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Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts.

Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) is the first complex of the oxidative phosphorylation system. It is the entry point for electrons into the respiratory chain by oxidation of NADH and transport of electrons to coenzyme-[Q.sub.10]. Complex I also has proton-transporting activity over the inner mitochondrial membrane to the intermembrane space. With a relative molecular mass of ~980 000, it is the largest complex of the respiratory chain. Complex I consists of 45 subunits (identified so far), forming a characteristic L-shaped configuration (1). The hydrophilic peripheral arm stretches out into the mitochondrial matrix and catalyzes the NADH oxidation and electron transport. The hydrophobic membrane arm is embedded in the inner mitochondrial membrane and contains the proton-transport activity. A deficiency of complex I is probably the most frequently encountered cause of mitochondrial disease, and mutations in several nuclear DNA-encoded and mitochondrial DNA-encoded subunits have been described to date (2). In addition, mutations in mitochondrial tRNAs, such as the m.3243A>G mutation in the mitochondrial [tRNA.sup.LEU(UUR)] usually result in complex I deficiency (2). The most commonly used technique for measuring complex I is a spectrophotometric assay measuring rotenone-sensitive NADH oxidation at 340 nm in tissue homogenate or mitochondria-enriched fractions from cultured fibroblasts (3, 4); however, the sensitivity and specificity of these assays are not optimal. One reason for this deficiency is the poor solubility of coenzyme-Q analogs. In addition, owing to the low sensitivity, relatively high concentrations of tissue extract are often required to detect complex I, resulting in turbidity of reaction mixtures. Here we describe a new sensitive and specific assay for complex I that is suitable for diagnostic purposes.

Materials and Methods

MATERIALS

Coenzyme [Q.sub.l], decylubiquinone, 2,6-dichloroindophenol (DCIP), [1] rotenone, and antimycin-A were obtained from Sigma. NADH and bovine serum albumin (BSA; fraction V, fatty acid free) were obtained from Roche. All other chemicals were of the highest purity commercially available. Spectrophotometric assays were performed on a Perkin-Elmer Lambda 25 UV/Vis spectrophotometer with an automatic water thermostattable 8-cell holder in disposable semimicro 10-mm acryl-cuvettes from Sarstedt.

PREPARATION OF MUSCLE-MITOCHONDRIA AND MITOCHONDRIA-ENRICHED FRACTIONS FROM CULTURED SKIN FIBROBLASTS

Human muscle tissue (musculus quadriceps or musculus semitendinosus) was homogenized as described (5). Fractions of 200 to 300 [micro]L of a frozen supernatant of muscle homogenate (centrifuged at 600g and 2 [degrees]C for 10 min) were thawed at 2-4 [degrees]C and centrifuged (10 min at 14 000g and 2 [degrees]C) in an Eppendorf 5402 centrifuge. We carefully removed the 14 000g supernatant and the fluffy layer with an Eppendorf pipettor. The mitochondrial pellet was resuspended in 150 [micro]L of 10 mmol/L Tris, pH 7.6, frozen in 25-[micro]L aliquots in liquid nitrogen, and kept at -80 [degrees]C.

We cultured human skin fibroblasts in M199 medium (Gibco) supplemented with 20% (vol/vol) fetal calf serum. Aliquots of 10 to 15 x [10.sup.6] cells were washed with ice-cold phosphate-buffered saline, frozen in liquid nitrogen, and kept at -80 [degrees]C until use. For isolation of mitochondria-enriched fractions, the pellets were thawed at 2-4 [degrees]C and suspended in 2.9 mL ice-cold 10 mmol/L Tris, pH 7.6. We disrupted the cells mechanically with a 5-mL glass/Teflon Potter-Elvehjem homogenizer (clearance, 0.025 mm), 8 strokes at 1800 rpm in melting ice. After homogenization, we added 0.6 mL ice-cold 1.5 mol/L sucrose and centrifuged the homogenate (10 min at 600g and 2 [degrees]C) in a Sorval-RC2B centrifuge. The 600g supernatant was centrifuged again (10 min at 14 000g and 2 [degrees]C), and the resulting supernatant was carefully removed. The mitochondrial pellet was resuspended in 0.5 mL of 10 mmol/L Tris, pH 7.6, frozen in 50-[micro]L aliquots in liquid nitrogen, and kept at -80 [degrees]C.

