Respiratory-chain enzyme activities in isolated mitochondria of lymphocytes from patients with Parkinson's disease: Preliminary study
Background: Evidence suggests that mitochondrial dysfunction stimulates the production of reactive oxygen species (ROS) that promote neural cell death in stroke and in Parkinson's disease. The sites of mitochondrial ROS production are not established but are generally believed to be located within the electron transport chain. Aims: We studied the mitochondrial respiratory chain enzymes function from human circulating lymphocytes. Setting and Design: Open study. Materials and Methods: Forty patients with Parkinson's disease (PD) with 30 age-matched control subjects were selected in this study. The patients had received no treatment before the study was conducted. Statistical Analysis: The data from patients and controls were compared using two-tailed student's t-test and values were expressed as means ± standard deviation (SD). Results: Respiratory complex I + III and IV activities were significantly lower (P <0.001) in patients than in control subjects. Conclusions: The use of lymphocytes for investigating the respiratory chain enzymes provides an easy, noninvasive method to assess mitochondrial function in patients with PD. Furthermore, our study supports the hypothesis that a biochemical defect in the respiratory chain may be involved in the pathogenesis of PD.
Keywords: Mitochondrial respiratory chain, oxidative phosphorylation, Parkinson′s disease
Parkinson's disease (PD) may be one of the most baffling and complex of the neurological disorders. Its cause remains a mystery but research in this area is active, with new and intriguing findings constantly being reported. Research into the etiology of PD has focused mainly on toxins that inhibit mitochondrial respiration. A definitive neuropathological diagnosis of PD requires loss of dopaminergic neurons in the substantia nigra and related brainstem nuclei and the presence of Lewy bodies in the remaining nerve cells. The contribution of genetic factors to the pathogenesis of PD is increasingly being recognized. a-synuclein ( SNCA ) was the first gene linked to PD, the protein being thought to play an essential role in synaptic transmission. An alanine to threonine conversion at amino acid 53 (A53T) was identified in a large Greek/Italian kindred with early-onset familial PD. In addition, alanine to proline (A30P) and glutamic acid to lysine (E46K) alterations in -synuclein have been identified in rare familial PD kindreds. Disease specificity of this defect has been demonstrated for the parkinsonian substantia nigra. Administration of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in both human and nonhuman primates. MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase B (EC 188.8.131.52) in glia; MPP+ is taken up into dopaminergic neurons by the dopamine reuptake pump. The finding that MPP+ inhibits mitochondrial complex I suggests that an energy failure resulting from inhibition of the mitochondrial respiratory chain is the most likely mechanism of neuronal death in MPTP-induced Parkinsonism More Details.
The mitochondrial respiratory chain consists of five enzymatic complexes located within the inner mitochondrial membrane. Four enzymes (complexes I-IV) transport electrons from NADH or succinate to oxygen and these complexes pump protons into the intermembrane space mitochondria to form an electrochemical gradient. The fifth enzyme (complex V) uses that electrochemical gradient to synthesize ATP from ADP. Decreased complex I activity has been reported in platelets and skeletal muscle, from patients with PD. Likewise, there have been reports of combined defects of the respiratory chain complexes.,,
In the present study, we assessed respiratory chain enzymes in lymphocytes from patients with PD.
Sodium salt of succinic acid, ADP, ATP, TritonX-100, BSA, Acetyl-CoA, oxaloacetic acid, dichlorophenolindophenol (DCIP), phenazine methosulphate (PMS), NADH, NAD were purchased from Sigma chemical Co, St. Louis. MO, U.S.A. All others chemicals used were of analytical reagent grade and obtained locally.
Patient and control subjects
There were age-matched 30 control subjects, who were apparently normal individuals free from neurological disorders. Patients with PD (n = 40 ) were selected on the basis of classic PD manifestations between January 2004 and December 2004. All patients were under antiparkinsonian-medication of levodopa. The staff and Research Society, ethics committee gave approval.
