Hypaque

Context.-Laboratory methods currently available for detecting mitochondrial respiratory dysfunction are laborintensive, require large amounts of isolated mitochondrial protein, invasive (require a skeletal muscle biopsy), and usually produce conflicting results.

Objective.-To develop a rapid, reliable, and noninvasive method for detecting oxidative phosphorylation activity without the need to isolate mitochondrial fractions.

Design.-Lymphocytes from 6 patients with mitochondrial disorders (3 with mitochondrial myopathy and 3 with Leber hereditary optic neuropathy) and 51 normal control subjects were incubated with 6µM resazurin without and with mitochondrial inhibition by amiodarone (200µM), and the fluorescence intensity resulting from resazurin reduction was monitored spectrofluorometrically over time. Mitochondrial respiratory activity was calculated as the difference between uninhibited and inhibited measurements.

Results.-Mitochondrial respiratory activity was established for 51 normal control subjects and was decreased in all 6 patients with mitochondrial syndromes. Mitochondrial respiratory activity values for patients 1 through 6 compared to the control group after 240 minutes' incubation with resazurin were 55%, 71%, 49%, 61%, 68%, and 59%, respectively (mean mitochondrial respiratory activity of patients, 13.6% or 60.5% of control mean; P

Conclusion.-This resazurin-based technique proved to be a fast and reproducible method for quantifying mitochondrial activity and identifying respiratory functional defects in patients with mitochondrial disorders.

(Arch Pathol Lab Med. 2005;129:1295-1298)

Biochemical confirmation of defective mitochondrial function usually involves measuring respiratory chain function directly by polarographic methods1 or by estimating the activity of individual respiratory chain enzyme complexes in mitochondria from tissue homogenates or cultured cells.2 These methods require a skeletal muscle biopsy yielding large amounts of mitochondrial protein (1-10 mg) and are labor-intensive.3 Plasma lactate and pyruvate levels are sometimes used as a screening test for detecting mitochondrial respiratory dysfunction.4 However, patients with some mitochondrial disorders, such as Leber hereditary optic neuropathy and chronic progressive external ophthalmoplegia, routinely have plasma lactate levels within normal limits.5

Circulating blood lymphocytes have been used to measure mitochondrial respiratory dysfunction in a group of myopathy patients by enzymatic6 and flow cytometric7 methods. They have also been used to measure adenosine triphosphate synthesis in patients with various mitochondrial disorders, including Leber hereditary optic neuropathy; mitochondrial encephalopathy, lactic acidosis, and strokelike episodes; chronic progressive external ophthalmoplegia; neuropathy, ataxia, and retinitis pigmentosa; and cytochrome C oxidase deficiency.8 The limitation of enzymatic studies is that most mitochondrial enzymes are unstable,9 they require a wide range of substrates,8 and they are usually not reliable for assessment of complex I activity.4,9 Flow cytometric methods, on the other hand, are laborious and require special machinery that might not be available in routine diagnostic laboratories.

Resazurin is a nontoxic redox-active dye traditionally used for evaluating the quality of raw milk and semen. Resazurin, which is blue and nonfluorescent, is reduced to resorufin, which is pink and highly fluorescent, and this conversion can be monitored by fluorometric measurement. A method was recently described for assessing mitochondrial respiratory function in rat liver mitochondria using resazurin reduction.10 Resazurin has also been used to assess mitochondrial metabolic activity in synaptosomes from spinal cord-injured animals" and neonatal rat cerebellum12 and in isolated yeast mitochondria.13 We describe a simplified, rapid method of measuring oxidative activity in humans using resazurin and circulating lymphocytes.

MATERIALS AND METHODS

Chemicals

Resazurin (Aldrich Chemical Company, Inc, Milwaukee, Wis) was prepared as an aqueous stock solution (4mM), sterilized by membrane filtration, and stored at -20°C until required. Analytic-grade oligomycin, antimycin A, and rotenone were obtained from Sigma Chemical Company (St Louis, Mo). Rotenone was dissolved in ethanol and stored at -20°C. Oligomycin and antimycin A were both dissolved in acetone at the required concentration. Amiodarone (Spectrum Chemicals & Laboratory Products, Gardena, Calif) was dissolved in methanol on the day of use.

Control Subjects and Patients

The control group consisted of 51 Middle-Eastern Arabs (30 men and 21 women; mean age, 34.04 ± 8.5 years), who visited the blood donor clinic at King Faisal Specialist Hospital and Research Center (Riyadh, Saudi Arabia). They answered an extensive questionnaire regarding their current health and medical history, and we chose healthy individuals with no apparent metabolic or genetic disorders.