COMPLEX I ASSAY

In the new complex I assay, DCIP is used as a terminal electron acceptor. Complex I oxidizes NADH, and the electrons produced reduce the artificial substrate decylubiquinone that subsequently delivers the electrons to DCIP. The reduction of DCIP can be followed spectrophotometrically at 600 run. As the electrons produced by other NADH-dehydrogenases are not accepted by decylubiquinone (3), reduction of DCIP is almost completely caused by complex I activity, resulting in very high rotenone-sensitive activity.

We measured complex I spectrophotometrically at 600 run in an incubation volume of 1.0 mL containing 25 mmol/L potassium phosphate, 3.5 g/L BSA, 60 [micro]mol/L DCIP, 70 [micro]mol/L decylubiquinone, 1.0 [micro]mol/L antimycine-A, and 0.2 mmol/L NADH, pH 7.8. Decylubiquinone and antimycine-A were dissolved in dimethyl sulfoxide (17.5 mmol/L and 1.0 mmol/L, respectively). We prepared a stock solution of 80 g/L BSA in 5 mmol/L potassium phosphate buffer, pH 7.4. Because BSA is a critical component of the complex I assay, we measured the concentration spectrophotometrically at 280 run (A280 1 g/L BSA = 0.667). The stock solution was diluted to 70 g/L and stored in 1-mL aliquots at -30 [degrees]C. Of this solution, 50 [micro]L was added to a final reaction volume of 1 mL. We preincubated an aliquot of 2.5 to 10 [micro]L mitochondrial suspension from muscle or 20 [micro]L mitochondriaenriched fraction from fibroblasts at 37 [degrees]C in 960 [micro]L incubation mixture without NADH. After 3 min, we added 20 [micro]L of 10 mmol/L NADH and measured the absorbance at 30-s intervals for 4 min at 37'C. After 4 min, we added 1.0 [micro]L rotenone (1 mmol/L in dimethyl sulfoxide) and measured the absorbance again at 30-s intervals for 4 min.

Complex I activity was expressed as mU/U complex II, mU/U complex IV, or U/g protein, in which 1 U complex I activity equals 1 [micro]mol DCIP reduced per min. Fibroblasts from 6 patients and muscle tissue samples from 3 patients with a diagnosed complex I deficiency were used to measure complex I to demonstrate the applicability of the assay to the diagnosis of complex I deficiency. The controls and patients have been described (5).

COMPLEX II ASSAY

During isolation of mitochondria from frozen samples of muscle or fibroblasts, part of the citrate synthase leaks out of the mitochondria and is lost, so citrate synthase cannot be used as a mitochondrial marker enzyme. For that reason, we used complex II and complex IV as mitochondrial marker enzymes. We measured complex II spectrophotometrically at 600 run as described, with some modifications (6). The 1.0-mL incubation volume contained 80 mmol/L potassium phosphate, 1 g/L BSA, 2 mmol/L EDTA, 0.2 mmol/L ATP, 10 mmol/L succinate, 0.3 mmol/L potassium cyanide (KCN), 80 [micro]mol/L DCIP, 50 [micro]mol/L decylubiquinone, 1 [micro]mol/L antimycine-A, and 3 [micro]mol/L rotenone, pH 7.8. We preincubated an aliquot of 5 [micro]L mitochondrial suspension from muscle or 10 [micro]L mitochondria-enriched fraction from fibroblasts at 37 [degrees]C in the incubation mixture without KCN and succinate. After 10 min, we added KCN and succinate to start the reaction and measured the absorbance at 1-min intervals for 5 min at 37 [degrees]C. Blanks were measured in the presence of 5 mmol/L malonate that was added before preincubation. Decylubiquinone (10 mmol/L) was dissolved in dimethyl sulfoxide. Antimycine-A (1 mmol/L) and rotenone (3 mmol/L) were dissolved in ethanol. For both assays, we used a molar absorptivity at 600 run of 19.1 (mmol/L)-1 cm-1 for DCIP.