Inclusion and exclusion criteria
Rigidity, a slowing of physical movement, resting tremor and gait impairment present in all patients was included in this study. Subjects were excluded from the study if hemoglobin levels were below the normal range. In the case of hemoglobin, values lower than 12 g/dl for males and 11 g/dl for females were excluded. Other exclusion criteria included any serious concomitant disease, including diabetes mellitus and significant gastrointestinal disease and ingestion of vitamin supplements.
Isolation of lymphocytes
Add 15-20 ml of heparinized blood to a 50 ml polypropylene conical tube. Add 15-20 ml 1 x PBS to the blood. Place an unplugged paseur pipette into the tube. Underlay with 10-15 ml isolymph using a syringe with a 22.5-guage needle (Note: place the needle beyond the bore of the pipette or you may get an air pocket that will prevent the flow of the isolymph; this may be remedied by sucking, lifting the pipette slightly) centrifuged at 300 x g for 30 min. with the brakes off. Remove the plasma layer to approximately 1 cm above the WBC interface. Using a circular method, starting form the outer part and moving towards the middle, harvest the WBC layer into a 50 ml polypropylene conical with a 10-25 ml-seriological pipette. (You should be able to look down the tube before and after to see the white layer removed.) When you are working with more than 10-15 ml of blood, you should only collect two WBC layers per 50 ml polypropylene tube before RBC lysis, this should help reduce clumping and is very important when working with buffers. Bring the volume to 45 ml with 1 x PBS. Harvest pellet centrifuged at 300 x g for 10 min. After removing the supernatant, resuspend the pellet gently by vortexing or running the bottom top the tube along a rack (it is important to resuspend the pellet to reduce clumping during RBC lysis). Add 30-40 ml ammonium chloride lysis buffers to the resuspended pellet and incubate in a 37°C waterbath for 5 min. Harvest the pellet at 1200 RPM for 5 min. Resuspend the pellet as above and combine the pellets into 45-50 ml 1 x PBS to wash at 1200 rpm for 7 min. Before harvesting, remove any clumping with a plugged Pasteur pipette; it usually sticks well to glass. Resuspend the pellet as above in 1 x PBS. Resuspend in volume to give approximately 1 x 10 6sub cells/ml.
Respiratory chain enzyme assay
Fresh lymphocyte pellets were homogenized by sonication in 20 mmol/L potassium phosphate buffer (pH 7.5) for 15s (three bursts of 5s each) at 30W on ice. The homogenate, containing 2-5 g/L protein, was kept on ice 4oC for more than one day or frozen at -20oC for any period of time lose respiratory chain enzyme activities at these protein concentrations.
Succinate cytochrome c reductase (EC 184.108.40.206, complexes II and III) was measured by monitoring the reduction of cytochrome c at 550 nm in the presence of succinate and enzyme (40-50 mL of lymphocyte homogenate). Assays for rotenone-sensitive NADH cytochrome c reductase (EC 220.127.116.11, Complexes I and III) were measured by monitoring the reduction of cytochrome c at 550 nm in the presence of NADH, rotenone and the enzyme (40-50 mL of lymphocyte homogenate). The rotenone-resistant activity was subtracted from the total NADH cytochrome c reductase activity to yield the activity of the rotenone-sensitive cytochrome c reductase.
Citrate synthase (EC 18.104.22.168) was measured by monitoring the change in absorbance at 412 nm caused by the reaction of 5,5'-dithiobis (2-nitrobenzoic acid) with the free coenzyme A formed by the condensation of acetyl-CoA with oxalacetate in the presence of the enzyme (5-10 mL of lymphocyte homogenate).
Succinate dehydrogenase (EC 22.214.171.124, complex II) was measured at 600 nm by monitoring the oxidation of succinate in the presence of the artificial electron acceptor, 2,6-dichiorophenol-indophenol and the enzyme (40-60 mL of lymphocyte homogenate).
