Resumen y valoración de actividades para el desarrollo del PIGARS en

2 .1 . Chemicals

All chemicals, unless otherwise stated, w ere purchased from the Sigma Chemical Co, Poole, Dorset, UK or Merck Ltd, Dagenham, Essex, UK.

2 .2 . Equipment

2 .2 .1 . Centrifugation

The following centrifuges and rotors were used at the following g forces, unless otherwise stated:

up to 3 0 0 0 X 9^3^: Beckman GPR bench-top centrifuge w ith G H -3 .7 horizontal

rotor (Beckman Ltd, High W ycom be, Bucks, UK);

3 0 0 0 -4 7 0 0 0 X 9^3^: Kontron T -124 high-speed centrifuge w ith A 8 .2 4 8 x 50m l fixed angle rotor (Kontron Instruments Ltd, W atford, Herts, UK);

above 4 7 0 0 0 x g^^^: MSE superspeed 75 ultra centrifuge w ith 6 x 38m l swing-out rotor (MSE Instruments Ltd, Crawley, Sussex, UK).

2 .2 .2 . Column chromatography

The peristaltic pump (P I), absorbance control unit (U V -1 ), fraction collector (Frac-100) and chart recorder ( R e d ) w ere supplied by Pharmacia Ltd (Milton Keynes, Bucks, UK),

2 .2 .3 . Electrophoresis

All agarose and polyacrylamide gel electrophoresis w as performed w ith BioRad 2 0 0 /2 .0 constant voltage power packs (BioRad Laboratories Ltd, Hemel Hem pstead, Herts, UK);

Horizontal submarine electrophoresis tanks (for agarose gels) w ere supplied by Uniscience Ltd (Banbury, Oxon, UK);

Vertical electrophoresis tanks (Mini-Protean II system; for acrylamide gels) w ere supplied by BioRad Laboratories Ltd (Hemel Hem pstead, Herts UK);

The PhastSystem horizontal electrophoresis unit w as supplied by Pharmacia Ltd (M ilton Keynes, Bucks, UK).

2 .2 .4 . Tissue Homogenisation

Uni-form 10m l glass/teflon homogenisers w ere supplied by Jencons Ltd (Leighton Buzzard, Beds, UK);

The Glas-Col motor was supplied by CamLab Ltd (Cambridge, Cambs, UK); Dounce type glass/glass homogenisers were supplied by W heaton Ltd (Millville, NJ, USA);

The cell disruption bomb (45m l volume) w as supplied by Parr Instrument Co (Moline, Illinois, USA).

2 .2 .5 . Spectrophotometry

All enzyme assays w ere performed on Hitachi U -3 2 1 0 (Hitachi Scientific Instrum ents, Wokingham, Berks, UK) or Kontron Uvikon 9 4 0 (Kontron Instrum ents, W atford, Herts, UK) split-beam spectrophotom eters.

2 .3 . Patients

All patient studies were performed w ith the full and informed consent of the patients and w ith the approval of the Joint Ethics Com m ittee of the National Hospital for Neurology and Neurosurgery and the Institute of Neurology, Queen Square, London, UK.

The diagnosis of Parkinson's disease w as based on the presence of an akinetic rigid syndrome w ith asym m etric onset, a resting trem or and a positive response to L-dopa. Patients w ho w ere untreated at the tim e of sample w ere followed up to ensure th at they subsequently demonstrated a good response to L-dopa.

2 .4 . Data analysis

Statistical analyses were performed w ith O xstat (version 4 .1 0 , release 1 .1 0 ) softw are, and all significance tests w ere performed using the M ann-W hitney U test. Figures w ere drawn using Sigmaplot (version 4 .0 2 ) graphing system software.