COMPLEX IV AND PROTEIN ASSAYS

We measured complex IV activity as described (7) and protein according to Lowry et al. (8).

Results

Lineweaver-Burk plots revealed Km values of 0.04 mmol/L for NADH and 0.017 mmol/L for DCIP (data not shown). The pH optimum was 7.8 (data not shown). In a concentration series experiment with decylubiquinone, the highest complex I activity was at 0.07 mmol/L decylubiquinone (data not shown). We tested coenzyme [Q.sub.l] as an alternative ubiquinone analog and found that in the presence of 0.07 mmol/L, the activity was ~80% of that measured with decylubiquinone, with similar rotenone sensitivities. Therefore, we continued with decylubiquinone as electron acceptor for complex I in the reaction mixture. Although in most complex I assays [Mg.sup.2+] and KCN are added, the latter to inhibit nonspecific NADH dehydrogenase activity (3), we observed no influence of [Mg.sup.2+] and KCN in our assay (data not shown).

In most complex I assays, pretreatment of the mitochondria, such as repeated freezing and thawing or sonication, is necessary to disrupt the mitochondrial membrane. In our assay, a simple osmotic shock in 10 mmol/L Tris * Cl, pH 7.6, followed by a single freeze-thaw cycle, was sufficient to measure optimal complex I activity. Repeated freezing and thawing did not improve this result, and sonication even decreased complex I activity (data not shown).

Complex I activity was linear for at least 4 min, with sample amounts varying between 0.25 [micro]g protein (containing 0.9 mU complex IV) and 3 [micro]g protein (11 mU complex IV; see Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vo153/issue4).

Because BSA is essential for measuring optimal complex I activity (3), we studied the effect of BSA on complex I activity in muscle mitochondria. We measured optimal complex I activity in the presence of BSA concentrations between 3.2 and 3.9 g/L, whereas the percentage rotenone sensitivity of complex I was near 100% at BSA concentrations between 2.1 and 4.9 g/L (Fig. 1). Similar results were obtained when decylubiquinone was replaced by coenzyme [Q.sub.1] in the reaction mixture (data not shown).

[FIGURE 1 OMITTED]

The [IC.sub.50] for rotenone in muscle mitochondria, which could be measured only in the presence of BSA, was 13.5 nmol/L (mean of 2 results; range, 10-17 nmol/L; Fig. 2). The presence of BSA in the reaction mixture is required not only for rotenone sensitivity of the assay (probably by solubilizing rotenone), but also for solubilization of decylubiquinone, as illustrated by the fact that in the absence of BSA, we observed an orange/yellow layer on the surface of the reaction mixture after centrifugation at 14000g. By spectrophotometric analysis at 278 run, we found that ~70% of the amount of decylubiquinone added to the mixture was present in this layer.

[FIGURE 2 OMITTED]

Intraassay imprecision (CV) was between 2% and 8%, and interassay imprecision was between 2% and 11% (Table 1).

To test the long-term reproducibility of the assay, complex I was measured repeatedly in 3 muscle mitochondrial samples over a period of 6 months. Mean (SD) complex I activity in these samples was 607 (47) U/L (n = 10; CV 8%), 795 (30) U/L (n = 6; CV 4%), and 888 (49) U/L (n = 6; CV 5%).

To compare this method to the method described by Fischer et al. (3), we used the 2 methods to measure complex I in mitochondria from muscle and fibroblasts. The mean (SD) activity measured in muscle mitochondria using the method of Fischer et al. (3) was 31% (5%; n = 7; range, 26%-38%; paired t-test P = 0.0005) and in mitochondria from fibroblasts was 17% (3%; n = 46; range, 10%-26%; paired t-test P = 7 x [10.sup.-29]) of the activities measured with our method, showing that the new method is 3-fold (for muscle) and more than 5-fold (for fibroblasts) more sensitive than the method of Fischer et al. (3).

We assessed the specificity of the complex I assay by measuring its rotenone sensitivity. The mean (SD) rotenone sensitivity in mitochondria from control muscle (n = 17) was 95% (5%), and in mitochondria from control fibroblasts (n = 46), it was 82% (9%).

