152
Biochimica et Biophysica Acta, 1097 (1991) 152-160 © 1991 Elsevier Science Publishers B.V. All rights reserved 0925-4439/91/$03.50 ADONIS 092544399100110B
BBADIS 61068
The neuromelanin of the human substantia nigra Ragnar Carstam 1, Carita Brinck 2, Annika Hindemith-Augustsson 3, Hans Rorsman l and Evald Rosengren 3 Department of Dermatology, University of Lund, Lund (Sweden), 2 Department of Ecology, Unit~ersityofLund, Lund (Sweden) and "~Department of Pharmacology, University ofLund, Lund (Sweden) (Received 7 January 1991)
Key words: Cysteinyldopamine; C'ysteinyldopa; Dopamine; Dopa; Parkinson's disease; (Aging)
The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with K M N O 4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyidopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.
Introduction
The nature of the dark brown pigment in the substantia nigra is still unknown. The pigment has been assumed to be an oxidation product of catechol derivatives and thus to be similar to the cutaneous melanins [1-4]. The melanin of the epidermis and the hair has dopa as a common precursor and is traditionally divided into two main classes: eumelanin which is black, insoluble and composed mainly of indole monomers; and pheomelanin which is brown, alkali-soluble and formed from oxidized cysteinyldopa products [5,6]. However, most of the melanins of the skin are co-polymers of the two main classes [7,8]. In the skin the formation of melanin is catalyzed by tyrosinase, which is a bi-functional enzyme oxygenating tyrosine to dopa and oxidizing dopa to dopaquinone [9], but the enzyme does not seem to be present in the substantia nigra [10]. Large amounts of dopa are formed in this brain
Abbreviations: AHPEA, 4-amino-3-hydroxyphenylethylamine; AHP, 4-amino-3-hydroxyphenylalanine; AHPAc, 4-amino-3-bydroxyphenylacetic acid; PTCA, pyrrole-2,3,5-tricarboxylic acid. Correspondence: H. Rorsman, Department of Dermatology, University of Lund, Lasarettet, S-221 85, Lund, Sweden.
nucleus by another enzyme, tyrosine hydroxylase, but the dopa produced is rapidly decarboxylated to dopamine. The quantities of dopa are consequently low, but large amounts of dopamine are present in the perikarya of the neurons of the pars compacta and the pars reticulata with the axons running to the nuclei of the corpus striatum [11]. It is known that the pigment of the substantia nigra contains much sulphur [12] and cysteine-containing catechol derivatives in the brain indicate that neuromelanin may contain oxidation products of catechols such as cysteinyldopamine [13,14]. Hydrolysis techniques make it possible to identify different types of melanin formed from catechol derivatives [15,16]. We have used such techniques for the investigation of the neuromelanin from the substantia nigra. We have synthetized 4-amino-3-hydroxyphenyletylamine (AHPEA) which is the product of reductive hydrolysis of cysteinyldopamine-melanin and also applied techniques for identification of pyrrole2,3,5-tricarboxylic acid (PTCA), the compound obtained from dopa- and dopamine-melanin at oxidative degradation. Certain characteristics of synthetic melanins formed from dopamine in the presence of cysteine will also be described. This investigation was in part presented at a workshop at the XIVth International Pigment Cell Conference in Kobe, on November 3rd 1990.
153 Materials and Methods
Materials The chemicals used were 3-hydroxytyramine (Sigma), cysteine (Merck), mushroom tyrosinase (3430 units/mg solid) (Sigma), catalase (11000 unit/mg protein) (Sigma), Catroniazid (Pharma-Stern, Hamburg, F.R.G.), H3PO 2 and HI (BDH Chemicals, England), 5-hydroxyindole-2-carboxylic acid (Aldrich Chemical, F.R.G.), u-3,4-dihydroxyphenyl-[3-14C]-alanine (10 tzCi) (Amersham, England). 5-S-Cysteinyldopa was prepared according to Agrup et al. [17], 4-amino-3-hydroxyphenylacetic acid (AHPAc) was prepared according to Karg et al. [18]. All other chemicals were of analytical grade.