2 .5 . Platelet preparation and subcellular fractionation

2 .5 .1 . Preparation of platelet homoaenates

The preparation of platelet homogenates w as performed at room tem perature to avoid platelet aggregation. 30m l of venous blood w as collected w ithout tourniquet via a 19-gauge butterfly into a plain syringe. The blood w as transferred to 25m l plastic universals and mixed 9:1 w ith 3 .8 % (w /v) tri-sodium citrate. The blood was centrifuged, to separate the platelet rich plasma (PRP) and red cells, at 2 0 0 x g for 20m ins. The PRP was carefully removed to clean

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universals and platelets pelleted by centrifugation at 1 0 0 0 x g^^^ for 30m ins. The platelet poor plasma w as poured off and the sides of the tube wiped clean to remove any serum proteins. The cell pellet w as gently resuspended w ith an equal volume (to the PRP) of a modified Tyrodes buffer (Appendix 1.1) and centrifuged at 1 0 0 0 x g^^^ for 1 5mins. The Tyrodes wash w as then repeated once more. The washed cell pellet w as resuspended w ith 7 5 0 //I of ice-cold homogenisation medium (Appendix 1 .2 ), split into tw o aliquots of 6 0 0 and

2 .5 .2 . Preparation of platelet mitochondriallv-enriched fraction

The preparation of the platelet pellet w as as in 2 .5 .1 except th at 60m l of venous blood w as required. The washed cell pellet w as resuspended w ith 6ml of ice-cold homogenisation medium (Appendix 1.2) and a 50/vl sub-sample removed for calculation of mitochondrial recovery. The cell suspension w as transferred to the cell disruption bomb which w as charged to 1200p si w ith oxygen free nitrogen gas (BOC Ltd, Guildford, Surrey, UK) and left on ice for 20m ins (Broekman e t a i 1 9 7 4 ). A fter this tim e the pressure w as released and the cell homogenate collected in a plastic universal. Any nuclei or unbroken cells w ere pelleted by centrifugation at 1 0 0 0 x g^^^ for 1 5mins at 4 ° C . The Post Nuclear Supernatant (PNS) w as transferred to a 30m l centrifuge tube on ice and the Nuclear Pellet (NP) w as resuspended w ith 6ml ice-cold homogenisation medium and re-homogenised in the cell disruption bomb. The second PNS w as pooled w ith the first, and the mitochondrially-enriched fraction (MEF) w as pelleted by centrifugation at 8 5 0 0 x g^^^ for lOm ins at 4 °C . The supernatant w as removed and the MEF w as resuspended w ith 600/yl homogenisation medium, split into tw o aliquots of 500 //I and 10 0//I, snap-frozen in liquid nitrogen and stored at -7 0 ° C .

2 .5 .3 . Electron microscopy

Electron microscopy w as performed by Dr Jenny Small, Departm ent of Neuroscience, Royal Free Hospital School of Medicine, London, UK. The platelet MEF from the equivalent of 30m l of whole blood w as gently resuspended w ith primary fixative (1 ml of PIPES (piperazine -N-N'-bis(2-ethanesulphonic acid)), 2 .5 % (v/v) glutaraldehyde, 2% (w /v) sucrose) and mixed on a whirly wheel overnight at 4 ° C . The MEF was pelleted at top speed in a microfuge for lO m ins, washed by gentle resuspension w ith PIPES buffer and re-pelleted. The pellet w as then post-fixed by resuspension w ith 1 ml of 1 % (w /v) osmium tetroxide and incubated at 4 ° C for 2 hrs, washed tw ice w ith 1 ml PIPES buffer and finally resuspended w ith 100/yl of PIPES buffer. This volume w as carefully pipetted onto W hatm ann No. 1 filter paper, to absorb the buffer, and the mitochondria w ere collected together w ith a small spatula. The pellet w as split into tw o, placed onto a 1 % agar plate, and then overlayed w ith a small drop of molten agar. Two small agar blocks containing the mitochondria w ere cut from the plate and placed in PIPES buffer. These blocks were dehydrated through a graded series of ethanol up to 1 0 0 % , immersed in propylene oxide and embedded in

araldite epoxy resin, in preparation for sectioning for electron microscopy. Ultra- thin sections, 90nm thick, w ere cut in a Reiche Ultracut microtome, placed onto 3 0 0 mesh copper grids, stained w ith 5 0 % methanolic uranyl acetate and aqueous lead citrate, and examined w ith a Jeol 1 0 0 C X transmission electron microscope.

2 .5 .4 . Blood cell counting

Samples of whole blood, platelet rich plasma, platelet poor plasma and Tyrodes w ash supernatants w ere counted on a Coulter Cell Counter (Coulter Electronics Ltd, Luton, Beds, UK).

2 .6 . Enzyme assays

All enzyme assays were performed at 3 0 ° C unless otherwise stated.