We tested whether the new complex I assay could be applied to cruder muscle preparations than those used in the experiments described above. In 5 control muscle samples, the mean (SD) complex I activity in 600g supernatants was 46% (6%; range, 43%-51%) of that in equivalent amounts of mitochondrial fractions, and the rotenone sensitivity was 71% (14%; range, 48%-86%). Finally, we tested whether the new complex I assay could be applied to the diagnosis of complex I deficiencies in both muscle and fibroblasts. First, we measured control values for mitochondria from muscle and fibroblasts (Table 2).

We examined fibroblasts from 6 patients carrying variations in different complex I genes and suffering from a previously established complex I deficiency (5). Using the new method, the enzyme deficiency could be confirmed in all 6 patients. In 3 of the patients, we measured complex I in muscle and also confirmed the deficiency in this tissue. The method showed lower results in all tested patients with complex I deficiencies than in any control subjects (Table 3).

Discussion

At present, the diagnosis of complex I deficiency is usually established using complex I assays that are based on the spectrophotometric measurement of rotenone-sensitive NADH oxidation in patient-derived tissue samples and cultured fibroblasts. In addition to complex I, muscle tissue and cultured fibroblasts contain several nonmitochondrial NADH-oxidizing dehydrogenases. Therefore, the use of tissue homogenates in complex I assays results in a relatively high rate of rotenone-insensitive NADH oxidation that interferes with the sensitivity of the complex I assay. Another disadvantage of measuring muscle homogenate is turbidity of the incubationmixture, which interferes with the spectrophotometric assay. For these reasons, Brooks and Krahenbuhl (9) developed a radiochemical assay for complex I in muscle by measuring [sup.3][H.sub.2]O production from [4B-[sup.3]H]-NADH oxidation, based on the stereospecificity of complex I for the 4B hydrogen atom of NADH.

Our new assay uses no radioactivity, is suitable for the diagnostic analysis of complex I in fibroblasts and muscle tissue, and uses DCIP as a final electron acceptor. DCIP has a molar absorptivity that is ~3 times higher than that of NADH: the molar absorptivity at 600 nm of DCIP is 19.1 [(mmol/L).sup.-1] [cm.sup.-1], whereas the molar absorptivity at 340 nm of NADH is 6.2 [(mmol/L).sup.-1] [cm.sup.-1]. Compared with the method described by Fischer et al. (3) that measures NADH oxidation, 3- to 5-fold more complex I activity is measured using our method.

DCIP has been used by others in a complex I assay (10), but that assay was not suitable for diagnostic purposes because of nonlinearity of the absorbance with time. In our assay, the addition of an optimal concentration of BSA to the reaction mixture, combined with the use of isolated mitochondrial preparations instead of crude sample homogenates, resulted in a complex I activity that was linear in time and had high rotenone sensitivity. BSA is essential, as it facilitates the solubilization of both rotenone and decylubiquinone. Rotenone and decylubiquinone are both hydrophobic and are practically insoluble in water; rotenone strongly and reversibly binds to BSA (11). Direct binding to BSA probably also plays a role in the solubilization of decylubiquinone, as it is known that BSA reversibly binds molecules with long alkyl chains (12).

The mean (SD) complex I activities measured in fibroblasts and expressed on complex IV activity were similar to those measured by Kramer et al. (4): 1100 (245; n = 46) vs 1200 (170; n = 15), respectively. The mean (SD) rotenone-sensitive activity in our assay was 82% (9%; n = 46) compared with 30% (range, 15%-50%) in the assay described by Kramer et al. (4). The mean (SD) rotenone sensitivity measured in digitonin- and Percoll-treated fibroblasts as described by Chretien et al. (13), 86 (19%; n = 22), was similar to our results. The radiochemical enzyme assay of Brooks and Krahenbuhl (9) gave a slightly lower rotenone sensitivity of 60%-80%. As both Chretien et al. (13) and Brooks and Krahenbuhl (9) measured complex I in crude lysates and expressed activities on protein base, it was not possible to directly compare the complex I activities measured by these 2 methods with our results.