Cytochrome c oxidase (EC 126.96.36.199, complex IV) was also determined spectrophotometrically by the decrease in absorbance at 550 nm of reduced cytochrome c in presence of the enzyme (30-50 mL of lymphocyte homogenate). Reduced cytochrome c was freshly prepared before each experiment by adding a few grains of sodium borohydride to a 10 g/L solution of the pigment in 10 mmol/L potassium phosphate buffer (pH 7.0). Addition of 0.1 mol/L HC1 stabilized the reduced cytochrome c and the excess borohydride was removed by centrifugation at 12000 x g for 4min. Incubation temperatures were 30oC for complexes I and III, II and III and II, IV and citrate synthase .
Protein content was measured by the method of Lowry et al with the use of human serum albumin as a standard.
Activities or respiratory chain complexes were reported as nanomoles of substrate per minute per milligram of protein. The ratio of complexes I and III to ll and III was used to estimate defects of complex I.
The data from patients and controls were compared using two-tailed student's t-test and values were expressed as means ± standard deviation (SD). Sigma stat version 3.0 was used for statistical analysis. P value of less than 0.05 was considered to indicate statistical significance.
The age ranges of patients with PD (52.2 ± 10.5 years) and control subjects (53.7 ± 12.2) were similar. The influence of aging on the results of the enzyme activities was ruled out. [Table - 1] summarizes the data for all five enzyme activities in lymphocytes from patients and control subjects. The activities of rotenone-sensitive NADH cytochrome c reductase (complexes I and III) and cytochrome c oxidase (complex IV) were significantly lower in patients than in control subjects (P <0.001) and the ratio of l and III to ll and III complexes (complex I) was significantly low ( P =0.014) in the patients group than in control subjects. However, the rest of the enzyme activities did not differ significantly between both groups.
Our results show that the activities of the respiratory chain complexes I and IV were significantly reduced in lymphocytes from patients with PD compared with age-matched control subjects. In addition, the specific activity of citrate synthase was similar in patients and control subjects, suggesting that equivalent amounts of mitochondria were present in the lymphocytes of both groups. Parker et al found a decrease of complex I activity (45% of mean in control subjects) in the platelets of PD patients, indicating that the complex I defect was present in an apparently unaffected tissue. Similarly, Shoffner et al and Bindoff et al , using skeletal muscle from patients with PD, reported low activities in complexes I and IV and variable findings in complexes II and Ill. Schapira et al and Mizuno et al found a marked reduction in complex I activity in substantia nigra obtained from PD patients postmortem. The mechanisms causing these defects and whether the defects are primary or secondary, remain unknown. The lack of a family history of PD in most cases of this disease seems to rule out a primary genetic defect involving a nuclear gene. It is possible that respiratory chain defects are secondary to the effects of environmental toxins that inhibit the respiratory chain and lead to increased free radical production.,, Such inhibition could ultimately lead to nonselective damage of respiratory chain components, and even to damage of mitochondrial DNA. Others reported functional defects of the respiratory chain are in platelets, muscle and brain of PD patients. Here, we report that such defects also occur in lymphocytes of patients with PD. These results, together with the MPTP hypothesis, support the view that a biochemical defect in the respiratory chain may be involved in the pathogenesis of PD. Our results also indicate that lymphocyte analysis provides an easy, noninvasive method for investigating respiratory chain enzymes and assessing mitochondrial function in patients with PD. In contrast to our results, Yoshino et al recently showed normal Complex I and Complex IV activities and a slightly decreased activity of Complex II in lymphocytes from PD patients. In addition, Mann et al reported normal platelet and skeletal muscle Complex I activities in PD. Thus, further work is needed at the molecular level to determine reasons for these discrepancies.
Strengths and limitations of the study and future research directions
This study suggests a relatively new approach to patients with PD. Mitochondrial respiratory chain enzymes are poorly studied in these patients. These enzymes can be used as measures of MRC associated with PD and can be beneficial to patient care, when controlled. But, as the number of samples is less in our study, extensive studies with larger number of samples are needed to confirm the exact role of these enzymes.
[Table - 1]