CONSIDERACIONES PARA DISEÑO DEL SISTEMA DE CAPTACIÒN

3 .1 . Aim

To compare activities of mitochondrial respiratory chain (MRC) enzymes in platelet homogenates of Parkinson's disease (PD) patients and controls.

3 .2 . Introduction

In 1 9 8 9 , Parker e t a! published data demonstrating a 5 5 % reduction in complex I activity of platelet mitochondria in patients w ith idiopathic Parkinson's disease compared to normal controls. The method of mitochondrial preparation involved the collection of platelets by plateletphoresis, homogenisation by nitrogen cavitation and preparation of mitochondrial fractions on density gradients. The whole procedure w as time consuming, taking up to tw o days (DiM onte, 1 9 9 1 ), and required specialised equipment for platelet collection, homogenisation and fractionation.

If confirm ed, this biochemical abnormality may be useful as a diagnostic test for PD, and further may be useful in identifying presymptomatic or at-risk cases. This would be important in terms of treatm ent since it has been suggested that the monoamine oxidase B inhibitor selegiline may retard the progression of the disease (Parkinson Study Group, 1 9 9 3 ). A routine diagnostic test has to be both quick and simple, and for this reason the possibility of detecting the complex I d efect in whole cell platelet homogenates w as investigated. The method only required 30m l of blood and the platelets w ere prepared in less than tw o hours. Since samples w ere stored frozen at -7 0 ° C prior to assay, the e ffe c t of storage tim e on complex I activity was determined. Complex I activity w as also compared in platelet homogenate samples after different membrane disruption techniques to maximise its activity. Mitochondrial respiratory chain enzyme activities w ere compared in platelet homogenates from 14 PD patients and 15 controls.

3 .3 . Results

3 .3 .1 . Cell counts through the platelet preparation

To determine the purity of platelets prepared from w hole blood and to quantify platelet recoveries, the number of blood cells in various samples throughout the platelet preparation w ere counted using a Coulter cell counter (for details of the platelet preparation see Methods 2 .5 .1 ). Nearly 9 0 % of the platelets in whole blood w ere recovered in the platelet rich plasma (PRP), and 9 5 % of these w ere pelleted at the first centrifugation. The Tyrodes buffer wash retained greater than 9 5 % of cells in the pellet. This data provides evidence th at the preparation achieved good recovery of highly pure platelets from whole blood; the platelets w ere greater than 9 9 .9 % pure and the total recovery from whole blood w as approxim ately 8 0 % .

3 .3 .2 . The effect of sample storage on complex I activity

Platelet homogenate samples from three normal controls w ere split into tw o aliquots. Complex I activity was measured on the day of preparation in one aliquot and 21 days later in the other. The mean specific activity of complex I in the samples assayed on the day of preparation w as 5 .3 4 ± 0 . 6 1 nmol/min/mg protein (m e a n ± S D ) and in the stored samples w as 5 . 2 6 ± 0 . 8 9 . This difference of less than 2 % w as well within the limits of assay variation. As complex I activity in platelet homogenates w as not affected by storage at -7 0 ° C for up to 21 days, all samples w ere subsequently assayed within 21 days of preparation.

3 .3 .3 . MRC function in PD and control platelet homogenates

There were no significant differences betw een the mean activities of complexes I, ll/lll, IV or citrate synthase (CS) in the PD patient (n = 14) and control (n = 1 5) groups (Table 3-1; Figure 3-1). To correct for any differences in mitochondrial numbers in whole tissue homogenates, all enzyme activities w ere divided by CS activity. This mitochondrial matrix enzyme is used as an indicator of mitochondrial numbers since its activity is not known to be affected in any disease state; the CS ratio of an enzym e's activity essentially represents that

enzymes activity per mitochondrial unit. There w ere no significant differences in the CS ratios of any of the MRC enzymes betw een the patient and control groups (Table 3-1; Figure 3 -2 ).