Chromatography Condition A. In the preparation of AHPEA we used semipreparative chromatography. We employed an LKB pump (LKB Bromma, Sweden) with a UV-detector Varichrom (Varian, CA, U.S.A.) set at 275 nm. A Bondapak column, C~s (Waters Associates, MA, U.S.A.), 5 /x, 300 × 7.8 mm i.d. was used. Mobile phase: 40 mg sodium octanesulphonic acid, 11 ml H3PO4/1 water (pH 1.75). The flow rate was 1.2 ml/min. Condition B1. The tissue contents of AHPEA were determined by high-performance liquid chromatography (HPLC) with UV-detection or with electrochemical detection. An LKB pump was employed with a UV-detector set at 275 nm. A Nucleosil column, C ls (Machery, Nagel & Co., Duren, S.F.R.), 5 ~, 250 x 5 mm i.d. was used. Mobile phase: 0.15% trifluoracetic acid. The flow rate was 1.2 ml/min. Condition B2. An LKB pump was employed with an electrochemical detector set at +0.67 mV vs. an Ag/AgC1 reference electrode. A Supelcosil column, OH
OH
H2NI
H2N/
Benzothiazolyl unit
AHPEA
KMn04
HO dihydroxyindole unit
H00C
HOOC
COOH
tt PTCA
Fig. 1. Degradation products from melanin containing cysteinyldopamine-derived benzothiazolyl units and dopamine-derived indole units.
LC 18 DB (Supelco, Bellafonte, PA, U.S.A.), 3 Ix, 150 x 4 . 6 mm i.d. was used. Mobile phase: 30 mg sodium octanesulphonic acid/l water, pH adjusted to 2.25 with H3PO 4. The flow rate was 1.2 ml/min. Condition C. Identification of AHPEA: the effluent from the HPLC column was introduced into a VG Trio 3 mass quadropole mass spectrometer. A Nucleosil column, SA 5 Iz 150 × 4.6 mm i.d. was used. Mobile phase: 5% MeOH in water, 0.3 M ammonium acetate (pH 5.5). The flow rate was 1 ml/min. Postcolumn addition of ammonium acetate buffer was employed to give a final buffer concentration of 0.3 M. The vaporizer capillary had a temperature of 210°C. A standard VG 11/253 data system configuration was employed to aquire mass spectral data. Condition D. The contents of PTCA were determined by HPLC. An LKB pump was employed with a UV-detector set at 270 nm. A Supelcosil column, LC 18 DB, 3 Iz, 150 × 4.6 mm i.d. was used. Mobile phase: 0.1 M KI-I2PO4,20% Methanol (pH 2.1). The flow rate was 1.2 ml/min.
HI-hydrolysis Reductive degradation in HI was performed according to Ito [16].
Potassium permanganate oxidation Oxidative degradation in potassium permanganate was performed according to Ito [16].
Preparation of 4-amino-3-hydroxyphenylethylamine (AHPEA) AHPEA was obtained by HI-hydrolysis of a melanin produced by enzymatic oxidation of dopamine in the presence of cysteine in the ratio 1.0/1.5, according to Ito [19]. 100/zg of the insoluble melanin was degraded by HI-hydrolysis and the resulting solution was put onto a semipreparative HPLC column (condition A). The elution was monitored by a UV-detector and the material having an UV maximum peak at 275 nm was collected [20]. A sample of the compound with the absorbance maximum at 275 nm was injected into a mass spectrometer by direct inlet and CI ionization (ammonia). The spectrum showed one major peak which correponded to (M + H) + m / z 153 for 4-amino3-hydroxyphenylethylamine. When the material from the semipreparative column corresponding to the peak was analyzed by LC-MS, in addition to a large peak at m / z 153 another one at m / z 149 was observed, probably representing the indole derivative of the original substance. Another sample of the compound isolated was treated with a monoamineoxidase preparation from kidney cortex of the guinea pig. Both kidneys from one animal were homogenized in 20 ml 0.5 M KH2PO 4 buffer (pH 7.4) with a Polytron homogenizer (Kine-
154 matics PT 10-35) setting 7 for 15 s. The suspension was centrifuged for 60 rain at 100000 ×g. The pellet was suspended in 5 ml of the phosphate buffer. 0.5 ml of the suspension containing high activity of monoamineoxidase was incubated in 1 ml 0.5 M KH2PO 4 buffer (pH 7.5) together with 3-4 /~g of the substance with UV-maximum at 275 nm obtained from semipreparative HPLC chromatography. The reaction was followed by HPLC (condition B2). During the incubation the peak corresponding to AHPEA gradually decreased and a peak corresponding to AHPAc [18] appeared. In a separate experiment the MAO inhibitor Catroniazid was added to a similar incubation mixture and the reaction was followed. In this case the AHPEA peak had a constant height and no peak could be observed at the place of AHPAc in the chromatogram.