Table 1 lists the clinical characteristics of the 6 subjects with unequivocal mitochondrial clinical syndromes. Three patients (patients 1, 2, and 3) had mitochondrial myopathy both clinically and on histologie examination of muscle biopsies. Mitochondrial DNA assessment revealed the 4.9-kb common deletion14 in muscle of patients 1 and 3, but muscle tissue was not available for molecular analysis in patient 2. Three patients (patients 4, 5, and 6) had variants of Leber hereditary optic neuropathy with bilateral optic nerve injury and mitochondrial DNA mutations confirmed by sequencing the entire mitochondrial genome, as previously described.15 All patients and control subjects signed informed consent approved by the King Faisal Specialist Hospital and Research Center Research Advisory Council and Research Ethics Committee.

Isolation of Lymphocytes From Peripheral Blood and Preparation of Cell Suspension

Blood (5 mL) was diluted with phosphate-buffered saline at a ratio of 1:1 within 1 hour of extraction and slowly layered onto a 15-mL screw-cap tube containing 4.5 mL Ficoll-Hypaque separating solution. The tubes were centrifuged for 20 minutes at 1800 rpm, after which the lymphocyte-containing layer was collected into a new centrifuge tube using a sterile pipette. The lymphocyte mix was then diluted in 10 mL phosphate-buffered saline and centrifuged for 10 minutes at 1200 rpm. The supernatant was discarded, 5 mL of hypotonie phosphate-buffered saline lysing buffer was added, the pellet was mixed gently in this buffer, and the mixture was allowed to sit for about 45 seconds. Five mL of 2× sodium chloride solution was added. The mixture was gently pipetted and then centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the pellet was suspended in RPMI 1640 medium (Invitrogen Corporation, Carlsbad, Calif) supplemented with L-glutamine. The optical density (OD^sub 660^) of the lymphocyte suspension was adjusted to 0.20 nm, which is equivalent to a cell density of approximately 5 × 10^sup 5^ cells/mL. Using this protocol, cell viability assessed by 0.2% trypan blue8 was 96% ± 2%.

Assessment of Changes in Resazurin Fluorescence

Varying concentrations of the mitochondrial inhibitors amiodarone (200µM-400µM), antimycin A (10µM-100µM), oligomycin (200µM-1000µM), and rotenone (20µM-100µM) were incubated with resazurin (6µM) and 200µL RPMI 1640 medium without cells. These concentrations are usually sufficient to inhibit 85% to 90% of mitochondrial respiration.16,17 Wells containing only resazurin and medium served as controls. Fluorescence intensity was measured (530 nm excitation and 590 nm emission) by an LS55 Luminescence Spectrometer (PerkinElmer Instruments, Shelton, Conn) fitted with a microplate reader. Each experimental condition was repeated in 5 different wells of a 96-well plate.

Resazurin Lymphocyte Assay

Lymphocyte mitochondrial respiratory activity was estimated in the following fashion: 10 wells of a 96-well plate were assigned to each patient and 6 wells were assigned to each control subject. Half of the wells assigned to each individual contained 200 µL of lymphocyte suspension (approximately 1 × 105 cells and 11.2 μg protein), and the other half contained 200 µL of lymphocyte suspension incubated with amiodarone at 37°C in a cell culture microplate incubator (Jitterbug model 130000, Boekel Scientific Inc, Feasterville, Pa) for 60 minutes. Resazurin (6µM) was then pipetted into all wells and the plate was incubated for an additional 240 minutes. Fluorescence intensity was measured as above at 0, 60, 120, 180, and 240 minutes after subtracting background measurements with only resazurin and media. These figures were averaged for each condition (without or with amiodarone) and individual (5 measurements for each patient and 3 measurements for each control subject) and were normalized for protein content. Mitochondrial respiratory activity (MRA) was estimated for each patient and control by subtracting the amiodarone measurements from those obtained without amiodarone.

RESULTS

Effects of Mitochondrial Inhibitors and Optimization of Assay Conditions

The changes in resazurin fluorescence after incubation with variable concentrations of inhibitors were assessed as described in the "Materials and Methods" section. The results of the 5 measurements at time 0 (F^sub 0^) and 240 minutes (F^sub 240^) were averaged, and the change in resazurin fluorescence was calculated as F^sub 240^ - F^sub 0^/F^sub 0^ × 100%. Table 2 shows that oligomycin, antimycin A, and rotenone increased the change in resazurin fluorescence measurements over 240 minutes to a level above that of resazurin alone. Amiodarone did not increase resazurin reduction, even at concentrations above the level required to inhibit mitochondrial respiration (200µM). Therefore, amiodarone was selected for subsequent experiments. Solvents for the inhibitors (acetone, methanol, and ethanol) had no effect on resazurin reduction at the concentrations used for the duration of these experiments (data not shown).

