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Department of Clinical Pharmacology, University of Berne, Murtenstrasse 35, CH-3010 Berne, Switzerland.
a Author for correspondence.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: The method was a radioenzymatic assay based on the preferential oxidation of the 4B hydrogen of NADH by complex I. We prepared tritiated isoforms of NADH for both the respective 4A-3H and 4B-3H positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product (3H2O) was collected after charcoal adsorption of unreacted NADH and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity.
Results: The assay was linear with time and protein for isolated mitochondria and tissue homogenates from rats and humans. The Vmax and Km values obtained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 µmol/L and 90 µmol · min-1 · mg protein-1, respectively. The assay was reproducible and useable for routine measurements in human skeletal muscle. The sensitivity was >10-fold higher than the sensitivities of spectrophotometric techniques.
Conclusions: The results of our studies demonstrate the successful development of a new assay for complex I that is rapid, easy to perform, and that enables the processing of multiple samples at one time.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Disorders of complex I are a frequent cause of mitochondrial diseases and can manifest with a wide variety of clinical symptoms, ranging from simple muscle lethargy to whole organ malfunctions (3). Recently, a deficiency of complex I has been implicated in neurodegenerative disorders, such as Parkinson disease (4).
For the diagnosis of complex I deficiency, detection of functional protein activity is preferable to a mutational screen of all 41 subunit transcripts or coding sequences. The technique used currently is a spectrophotometric assay, which measures the oxidation of NADH to NAD+ at 340 nm by skeletal muscle homogenate prepared from patient biopsies (5)(6)(7). The disadvantage with this method has been its insensitivity. One reason for the insensitivity is that it requires large quantities of tissue, which make the assay solution turbid and difficult to analyze by optical methods. Another of the reasons for the low sensitivity of the spectrophotometric assay is that NADH is oxidized not only by complex I but also by the many other dehydrogenases located in skeletal muscle or other tissues.
It is well established that dehydrogenases such as complex I exhibit stereospecificity regarding the oxidation of NADH. Studies with submitochondrial particles from beef heart and rat liver have revealed a specificity of the respiratory chain complex I for the 4B hydrogen atom of NADH (8). At the same time, it was found that most other NADH dehydrogenases (microsomal, mitochondrial outer membrane, malic acid, and cytochrome c reductase) were 4A-3H specific.
Because, as mentioned above, one of the disadvantages of the spectrophotometric assays is the high background activity of dehydrogenases other than complex I, the availability of [4B-3H]NADH could potentially produce a more specific assay. We therefore prepared radioactive isoforms of NADH tritiated in the respective 4A-3H or 4B-3H positions and tested both as suitable substrates for complex I activity. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product (3H2O) was collected after charcoal adsorption of unreacted 3H-NADH and measured as an indicator of the amount of NADH oxidation. Sensitivity to the inhibitor rotenone was used as the indicator of complex I specific activity (9).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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preparation of 4a and 4b stereoisomers of nadh
Tritiated [4-3H]NAD-ammonium salt was
obtained from Amersham at a specific activity of 1.35 Ci/mmol. The
respective isoforms of NADH were prepared enzymatically from
stereospecific dehydrogenases and then purified on an anion-exchange
column, as described previously by Fisher and Guillory (10).
[4A-3H]NADH was synthesized with 1 mg of the enzyme L-glutamate dehydrogenase (45 U/mg; Sigma) and 60 µmol L-glutamic acid (monosodium salt). The reaction was carried out in a mixture containing 40 µmol of Tris-HCl, 112 µmol of sucrose, and 360 µmol of hydrazine hydrate. Five micromoles of unlabeled and 15 nmol of tritiated NAD+ were added to a total volume of 1.25 mL (final specific activity, 4 mCi/mmol) and incubated at room temperature for 1.5 h.
[4B-3H]NADH was synthesized with 200 µmol of ethanol and 0.2 mg of alcohol dehydrogenase (305 U/mg; Fluka) in a mixture containing 24 µmol of Tris-HCl, 70 µmol of sucrose, and 240 µmol of hydrazine hydrate. Five micromoles of unlabeled and 15 nmol of tritiated NAD+ were added to a total volume of 2.5 mL (final specific activity, 4 mCi/mmol) and incubated at room temperature for 1 h. Reactions were stopped by heating to 95 °C for 3 min, the protein was pelleted by centrifugation at 15 000g for 5 min, and the supernatant was used for isolation of the respective reaction products.
The product from each of the enzyme reactions was loaded onto an A-25 DEAE Sephadex (Amersham Pharmacia) column (1 x 4.5 cm) equilibrated with starting buffer (25 mmol/L Tris-HCl, pH 8). The column was washed with 100 mL of water, and then unreacted NAD+ was eluted with incremental concentrations (20 mL of 0.05 mol/L, 20 mL of 0.1 mol/L, and 10 mL of 0.2 mol/L) of Tris-HCl, pH 7.