TABLE 1
Analytical data of synthetic melanin formed from dopamine Melanin was formed by incubation of dopamine with tyrosinase at different cysteine concentrations and in the presence of catalase. The molar ratio of cysteine (C) and dopamine (DA) in the incubate and the ratio of sulphur to nitrogen in the formed insoluble melanin are given. The values for products obtained after HI-hydrolysis (AHPEA, DA) and after degradation by KMNO 4 (PTCA), are also given. 1 mg melanin was used for analysis. Data represent mean values-+ S.D. for 10 incubations. Values for PTCA, DA and AHPEA are in p~g. C/DA
S/N
PTCA
DA
AHPEA
0 0.25 0.50 1.00 1.5
0.04 0.23 0.30 0.35 0.42
0.27 _+0.018 0.31 _+0.019 0.27 _+0.014 0.017_+0.004 0.011 + 0.001
67_+2 86_+7 72_+7 27_+2 25 _+3
122_+ 8 123_+10 211_+11 235 + 24
Preparation of pyrroIe-2,3,5-tricarboxylic acid (PTCA) As a standard for determination of PTCA we prepared the compound by permanganate oxidation of 5-hydroxyindole-2-carboxylic acid in 1 M H2SO 4 according to the method of Ito and Fujita [16]. The main lipid soluble product obtained after permanganate degradation was analyzed by HPLC-diode array. The substance was found to have an absorption maximum at 270 nm. On analysis with LC-MS and CI (ammonia) a major peak was obtained at m / z 200, (M + H) +, and a smaller one at m/z 217, (M + NH4) +, as could be expected for a pyrrole-tricarboxylic acid (Fig. 4). For quantification of PTCA the compound was prepared by KMNO 4 degradation of a melanin prepared from [14C]dopa. L-3,4-Dihydroxyphenyl-[3-14C]-alanine (10 /~Ci) was incubated together with 0.5 mmol unlabelled L-dopa, 1 mg mushroom tyrosinase and 0.25 mg catalase in 20 ml 0.05 M phosphate buffer (pH 6.8), for 4 days. The black melanin formed was spun down and dried. It was degraded to give PTCA according to the method of Ito and Fujita [16] and the pyrrole further purified with HPLC (condition D). The amount of pyrrole-2,3,5-tricarboxylic acid isolated was determined by measuring its radioactivity and the sample was then used as a standard in the quantification of PTCA
Preparation of melanins from dopamine and dopamine plus cysteine Since we suspected that the pigment of the substantia nigra was a polymer formed by the oxidation of dopamine in the presence of cysteine, we performed some model experiments on such melanins. Preparations were made according to the method of Ito [19] and the incubation conditions are presented in Table I. The samples were freeze-dried. Sulphur and nitrogen contents of the dried melanins were determined. The solubility of the different melanins were tested using buffers with different pH values. Melanins from dopa,
dopa plus cysteine, dopa plus glutathione and dopamine plus glutathione were prepared in an analogous way. The yields of PTCA and AHPEA from different melanins were determined after degradation by the methods of Ito and Fujita [16]. Table I gives the results of oxidative degradation and HI-hydrolysis of melanins formed by oxidation of dopamine alone and from dopamine with various amount of cysteine.