2 .6 .1 . NADH-CoQ^ oxidoreductase

The assay of NADH CoQ^ oxidoreductase w as used to measure the activity of complex I of the mitochondrial electron transport chain. The assay was performed according to the method of Ragan et a! (1 9 8 7 ). The assay measures the NADH dependent reduction of ubiquinone at 3 4 0 n m . The assay mixture consisted of identical cuvettes containing a final concentration of 2 0 m M potassium phosphate buffer pHS, 150/yM NAD H, Im M KCN and sample in 1 ml. The reaction w as initiated by the addition of 50//IVI CoQ^ (see Methods 2 .6 .1 .1 ) to the sample cuvette. The rate of NADH oxidation w as measured, then rotenone w as added to a final concentration of 1 0//M and the rotenone insensitive rate w as measured. The mitochondrial complex I activity w as taken as that which w as sensitive to rotenone. A molar extinction coefficient for NAD H of 6.81 X 10^ w as used to allow for the contribution of reduced CoQ^ to the absorbance at 3 40 n m (Ragan a t al, 1 9 8 7 ). The assay required approximately 1 50/yg of platelet homogenate and 100//g of platelet MEF. Enzyme activity was expressed as nmol NADH oxidised per minute per mg of protein.

The assay w as later modified to optimise activity in platelet mitochondrial fractions. The modifications were as follows; 2 0 m M potassium phosphate buffer p H 7.2; 8 m M MgCI^ w as included; 2.5m g /m l essentially fatty-acid free BSA w as included. In all other respects the assay w as performed as before, and required approximately 90/yg of platelet homogenate and 60/yg of platelet MEF.

2 .6 .1 .1 . Calculation of ubiauinone-1 concentration

The ubiquinone-1 (CoQ^) w as a kind gift of the Eisai Chemical Co, Tokyo, Japan. A dilution of the stock w as made in ethanol and the absorbance of ubiquinone-1 w as read at 2 75 n m . An excess of sodium borohydride w as added to the reference cuvette to com pletely reduce the ubiquinone to ubiquinol, and the absorbance change w as noted. The molar extinction coefficient of ubiquinone-1 w as taken as 1 2 .2 5 x 10^ (Redfearn, 1 9 6 7 ), and the volume of ubiquinone required to give a final cuvette concentration in the assay of 50/yM was calculated (usually approximately 10//I).

2 .6 .2 . NADH ferricvanide oxidoreductase

The assay of NADH ferricyanide reductase activity w as performed according to the method of King and Howard (1 9 6 7 ). The assay measures the oxidation of NADH at 3 4 0 n m , w ith potassium ferricyanide as electron acceptor. The assay mixture consisted of identical cuvettes containing final concentrations of 5 0 m M potassium phosphate buffer p H 7 .4 , 0 .1 % (v/v) Triton X -1 0 0 , 0 . 1 7 m M NADH and 0 .6 m M potassium ferricyanide in 1 ml. The reaction w as initiated by the addition of sample to the sample cuvette, and the change in absorbance at 3 40 n m was monitored. Enzyme activity w as expressed as nmol NADH oxidised per minute per mg of protein, calculated using a molar extinction coefficient for NADH of 6 .2 2 x 1 0 ^

2 .6 .3 . Succinate cytochrome c oxidoreductase

The assay of succinate cytochrome c oxidoreductase measures the activity of complexes II and III of the mitochondrial electron transport chain. The assay w as performed according to the method of King (1 9 6 7 ). The assay measures the succinate dependent reduction of cytochrome c at 5 5 0 n m . The assay mixture consisted of identical cuvettes containing final concentrations of 0.1 (VI potassium phosphate buffer p H 7 .4, O.SmM EDTA (di-K), 0.1 m M cytochrome c, Im M sodium azide, 2.5m g /m l essentially fa tty acid free BSA and sample in 1 ml. A mixture of sample plus 4 0//I of 5 0 0 m M sodium succinate (substrate) and 10//I of lO O m M sodium azide w as pre-incubated at 3 7 °C for 5mins to activate the enzyme. This mixture w as then used to initiate the reaction. The final concentration of sodium succinate in the sample cuvette w as 2 0 m M . The mitochondrial complex ll/lll activity w as taken as that which w as sensitive to the addition of antimycin A (final concentration of 0 .0 2 m M ). The assay required approxim ately 70/yg of platelet homogenate and 50/vg of platelet MEF. Enzyme activity w as expressed as nmol cytochrome c reduced per minute per mg of protein, calculated using a molar extinction coefficient for reduced cytochrome c of 1 9 .2 X 10%.