We then proceeded with optimization of other assay conditions, such as resazurin concentration, optimal assay temperature, and time. Incubated lymphocytes yielded a maximum measurable rate of resazurin reduction at a resazurin concentration of 6µM, while higher concentrations of resazurin (10µM, 20µM, and 30µM) decreased the rate of reduction by 5%, 13%, and 20%, respectively, as compared to the rate obtained with 6µM resazurin. Following this determination, resazurin (6µM) was incubated with lymphocytes at different temperatures (0°C, 20°C, 30°C, and 37°C), in order to determine the optimal assay temperature. Although lymphocytes were able to reduce resazurin at a very low speed at 0°C, the reduction rate was highest at 37°C. Additionally, resazurin (6µM) was reduced by lymphocytes during a 240-minute incubation period in a linear manner at 37°C (results not shown). The optimal assay conditions were comparable to those reported previously.8,10-13,18

Resazurin Lymphocyte Assay

Figure 1 graphs the mean normalized (for background fluorescence and protein concentration) resazurin fluorescence measurements for control group lymphocytes after incubation for 240 minutes without (no-amiodarone curve) and with (amiodarone curve) mitochondrial inhibition using amiodarone (200µM). Mitochondrial respiratory activity (MRA curve) was calculated by subtracting values obtained with amiodarone from those obtained without amiodarone.

Figure 2 graphs MRA values for the control group to those of 6 patients with mitochondrial syndromes at various time points. Mitochondrial respiratory activity values for patients 1 through 6 compared to the control group after 240 minutes' incubation were 55%, 71%, 49%, 61%, 68%, and 59%, respectively (mean MRA of patients, 13.6% or 60.5% of control mean; P

COMMENT

To our knowledge, estimation of mitochondrial respiratory function by in vitro reduction of resazurin in freshly extracted blood lymphocytes has not been reported previously. The evaluation of organelles such as mitochondria with resazurin was reported recently,10 and the utilization of other testing protocols in lymphocytes to screen mitochondrial disorders has also been described.8 These studies suggested that resazurin could be used to quantify lymphocyte mitochondrial activity.

The experimental conditions for this assay, such as the incubation temperature and assay time, have been initially adapted from published methods using mitochondrial fractions8,10-12,18 and were tested here for optimal conditions with lymphocytes. Resazurin (6µM) proved an ideal concentration, since lower concentrations (1µM and 3µM) led to fast reduction of resazurin and higher concentrations (20µM-30µM) slowed the reduction rate. The fast reduction of low resazurin concentrations created a reaction that went to completion too rapidly, while higher concentrations made fluorescence changes too slow to measure over time.

Inhibitors of mitochondrial activity are necessary to ensure that the metabolic activity observed in lymphocytes is due to mitochondrial function. In contrast to a previous report,1" our experiments proved that a number of commonly used mitochondrial inhibitors (antimycin A, rotenone, and oligomycin) caused resazurin reduction. Among the inhibitors we tested, only amiodarone, a complex I and V inhibitor, did not reduce resazurin at concentrations necessary for mitochondrial inhibition. Therefore, amiodarone is the only suitable inhibitor evaluated for this purpose so far.

Once the experimental parameters were established, we then proceeded with testing the validity of this method in patients with established oxidative phosphorylation disorders and in normal control subjects. The results conclusively showed differentiation between these 2 groups.

This study provides evidence that resazurin blue is a sensitive indicator of mitochondrial function that is compatible with blood lymphocytes. The benefits of using resazurin for measuring mitochondrial function in lymphocytes include (1) no need for an invasive procedure, such as muscle biopsy (the tissue of choice to characterize respiratory chain activity), because only a blood sample is required; (2) no requirement for a large volume of blood because 5 mL of venous blood is sufficient; (3) resazurin is nontoxic and its reduction can be assessed multiple times from a single sample; (4) the protocol is simple and does not require highly trained personnel; and (5) the measurements are adaptable for high throughput.

