduction is observed due to an excessively high sample concentration in the reaction mixture, samples are diluted to achieve a linear reaction. The results described in this report were obtained using a Kone 20XTi automated spectrophotometer (Siemens Medical Solutions Diagnostics, Breda, the Netherlands). A full report of the automated complex I assay will be submitted for publication once the validation procedure has been completed.
A final note is that the reduced form of DQ, decylubiquinol, has recently been demonstrated to strongly inhibit complex I activity (2), a phenomenon also observed for other CoQ analogs(2)(3). Therefore we emphasize that the method described here should preferably be used for preparations of isolated mitochondria, because in less pure preparations other cellular NADH dehydrogenases will readily reduce quinines, leading to underestimation of complex I activity.
Acknowledgments
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the design of the study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
Footnotes
1 Nonstandard abbreviations: DCIP, 2,6-dichloroindophenol; DQ, decylubiquinone; CoQ, coenzyme Q. 
References
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