3 .3 .3 .1 . PD patients

Fourteen PD patients w ere included in the study. The mean age w as 59.1 ± 1 0 .2 years (range 4 5 -7 4 years). The mean age at onset of disease w as 48.1 years (range 3 2 -6 3 years) and the mean duration of disease w as 1 0 .9 years (range 2 -2 2 years). Complex I activity did not significantly correlate w ith age at disease onset, but just reached statistical significance (p = 0 .0 5 ) when correlated w ith disease duration (Figure 3-3 ).

Two patients w ere untreated and had not taken any L-dopa at the tim e of sample. The specific activity of complex I in these tw o patients w as 3 .4 4 and 4 .9 9 nmol/min/mg protein, as compared to a group mean of 3.51 ± 0 .7 9 nm ol/min/mg protein (mean ± SD).

3 .3 .3 .2 . Controls

Fifteen controls w ere included in the study. The mean age w as 5 8 .3 ± 9 .9 years (range 4 8 -7 7 ). Nine of the controls w ere normal, healthy individuals and six w ere disease controls who showed no evidence of primary neurodegenerative disease. This group included patients w ith multiple sclerosis (1), Guillan-Barre syndrome (1), carcinoma (3) and spastic paraparesis due to cord compression (1). Since there w as no significant difference in complex I specific activity (3 .5 3 ± 0 .7 2 and 3 .6 5 ± 1.11 nm ol/min/mg protein; p > 0 .1 ) or the CS corrected ratio of complex I activity (xlOO; 4 .2 0 ± 1 .0 3 and 4 .2 1 ± 1 .3 2 ; p > 0 .1 ) betw een the normal control and disease control groups, they were pooled to form one group.

3 .3 .3 .3 . Complex I activity and age

Several studies have demonstrated that mitochondrial respiratory chain function decreases w ith age (see Introduction 1 .4 .5 ), and therefore patient and control groups w ere carefully age-matched in this study. Complex I activity showed no significant correlation w ith age in platelet homogenate samples of PD patients or controls (Figure 3-4 ).

3 .3 .4 . MRC function in LHON patients platelet homogenates

Mitochondrial enzyme activities in platelet homogenates from three patients w ith Leber's hereditary optic neuropathy (LHON) w ere compared to controls. Two patients w ith the N D 4 gene mutation had complex I activities 8 9 % and 1 0 1 % of the control mean; one patient w ith the ND1 gene mutation had a complex I activity 3 9 % of the control mean (Table 3-2 ).

Specific Activity

complex 1 complex ll/lll complex IV CS

PD 3 .51 ± 0 . 7 9 9 . 9 6 ± 2 . 0 0 0 . 5 0 ± 0 . 1 3 8 8 . 1 3 ± 1 6 . 9 0 C 3 . 5 8 ± 0 . 8 6 9 . 2 2 ± 2 . 3 0 0 . 4 4 ± 0 . 1 4 8 6 . 1 7 ± 1 2 . 5 8

CS corrected ratios

complex 1 complex ll/lll complex IV

(XlOO) (x IO ) (XlOO)

PD 4.1 6 ± 1 . 3 2 1 . 1 4 ± 0 . 1 5 0 . 5 6 4 ± 0 . 1 0 8 C 4 .2 1 ± 1 .1 1 1 .0 8 ± 0 . 2 7 0 . 5 2 3 ± 0 . 1 6 8

Table 3 -1 . Mitochondrial enzyme activities in PD and control platelet homogenates.

Activities expressed as nmol/min/mg protein, except com plex IV (k/min/mg protein). All activities mean ± SD.

I/CS (XlOO)

Controls (n = 1 5) 4.2 1 ± 1.11

Patient 1 (ND4) 3 .7 3

Patient 2 (ND4) 4 .2 5

Patient 3 (ND1 ) 1 .6 3

Table 3 -2 . Citrate synthase corrected complex I activity in platelet homogenates of LHON patients.

I/CS (citrate synthase corrected complex I activity); N D 4/N D 1 (patients harbouring LHON-associated point mutations in the N D 4 (n t1 1 7 7 8 ) / ND1 (n t3 4 6 0 ) complex I genes).

S p e c ific