Preparation and analysis of the substantia nigra The substantia nigra and pieces of the frontal cortex were obtained at autopsies performed at the department of Pathology, Lund University Hospital, Lund. The patients had no history of Parkinsons disease, nor of medication known to interfere with dopaminergic neurons. The tissues were excised and then frozen. Crude samples of 1-2 mg of the frozen tissue from the substantia nigra and the frontal cortex were analyzed for PTCA and AHPEA according to the method of Ito and Fujita. Neuromelanin was also isolated, as follows, from the substantia nigra for further analysis of the compounds with the chromatographic properties of PTCA and AHPEA. Frozen tissue (approx. 1 g) was homogenized with twelve strokes in a Dounce glass-glass homogenizer with 10 ml of a solution containing 2% sodium cholate in 30% ethanol. The homogenate was centrifuged for 60 rain at 100000×g and the colorless supernatant was discarded. The pellet was suspended in 10 ml of a solution containing 10% glycerol and 2% sodium dodecyl sulphate (SDS) in water and homogenized with five strokes in a Dounce glass-glass homogenizer. The suspension was heated to 60°C for 30 min and centrifuged for 60 min at 3000 × g and the colorless supernatant was then discarded. The pellet was not uniformly colored, a black substance was concentrated at the bottom
155 m/z:149
m/z:153
8:54
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8:20 10:00
rain
Fig. 2. Mass fragmentograms of: A, 3-amino-4-hydroxyphenylethylamine(7.45) and 4-amino-3-hydroxyphenylethylamine(8.54); and B, HI-hydrolysate of human substantia nigra melanin.
100
100
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;
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m/z
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Fig. 3. Mass spectra of: 3-amino-4-hydroxy-phenylethylamine, (AI) and 4-amino-3-hydroxyphenylethylamine, (A2)" HI-hydrolysate of human substantia nigra melanin, peak at 7.44-7.46 min, (B1) and peak at 8.55 minutes, (B2).
156 of the tube, covered by a light brown layer, in turn covered by a colorless gelatinous substance. This substance was carefully sucked off using a Pasteur pipette connected to a water suction device. The residue was then washed twice with 97% ethanol and twice with water to get rid of the light brown layer. The black pellet remaining was analysed for solubility and for degradation products. For investigation of the presence of pheomelanin units in the isolated pigment the pellet was hydrolysed in HI according to the method of Ito and Fujita [16]. After hydrolysing and evaporation the residue was dissolved in water and analysed on reverse phase HPLC with two mobile phases. Samples were injected onto a HPLC column (conditions B1). The peaks corresponding to the standard AHPEA were collected, usually in 3-400 /xl. A sample of 100 /xl was injected onto another HPLC column (conditions B2) to establish that the peak corresponded with AHPEA. In another set of experiments four hydrolysates were combined and placed in a round-bottomed bottle connected to a Buchi rotary evaporator and evaporated to almost dryness. The residue was dissolved in 0.5 ml water and evaporated to dryness, again dissolved in 0.5 ml water and injected onto a HPLC column (condition B1) and the eluate was followed with a UV-detector set at 275 nm. The material with absorption at 275 nm was collected and the combined samples evaporated to dryness. The samples were anlaysed by means of LCMS (condition C). The sample was dissolved in the eluent. Mass fragmentography using the m / z 153 and m / z 149 was carried out. Melanin synthesized from cysteine and dopamine in ratio 1 : 1 was treated in the same way as the substantia nigra melanin sample and the hydrolysation products were used as references. (Figs. 2 and 3) In six experiments the pellet was treated with KMNO 4 and analysed according to Ito and Fujita [16]. Results