In conclusion, resazurin reduction is a promising in vitro screening assay for mitochondrial disorders. For this assay to be adopted for general screening, the following factors have to be tested: (1) more patients with various mitochondrial diseases; (2) patients with nonmitochondrial diseases to assure that the results are specific; (3) more normal control subjects from a wider age range; and (4) additional inhibitors for different respiratory chain complexes in order to assess specific respiratory chain complexes.

References

1. Wenchich L, Drahota Z, Honzik T, et al. Polarographic evaluation of mitochondrial enzymes activity in isolated mitochondria and in pcrmeabilized human muscle cells with inherited mitochondrial defects. PhysiolRes. 2003;S2:781-788.

2. Trounce IA, Kim YL, )un AS, Wallace DC. Assessment of mitochondrial ox- . idative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol. 1996;264:484-509.

3. Taylor DJ, Turnbull DM. Organelle diseases. In: Applegarth DA, Dimmick JE, HaM JG, cds. Organelle Diseases: Clinical Features, Diagnosis, and Management. New York, NY: Chapman & Hall; 1997:341-350.

4. Munnich A, RotigA, Chretien D, Saudubray JM, Cormier V, Rustin P. Clinical presentations and laboratory investigations in respiratory chain deficiency. Eur J Pediatr. 1996;155:262-274.

5. Robinson B, ed. The Metabolic and Molecular Basis of Inherited Diseases. New York, NY: McGraw-Hill; 2001:2.

6. Sukhorukov VS, Nartsissov RP, Petrichuk SV, et al. Comparative diagnostic value of the analysis of the skeletal muscle and lymphocytes in mitochondrial diseases [in Russian]. Arkh Patol. 2000;62:19-21.

7. Kunz D, Luley C, Winkler K, Lins H, Kunz WS. Flow cytometric detection of mitochondrial dysfunction in subpopulations of human mononuclear cells. Anal Biochem. 1997;246:218-224.

8. Marriage BJ, Clandinin MT, MacDonald IM, Glerum DM. The use of lymphocytes to screen for oxidative phosphorylation disorders. Anal Biochem. 2003; 313:137-144.

9. Rustin P, Chretien D, Bourgeron T, et al. Biochemical and molecular investigations in respiratory chain deficiencies. CHn Chim Acta. 1994;228:35-51.

10. Zhang HX, Du GH, Zhang JT. Assay of mitochondrial functions by resazurin in vitro. Acta Pharmacol Sin. 2004;25:385-389.

11. Azbill RD, Mu X, Bruce-Keller AJ, Mattson MP, Springer JE. Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury. Brain Res. 1997;765:283-290.

12. White MJ, DiCaprio MJ, Greenberg DA. Assessment of neuronal viability with Alamar blue in cortical and granule cell cultures. J Neurosci Methods. 1996; 70:195-200.

13. Visser W, Scheffers WA, Batenburg-van der Vegte WH, van Dijken ]P. Oxygen requirements of yeasts. Appl Environ Microbiol. 1990;56:3785-3792.

14. Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M, DiMauro S. A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA. Science. 1989;244:346-349.

15. Bosley TM, Abu-Amero KK, Ozand PT. Mitochondrial DNA nucleotide changes in non-arteritic ischemie optic neuropathy. Neurology. 2004;63:1305-1308.

16. Birch-Machin MA, Turnbull DJ. Spectrophotometric measurement of the activities of individual complexes I-IV. In: Pon L, Schon E, eds. Mitochondria. VoI 65. San Diego, Calif: Academic Press; 2001:97-117.

17. Fromenty B, Fisch C, Berson A, Letteron P, Larrcy D, Pessayre D. Dual effect of amiodarone on mitochondrial respiration: initial protonophoric uncoupling effect fol lowed by inhibition of the respiratory chain at the levels of complex I and complex II. I Pharmacol Exp Ther. 1990;255:1377-1384.

18. Chretien D, Benit P, Chol M, et al. Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts. Biochem Biophys Res Commun. 2003;301:222-224.

Khaled K. Abu-Amero, PhD; Thomas M. Bosley, MD

Accepted for publication June 15, 2005.

From the Departments of Genetics (Dr Abu-Amero) and Neuroscience (Dr Bosley), King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Khaled K. Abu-Amero, PhD, Department of Genetics, King Faisal Specialist Hospital and Research Centre, PO Box 3354 (MBC 03), Riyadh 11211, Saudi Arabia (e-mail: kamero@kfshrc.edu.sa).

Copyright College of American Pathologists Oct 2005
Provided by ProQuest Information and Learning Company. All rights Reserved