The remaining bound 3H-NADH isomer was eluted with 0.5 mol/L Tris-HCl, pH 7, in 1-mL fractions. The fractions were checked spectrophotometrically at 340 nm, and the final concentration of the 3H-NADH isomer was established against a calibration curve of unlabeled NADH in elution buffer. The specific activity of the product was assessed by scintillation counting in a BETA V liquid scintillation counter (Kontron Instruments) with 5 mL of IRGA-safe plus (Packard) scintillation fluid in polyethylene vials. [4A-3H]NADH and [4B-3H]NADH were then diluted with commercially available NADH and water to a specific activity of 1 mCi/mmol and a concentration of 1 mmol/L. As shown in the Results, unlabeled [4A]NADH cannot be differentiated from [4B]NADH by complex I, allowing us to use commercial NADH for dilution of the respective [3H]NADH isotopes. Aliquots were stored at either 4 or -20 °C. NADH stored at 4 °C degraded over periods longer than 3 weeks, but stocks stored at -20 °C were still stable after storage for at least 4 months. The column was refreshed between runs with 0.25 mol/L HCl and then rinsed with 100 mL of H2O and titrated back to pH 8 with starting buffer.
Unlabeled [4A]NADH and [4B]NADH was prepared by the same method as described for the respective tritiated isoforms for initial experiments to demonstrate that complex I is not able to differentiate between these two isoforms.
preparation of mitochondria
Mitochondria were prepared from rat skeletal muscle as described
by Hoppel et al. (11). The final yield was 5.5 mg of
mitochondrial protein per gram of skeletal muscle wet weight as
determined by the Bradford protein assay (12). Mitochondria
were stored in 25 mmol/L potassium phosphate buffer at -20 °C until
needed. Usually, 2 µg of mitochondrial protein was used per assay
unless otherwise specified.
preparation of homogenate
Skeletal muscle samples from human biopsies or rats were stored
frozen at -70 °C until needed and then were homogenized with a
motorized glass/glass homogenizer (Eurostar) at a concentration of
20 g tissue/L in 25 mmol/L potassium phosphate buffer until
complete tissue disruption was achieved. There was no change in
activity observed after freezing and thawing. The standard assay
measured 40 µg of tissue per assay unless otherwise specified.
enzyme assays
Complex I activity was measured in a 50 mmol/L potassium phosphate
buffer, pH 6.5, containing 5 mmol/L MgCl2, 2.5
g/L bovine serum albumin, 100 µmol/L decylubiquinone, 0.02
g/L antimycin A, and 0.0125 g/L KCN. Buffer was made fresh each
day and used within 4 h. KCN and antimycin A were added to prevent
backflow of electrons along the respiratory chain.
3H-NADH (specific activity 1 mCi/mmol) in a final concentration of 100 µmol/L in either its 4A-3H or 4B-3H form was added to the buffer and preincubated at 37 °C for 3 min in a final volume of 98 µL. Control reactions contained 2 µmol/L rotenone as a specific inhibitor of complex I activity. The reaction was started by addition of 2 µL of enzyme preparation.
Reactions were allowed to proceed for 5 min before being stopped by the addition of 1 mL of 75 g/L activated charcoal in water and immediately vortex-mixed for 20 s. Samples were centrifuged at maximal speed on a bench top centrifuge for 5 min, and duplicate 300-µL aliquots were taken for counting with 5 mL of scintillation fluid.
Spectrophotometric determination of the activity of complex I was performed as described previously (6) but with 100 µmol/L decylubiquinone instead of ubiquinone-1 as the electron acceptor.
data presentation and statistical analysis
For all points represented in the graphs, the averaged value from
triplicate reactions in the presence of rotenone was subtracted from a
minimum of three individual reactions in the absence of the inhibitor.
Activities were adjusted to µmol of NADH oxidized per minute per
milligram of protein or tissue and taken as only that portion of
activity inhibitable by rotenone, unless otherwise specified.
Data are given as mean ± SD. Michaelis-Menten kinetics were fitted using nonlinear regression analysis (Sigmaplot for Windows 4.0; Jandel Scientific).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The synthesis of the two radioactive isomers of NADH was achieved enzymatically from tritiated NAD+ and either L-glutamate dehydrogenase (4A-3H) or alcohol dehydrogenase (4B-3H) as described in Materials and Methods. The reaction product was adsorbed onto a DEAE Sephadex anion-exchange column, and the tritiated NADH was eluted with 0.5 mol/L Tris buffer, pH 7. The concentrations of the resulting fractions were typically 0.5–1 mmol/L, and the specific activity was 4 mCi/mmol NADH.