Observation on the synthetic melanins In the course of our work with isolation of the pigment of the substantia nigra it became evident that the pigment had properties different from the melanins of the skin, hair and eyes. Therefore a series of model experiments with synthetic melanins and their degradation products were performed and the result of this work provides a basis for our investigation on the pigment of the substantia nigra. Incubation of dopamine with tyrosinase gave an insoluble totally black pigment that could easily be spun down. At incubation of dopamine together with low concentrations of cysteine, black precipitates were obtained. At high concentrations of cysteine only small amounts of insoluble material were formed and the
supernatants after centrifugation were intensely colored. The precipitates with high concentrations of cysteine were brownish black. The ratio S / N in the insoluble melanin increased with the ratio of cysteine/ dopamine in the incubation mixture. When the ratio of cysteine/dopamine in the incubate was higher than 1.5 no melanin could be precipitated. Dopamine-cysteine melanin precipitated at neutral pH, but was soluble in alkali like dopa-cysteine melanin [6]. Melanin obtained by enzymatic oxidation of dopamine was degraded with KMNO 4. The product formed from dopamine-melanin had the same chromatographic properties as and could not be separated from, the corresponding compound obtained from 5hydroxyindole-2-carboxylic acid, at LC-MS, in several systems, indicating that PTCA prepared from dopamine has its three carboxyl groups in positions 2, 3 and 5. Table I shows that the yields of PTCA after KMNO 4 degradation of the melanin formed from dopamine was 0.03% which is somewhat lower than that observed by Ito and Fujita. Only small amounts of PTCA were obtained from melanin formed in the presence of large quantities of cysteine. In spite of the low yield, the method was found to be adequate for quantification of the eumelanin. The yields of AHPEA formed from dopamine oxidized in the presence of cysteine were much higher than those of PTCA from eumelanin. The original amount of pheo- and eumelanin was estimated by multiplying with a factor of 4 and 3000, respectively, for the two compounds. HI-hydrolysis of all dopamine melanins gave considerable amounts of dopamine. Thus the yield of dopamine from HI-hydrolysis of the melanin formed from dopamine was 7 percent and the yields from the sulphur containing melanins were also high. The dopamine found after HI-hydrolysis could not be explained by absorption of the amine onto the polymer, since careful washing of the melanin with 6 M HCI did not decrease the amounts obtained. The finding indicates instead that the melanins formed from dopamine and cysteinyldopamine contain a substantial amount of uncyclized monomers. The yield of dopa from dopamelanin prepared and analyzed in the same way was only 2 percent. Dopamine-melanin thus contains more uncyclized units of catechol derivatives than dopamelanin, which may be related to the fact that the amino group in dopamine is ionized at physiological pH, a fact which is less favorable for the formation of indoles. Dopamelanin, like dopaminemelanin was not soluble in buffers. Pigment formed from dopa plus glutathione or dopamine plus glutathione were soluble in all buffers used, pH 1-13.
Observations on the substantia nigra neuromelanin The results of K / V I N O 4 degradation and of HI-hydrolysis of the substantia nigra tissues without prior
157 2O0
TABLE II
100'
(M+H) +
Composition of substantia nigra neuromelanin as reflected in yields of degradation products
217
80.
Samples of substanta nigra tissue were hydrolysed in HI and oxidized by K/VINO4 (Materials and Methods). PTCA is a degradation product from eumelanin, yield approx 0.03% of original amount eumelanin. AHPEA, AHP and AHPAc from pheomelanin, yield approx 25%. Data from nine samples. All values in /zg/g.
(M+NH4)+ 60 ¸
40 ¸ HOOC
Compound
PTCA
AHPEA
DA
AHP
0.15+_0.06
200+6
2 2 + _ 4 Trace
AHPAc
_•COOH COOH
H
20
n.d.