The method we developed is also suitable for measurements of complex I in 600g supernatants from muscle. The sensitivity and specificity appeared to be lower than observed with mitochondrial fractions.

This complex I assay has clear advantages over the commonly used assays because it is nonradioactive; shows high sensitivity, precision, and rotenone sensitivity; and can be performed on a simple spectrophotometer. The method is applicable to the analysis of muscle samples and is also suitable for measuring complex I in cultured fibroblasts.

This work was supported by a grant from the European Union Framework Program 6 (MITOCIRCLE). Financial disclosure: None declared.

Received August 28, 2006; accepted January 18, 2007. Previously published online at DOI: 10.1373/clinchem.2006.078873

References

(1.) Carroll J, Fearnley IM, Skehel JM, Shannon RJ, Hirst J, Walker JE. Bovine complex I is a complex of 45 different subunits. J Biol Chem 2006;281:32724-7.

(2.) Janssen RJ, Nijtmans LG, van den Heuvel LP, Smeitink JA. Mitochondrial complex I: structure, function and pathology. J Inherit Metab Dis 2006;29:499-515.

(3.) Fischer JC, Ruitenbeek W, Trijbels JMF, Veerkamp JH, Stadhouders AM, Sengers RCA, et al. Estimation of NADH oxidation in human skeletal muscle mitochondria. Clin Chim Acta 1986;155: 263-73.

(4.) Kramer KA, Oglesbee D, Hartman SJ, Huey J, Anderson B, Magera MJ, et al. Automated spectrophotometric analysis of mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts. Clin Chem 2005;51:2110-6.

(5.) Janssen AJM, Trijbels JMF, Sengers RCA, Wintjes LTM, Ruitenbeek W, Smeitink JAM, et al. Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology. Clin Chem 2006;52:860-71.

(6.) Rustin P, Chretien D, Bourgeron T, Gerard B, Rotig A, Saudubray JM, et al. Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 1994;228:35-51.

(7.) Cooperstein SJ, Lazarow A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem 1951;189: 665-70.

(8.) Lowry OH, Rosebrough NJ, Farr AL, Randell RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.

(9.) Brooks H, Krahenbuhl S. Development of a new assay for complex I of the respiratory chain. Clin Chem 2000;46:345-50.

(10.) Jewess PJ, Devonshire AL. Kinetic microplate-based assays for inhibitors of mitochondrial NADH:ubiquinone oxidoreductase (complex I) and succinate:cytochrome c oxidoreductase. Anal Biochem 1999;272:56-63.

(11.) Panov A, Dikalov S, Shalbuyeva N, Taylor G, Sheror T, Greenamyre JT. Rotenone model of Parkinson disease: multiple brain mitochondria dysfunctions after short term systemic rotenone intoxication. J Biol Chem 2005;280:42026-35.

(12.) Spector A, Fletcher J, Ashbrook J. Analysis of long-chain free fatty acid binding to bovine serum albumin by determination of stepwise equilibrium constants. Biochemistry 1971;10:3229-32.

(13.) Chretien D, Benit P, Chol M, Lebon S, Rotig A, Munnich A, et al. Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts. Biochem Biophys Res Commun 2003;301:222-4.

[1] Nonstandard abbreviations: DCIP, 2,6-dichloroindophenol; BSA, bovine serum albumin; KCN, potassium cyanide.

ANTOON J.M. JANSSEN, FRANS J.M. TRIJBELS, ROB C.A. SENGERS, [[dagger]] JAN A.M. SMEITINK, LAMBERT P. VAN DEN HEUVEL, LIESBETH T.M. WINTJES, BERENDIEN J.M. STOLTENBORG-HOGENKAMP, and RICHARD J.T. RODENBURG *

Department of Pediatrics and Laboratory of Pediatrics and Neurology, The Nijmegen Centre for Mitochondrial Disorders at the Radboud University Medical Centre, Nijmegen, The Netherlands.

[[dagger]] Dr. Sengers died on April 6, 2006.