2 .6 .4 . Cytochrome c oxidase

The assay of cytochrome c oxidase measures the activity of complex IV of the mitochondrial electron transport chain. The assay w as performed according to the method of W harton and Tzagoloff (1 9 6 7 ). The assay measures the oxidation of reduced cytochrome c at 5 50n m . The assay mixture consisted of identical cuvettes containing a final concentration of 5 0 m M potassium phosphate buffer p H 7 .0 and bOjjW\ reduced cytochrome c in 1 ml. Im M potassium ferricyanide w as added to the reference cuvette to fully oxidise the reduced cytochrome c, and the reaction w as initiated by the addition of sample to the sample cuvette. The assay is first order w ith respect to cytochrome c, so the pseudo first order rate constant k w as calculated. Enzyme activity w as expressed as k per minute per mg of protein. The assay required approxim ately 3 0 0 /jq of platelet homogenate and 200/yg of platelet MEF.

2 .6 .4 .1 . Preparation of reduced cytochrome c

A 1 % (w /v) solution of cytochrome c (from horse heart; Boehringer Mannheim Ltd, Lewes, Sussex, UK) w as fully reduced by the addition of an excess of ascorbate. The reduced cytochrome c solution w as transferred to size 1 dialysis tubing (Medicell International Ltd, London, UK) which had been boiled in ddH^O for 3hrs. It w as subsequently dialysed against three changes of 51 of lO m M potassium phosphate buffer p H 7 .0 at 4 ° C , over at least 3 6 hrs, to remove excess ascorbate, and stored in aliquots at -2 0 ° C . To check th at no excess ascorbate remained it w as shown that oxidised cytochrom e c added to the dialysed reduced sample w as not reduced, and to check th at the cytochrom e c w as fully reduced, it w as shown that the addition of more ascorbate failed to reduce the cytochrome c further.

2 .6 .5 . Citrate synthase

The assay of citrate synthase, an enzyme of the mitochondrial m atrix, w as performed according to the method of Coore e t a / (1 9 7 1 ). The enzyme catalyses the condensation of acetyl-CoA and oxaloacetate to form citrate. This reaction produces CoenzymeA whose free thiol group combines w ith DTNB (5-5'-dith io- bis(2-nitrobenzoic acid), resulting in an increase in absorbance at 4 1 2nm . The assay mixture consisted of identical cuvettes containing a final concentration of lO O m M Tris pHS.O, 200/vM acetyl-CoA, 200/yM DTNB, 0 .1 % (v/v) Triton X -1 0 0 and sample in 1 ml. The reaction was initiated by the addition of 1 0 0 //M oxaloacetate to the sample cuvette. The assay required approximately 15^g of platelet homogenate and 10//g of platelet MEF. Enzyme activity w as expressed as nmol DTNB reduced per minute per mg of protein, and w as calculated using a molar extinction coefficient of 1 3 .6 x 10^ for the DTNB-CoA-SH complex.

2 .6 .6 . Lactate dehydrogenase

Lactate dehydrogenase activity, a specific marker of the cytosol, w as measured according to the method of Clark and Nicklas (1 9 7 0 ). This enzyme catalyses the reduction of pyruvate to lactate and the assay measures the oxidation of NADH at 3 40 n m in the presence of pyruvate. The assay mixture consisted of identical cuvettes containing a final concentration of lO O m M potassium phosphate buffer

p H 7 .4 , 0 .2 4 m M NAD H, 0 .5 % (v/v) Triton X -1 0 0 and sample in 1ml. The reaction w as initiated by the addition of Im M sodium pyruvate to the sample cuvette. Enzyme activity w as expressed as nmol NADH oxidised per minute per mg of protein, and w as calculated using a molar extinction coefficient for NADH of 6 .2 2 X 10%.