Using isolated rat skeletal muscle mitochondria, we optimized assay
conditions for temperature (data not shown) and pH (Fig. 1
). The highest activity without loss of sensitivity to rotenone
was observed at pH 6.5. This pH was therefore chosen for all further
experiments. The optimal temperature was 37 °C, with increasing
activity over the range investigated (20–37 °C). As shown in Fig. 2
A, the reaction was linear for 10 min with isolated mitochondria
as the enzyme source, and a similar result was obtained with rat
skeletal muscle homogenate (Fig. 2B
). With an incubation time of 5 min,
the reaction was linear for up to 4 µg of mitochondrial protein or 80
µg of skeletal muscle. Similar results were obtained for isolated rat
liver mitochondria and liver homogenate (data not shown). Replacing the
phosphate buffer with Tris-HCl did not produce higher activities (data
not shown).
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Activity at all times was taken as a subtraction of the rotenone-insensitive portion from the total observed; i.e., total activity observed minus activity in the presence of 2 µmol/L rotenone. The background at zero time or in the absence of enzyme was directly proportional to the total tritium added to the reaction and represented 5–10% of the total radioactivity used. This background typically represented 20–40% of the final activity (after charcoal adsorption) in the presence of enzyme and after incubation for 5–10 min. Rotenone-sensitive NADH oxidation was between 60% and 80% of the total activity after subtraction of background for both isolated mitochondria and muscle homogenate.
Complex I is a multisubstrate enzyme, requiring a suitable quinone as
an electron acceptor. Michaelis-Menten kinetics were observed for both
decylubiquinone (Fig. 3
) and [4B-3H]NADH (Fig. 4
). The corresponding Km values
were 48 µmol/L for decylubiquinone and 35 µmol/L for
[4B-3H]NADH, and the
Vmax values were 89 and 90
µmol · min-1 · mg
protein-1 for decylubiquinone and
[4B-3H]NADH, respectively. When
[4A-3H]NADH was used as a substrate,
rotenone-insensitive but no rotenone-sensitive activity could be
observed with isolated mitochondria, demonstrating the specificity of
the assay (Fig. 4
). However, as shown directly by spectrophotometric
analysis, both 4A-NADH and 4B-NADH are substrates for complex I and
produce the same activity (data not shown). These experiments
demonstrate that complex I specifically uses the hydrogen of NADH that
is located in the 4B position.
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Studies of variability in the assay were conducted with multiple homogenates of the same sample (rat skeletal muscle) on different days, and the standard deviation was taken as a gauge of the reproducibility of the assay. Five separate homogenates were prepared individually on 3 different days, with the mean activity of complex I showing an overall variation of 6% of the mean (2.66 ± 0.17 U/g tissue). On the 3 individual days, the mean activities were 2.48 ± 0.95, 2.79 ± 0.79, and 2.71 ± 0.59 U/g tissue, respectively.
Simultaneous determination of complex I activity in rat skeletal muscle (3.1 ± 1.2 U/g) and mitochondria isolated from the same muscle (76 ± 13 U/g protein) allowed us to estimate the amount of mitochondrial protein per gram of muscle. According to these activities, 1 g of skeletal muscle yielded 42 mg of mitochondrial protein.
Given that the overall aim of this project was to establish a workable method for human tissue homogenate, we assayed complex I activity in frozen tissue samples (stored for 1 month to 2 years at -70 °C) of different adult patients. The reaction demonstrated Michaelis-Menten kinetics with similar kinetic constants as found in rat skeletal muscle. The reaction was linear at 5 min for up to 60 µg of tissue per assay and at 10 min for up to 40 µg of tissue per assay. Maximal activities were determined for seven different human tissue homogenates, using the conditions worked out for rat tissues. The activities were close to those in rat skeletal muscle homogenate, averaging 4.2 ± 2.4 U/g tissue. The same tissues were also analyzed by the spectrophotometric method, which yielded an activity of 3.2 ± 1.8 U/g tissue. Unfortunately, no skeletal muscle samples of patients with complex I deficiency were available for testing.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We developed an approach to determining complex I activity that is entirely different from the spectrophotometric methods used currently. This approach is based on the ability of the NADH dehydrogenases to differentiate between the two hydrogens in position 4 of the dihydropyridine ring of NADH. This specificity of the NADH dehydrogenases is most likely related to steric hindrance of the rotation of the dihydropyridine ring, which prevents free access of both hydrogens to the active center of the respective dehydrogenase. Regarding unlabeled NADH, the existence of the 4A- and 4B-isoforms is functionally not important because both positions contain hydrogen atoms. We have shown this directly with the spectrophotometric method, which indicated identical activities for complex I for both the 4A- and 4B-NADH isoforms. Therefore, [4B-3H]NADH can be diluted with commercial NADH for the assay of complex I activity as described by us. As demonstrated by our experiments, the existence of the 4A- and the 4B-NADH isoforms becomes an important diagnostic tool, however, when one of the hydrogens is labeled and can therefore be differentiated from the other one.