* Address 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@cukz.umcn.nl.
Table 1. Intra- and interassay imprecision for complex I in
mitochondrial fractions from muscle and cultured fibroblasts. (a)

 Complex I (U/L)

Intraassay imprecision Experiment 1 Experiment 2 Experiment 3

Muscle mitochondria
10 [micro]L 240 267 262
10 [micro]L, 1:1 dilution 248 234 276
10 [micro]L, 1:2 dilution 252 249 241

Fibroblast mitochondria
20 [micro]L 117 131 124
20 [micro]L, 1:1 dilution 124 118 130
20 [micro]L, 1:2 dilution 132 129 123

Interassay imprecision

Muscle mitochondria 1 2 3
10 [micro]L 251 256 263
10 [micro]L, 1:1 dilution 280 252 280
10 [micro]L, 1:2 dilution 249 243 297

Fibroblast mitochondria
20 [micro]L 124 131 110
20 [micro]L, 1:1 dilution 124 134 116
20 [micro]L, 1:2 dilution 129 147 123

Intraassay imprecision Mean SD CV, %

Muscle mitochondria
10 [micro]L 256 14 6
10 [micro]L, 1:1 dilution 253 21 8
10 [micro]L, 1:2 dilution 247 6 2

Fibroblast mitochondria
20 [micro]L 124 7 6
20 [micro]L, 1:1 dilution 124 6 5
20 [micro]L, 1:2 dilution 128 5 4

Interassay imprecision

Muscle mitochondria
10 [micro]L 257 6 2
10 [micro]L, 1:1 dilution 271 16 6
10 [micro]L, 1:2 dilution 263 30 11

Fibroblast mitochondria
20 [micro]L 122 11 9
20 [micro]L, 1:1 dilution 125 9 7
20 [micro]L, 1:2 dilution 133 13 9

(a) Complex I activities are expressed as U/L undiluted sample.
Intra- and interassay imprecision were determined with undiluted,
1:1 diluted, and 1:2 diluted mitochondrial fractions from muscle
and cultured fibroblasts. Intraassay imprecision was determined by
measuring complex I activities in 3-fold on the same day, and
interassay imprecision was determined by measuring complex I
activities on 3 different days. All incubations were performed in
duplicate. The protein contents of the undiluted muscle and
fibroblast mitochondrial fractions were 0.26 and 0.88 g/L,
respectively.

Table 2. Control values for complex I in mitochondrial
fractions from muscle and cultured fibroblasts. (a)

 Complex I

 mU/U CII mU/U C IV

Muscle

Mean (n = 17) 1140 343
SD 180 63
Observed range 783-1497 270-475
Mean (2 SD) 1140 (360) 343 (126)

Fibroblasts

Mean (n = 46) 1161 1100
SD 237 245
Observed range 720-1708 678-1675
Mean (2 SD) 1161 (474) 1100 (490)

(a) All incubations were performed in duplicate.

Table 3. Complex I in muscle and fibroblasts of 6 patients with a
previously established complex I deficiency.

 Complex 1 by our
 method (b)

Patient (a) mU/U CII mU/U C IV Complex I by the
 method of Fischer
 et al. (3), mU/U CS (c)

Muscle
1 154 48 14
3 510 144 16
5 465 157 24
Control mean (2 SD) 1140 (360) 343 (126) 85 (40)
Observed range 783-1497 270-475 53-163
n 17 17 43

Fibroblasts
1 (NDUFS2) 255 179 29
2 (NDUFS4) 240 145 64
3 (NDUFS7) 484 459 65
5 (NDUFS7) 438 467 26
7 (NDUFV1) 426 416 85
8 (MT-ND2) 339 333 42
Control mean (2 SD) 1161 (474) 1100 (490) 188 (104)
Observed range 720-1708 678-1675 110-260
n 46 46 14

(a) Patient numbering is the same as in Janssen et al. (5).

(b) Complex I was measured in mitochondrial fractions.

(c) Complex I was measured in 600g supernatants of muscle and
mitochondrial fractions from fibroblasts.
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Title Annotation:Endocrinology and Metabolism
Author:Janssen, Antoon J.M.; Trijbels, Frans J.M.; Sengers, Rob C.A.; Smeitink, Jan A.M.; van den Heuvel, L
Publication:Clinical Chemistry
Date:Apr 1, 2007
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