2 .6 .7 . Esterase

Esterase activity w as measured as a specific endoplasmic reticulum marker, according to the method of Beaufay e t a! (1 9 7 4 ). The assay measures the production of o-nitrophenol from o-nitrophenyl acetate, at 4 2 0 n m . The assay mixture consisted of identical cuvettes containing a final concentration of 2 0 m M potassium phosphate buffer p H 7 .4, Im M EDTA (di-K), 0 .1 % (v/v) Triton X -1 0 0 and 3 m M o-nitrophenyl acetate in 1 ml. The reaction w as initiated by the addition of sample to the test cuvette. Enzyme activity w as expressed as nmol o-nitrophenol produced per minute per mg of protein, using a molar extinction coefficient for o-nitrophenol of 3 .0 6 x 10^.

2 .6 .8 . Acid Phosphatase

Acid phosphatase activity was measured as a specific lysosomal marker, according to the method of Hubscher and W est (1 9 6 5 ) w ith m odifications, at 3 7 °C. Identical samples were pre-incubated in a volume of 625/yl in a final concentration of 9 0 m M sodium acetate buffer p H 5 .0 , I.S m M EDTA (di-Na), 0 .2 % (v/v) Triton X -1 0 0 and 2 0 m M sodium fluoride (blanks only). The reaction mixtures were incubated at 3 7 °C for 10mins to ensure fluoride inhibition of the lysosomal enzyme in the blank assays. The reaction w as initiated by the addition of 50/yl of 10m M 1 -napthyl phosphate to both test and blank samples which w ere incubated at 3 7 °C for 2hrs. The reaction w as term inated by the addition of 400/vl of 5 3 m M fast red ITR dye (in 2 0 % (v/v) dimethyl formamide) and 3ml stop mix (3 5 % (v/v) ethanol, 6 0 % (v/v) ethyl acetate, 0 .5 % (w /v) trichloracetic acid). The samples w ere centrifuged at 1 5 0 0 x g^^^ for 20m ins in an MSE Centaur 3 centrifuge, and their absorbances read at 5 40 n m against a no-enzyme blank reference. Blank absorbances w ere subtracted from test. Enzyme activity w as expressed as nmol diazo-dye complex formed per minute per mg of protein, using a molar extinction coefficient for the diazo-dye complex of 1 2 .5 3 8 x 10^.

2 .6 .9 . R-thromboalobulin

(S-thromboglobulin, a specific marker of platelet alpha granules (Kaplan e t a!, 1 9 7 9 ), w as measured using a com mercially available enzyme-linked immunosorbent assay (ELISA) kit (Diagnostica Stago, Asnieres sur Seine, France) by Dr Ron Hutton, Departm ent of Haematology, Royal Free Hospital School of Medicine, London, UK.

2 .6 .1 0 . Serotonin

Serotonin, a specific marker of platelet dense granules (Tranzer e t a!, 1 9 6 6 ), w as measured using a commercially available ELISA kit (Immunotech International, Marseille, France) by Dr Manuel Baradas, Royal Free Hospital School of Medicine, London, UK.

2 .7 . Affinity purification of complex I antibodies

2 .7 .1 . Production of antisera

All procedures w ere performed by the staff of the CBU, Royal Free Hospital, London, UK. The initial immunisation of adult fem ale N ew Zealand half-lop rabbits, w as by injection at 4 sub-cutaneous sites of 1.5m g purified native bovine heart complex I holoenzyme (kindly provided by Dr JM Cooper, Royal Free Hospital School of Medicine, London, UK) mixed 1 :2 .5 w ith Freund's com plete adjuvant in a final volume of 1 ml. Booster immunisations at approxim ately 4 w eek intervals were performed as above except that 0.5m g antigen w as injected in Freund's incomplete adjuvant. Up to 20m l of blood was removed at 4 w eek intervals from the ear vein and animals w ere killed by cardiac exsanguination under deep terminal anaesthesia. Blood w as collected in glass universals, incubated at 3 7 °C for 1 hr and then at 4 ° C overnight. The blood clot w as compressed by centrifugation at 3 0 0 0 x g^^^ for 15m ins and the serum removed. Any contaminating red blood cells w ere removed by centrifugation as above. Serum w as stored in aliquots at -2 0 ° C , and current stock w as maintained at 4 ° C in the presence of 0 .0 5 % (w /v) sodium azide.

2 .7 .2 . Preparation of complex I affinity column

Complex I w as covalently coupled to the gel matrix according to the method of