The 4A- and 4B-isoforms must be differentiated from the 
- and
ß-isoforms of NADH, which are commercially available. The - and
ß-isoforms of NADH are true stereoisomers, differing in the position
of the adenosine moiety in relation to the plane of the sugar ring.
This position is above the plane of the sugar ring in the case of the
-isomer and below the plane in the case of the ß-isomer. In all
cases, we used the more common ß-NADH isomer.
Our studies show that [4B-3H]NADH can be used reliably as a substrate for the measurement of complex I activity. We have demonstrated that the assay is linear with time and protein or tissue content for isolated mitochondria and tissue homogenates from rats and humans, and that the results are reproducible. The reactions of the two substrates can be described by Michaelis-Menten kinetics, and the Km values are in the expected range. Previous reports of the activity of complex I have varied according to the enzyme preparations and the ubiquinone species used. Values range from 0.3–1 µmol · min-1 · mg protein-1 for submitochondrial particles from bovine heart (13)(14) or yeast (15) to 0.082 µmol · min-1 · mg protein-1 for human skeletal muscle mitochondria (7). This value is close to the Vmax obtained with rat skeletal muscle mitochondria in our studies (0.090 µmol · min-1 · mg protein-1). The ubiquinone species used is another source of variation of the Vmax for this enzyme reaction. The longer chain ubiquinones are problematic because of their insolubility in water (7), and other ubiquinones are problematic because they render complex I activity less sensitive to rotenone (16). Because we (17) and others (14)(15)(18) have used decylubiquinone successfully for the spectrophotometric determination of complex I activity, we considered it a suitable substrate for the current study.
The background was consistently 5–10% of the radioactivity added to the assay and was not dependent on the reaction time, the amount of enzyme present, or the concentration of unlabeled NADH. It therefore most likely represents an impurity of the synthesized isotope. In support of this assumption, the background rose over the period of storage, parallel with the decay of NADH. After preparation, 3H-NADH should therefore be stored in aliquots at -20 °C. Under these conditions, 3H-NADH is stable for months and the background remains low.
After subtraction of the background, the rotenone-sensitive
[4B-3H]NADH activity accounted for 60–80% of
the total activity observed, depending on the enzyme preparation
investigated. The remaining activity can be explained by incomplete
inhibition by rotenone and/or antimycin and cyanide, or by the
presence of other dehydrogenases that use
[4B-3H]NADH as a substrate (e.g., -lipoyl
dehydrogenase) (8). When compared with the
spectrophotometric methods, rotenone is a more efficient inhibitor with
[4B-3H]NADH as the substrate, in particular
with tissue homogenates as the enzyme source. This finding represents
an advantage of the current assay over the spectrophotometric methods,
and can be explained by the specificity of complex I for
[4B-3H]NADH.
In contrast to [4B-3H]NADH, [4A-3H]NADH was not a substrate for complex I because no rotenone-sensitive activity was observed with isolated mitochondria as the enzyme source. On the other hand, a small rotenone-insensitive activity was detectable with isolated mitochondria as the enzyme source. This activity can be explained by mitochondrial dehydrogenases that use [4A-3H]NADH as a substrate, e.g., 3-phosphoglycerol dehydrogenase and malate dehydrogenase (8).
A disadvantage of the new assay of complex I activity is the initial preparation of [4B-3H]NADH. However, once set up, the column can be stored under ethanol and refreshed before each new run. Preparation of [4B-3H]NADH can be achieved easily within 1 day, along with the determination of the concentration of NADH and its specific activity. As discussed above, [4B-3H]NADH is stable for months if stored at -20 °C.
Based on the results of our study, we believe that this novel assay for complex I has several advantages over the currently used spectrophotometric assays and could therefore potentially replace these methods, in particular for the determination of complex I activity in tissue homogenates.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  A. J.M. Janssen, F. J.M. Trijbels, R. C.A. Sengers, J. A.M. Smeitink, L. P. van den Heuvel, L. T.M. Wintjes, B. J.M. Stoltenborg-Hogenkamp, and R. J.T. Rodenburg Spectrophotometric Assay for Complex I of the Respiratory Chain in Tissue Samples and Cultured Fibroblasts Clin. Chem., April 1, 2007; 53(4): 729 - 734. [Abstract] [Full Text] [PDF]
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) of 2
µmol/L rotenone. The highest rotenone-sensitive activity was at pH
6.5.