0
i
i
i
~9o
,
L
2O0
zi0
i
z~0
2~o
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m/z
isolation of the pigment are summarised in Table II. Significant amounts of PTCA, the degradation product of indolic melanins, were found when the tissues obtained from the substantia nigra was treated with K_MNO4. AHPEA, the product at HI-hydrolysis of melanin formed from dopamine in the presence of cysteine, was also found in the hydrolysates from the substantia nigra. The amounts of PTCA and AHPEA roughly corresponds to those of synthetic melanins prepared from cysteine and dopamine in molar ratios between 1/2 and 1/1, see Table I. Trace quantities of AHP indicating the presence of units formed by oxidation of cysteinyldopa were also detected. No products from cysteinyldopac-melanin were found in the HI-hydrolysate. In samples of the frontal cortex treated in the same way as the substantia nigra, none of the degradation products could be found. The isolation of neuromelanin from substantia nigra met with several obstacles since it was difficult to separate from brain lipids. However neuromelanin could be cleared from contaminating substances if organic solvents were used together with a detergent. We found the following procedure for the isolation of the neuromelanin most effective: Extraction of lipids and proteins by 2% sodium cholate in 30% ethanol followed by extraction with 2% SDS in 10% glycerol. With this extraction procedure the neuromelanin was obtained as a fine black pellet. At HI-hydrolysis of the pellet and following HPLCanalyses with UV-detection or electrochemical detection, a predominant peak corresponding to AHPEA was found (Fig. 5). Small quantities of AHP, but not AHPAc, was observed indicating that the pigment polymer contained small amounts of cysteinyldopa derived units, but no cysteinyldopae derived units. A large peak corresponding to dopamine was seen. The quantity of dopa was 1% of that of AHPEA in the substantia nigra neuromelanin. The mass-fragmentogram during the LC run after loading the sample from the isolated substantia nigra pigment onto LC-mass apparatus, exhibited fragments at m/z 153 and m/z
Fig. 4. Ammonium CI mass spectrum of pyrrole-2,3,5-tricarboxylic acid (PTCA).
149. The retention time of the substance responsible for these ions were identical to that of authentic AHPEA. After degradation with IQV[NO4 of the isolated substantia nigra neuromelanin we obtained a substance with the same optical absorption as PTCA and with the same properties as PTCA in several chromatographic systems (Fig. 6).
B
A
i
0
I
I
5
10
rain
I
I
I
0
5
10
rain
Fig. 5. HPLC-chromatogram of HI-hydrolysis products of: A, authentic AHPEA; and B, neuromelanin isolated from the substantia nigra. Arrows indicate AHPEA.
158
B
A
~TCA
5
1'0
1'5 min
0
5
f0
1'5 rain
Fig. 6. HPLC-chromatogram of KMNO4 dcgradation products of: A,
melanin prepared from dopamine and cysteine (1/1); and B, neuromelanin isolated from the substantia nigra.
Solubility of the substantia nigra neuromelanin In three experiments the solubility of the neuromeianin in the pellet was tested. The pellets were dissolved in 0.1 M NaOH. After centrifugation of the sample for 5 rain at 3000 x g, a yellow-brown supernatant was obtained. The supernatant was transferred to another test-tube and acidified to pH 1 with 1 M HC1 and the test tube was left for 10 min. At that time a yellow-brown precipitate had appeared. It could be pelleted by centrifugation at 3000 x g for 10 min. The yellow-brown sediment was again dissolved in 1 M NaOH.
Quantification of the neuromelanin from the substantia nigra Absorption measurement showed a general absorption curve without any distinct maxima. A similar spectrum was obtained if synthetic melanin prepared from dopamine in the presence of cysteine ( 1 / 1 ) was dissolved in alkali. The amounts of neuromelanin from 1 g substantia nigra were estimated to be 1.2, 1.5 and 2.0 mg from the A determined in three cases, these numbers are however rough estimates due to the unfavourable spectrophotometric properties of the materials. Discussion
The finding of A H P E A after HI-hydrolysis of the pigment of the substantia nigra shows that 5-Scysteinyldopamine, a compound previously demonstrated in this part of the brain can further be oxidized to form benzothiazines which are polymerized to neu-
romelanin. Results from mass spectometry with pyrolytic degradation seems to be in accordance with our results (L. Zecca, personal communication). Analysis of the HI-hydrolysates of the neuromelanin from the substantia nigra also showed the presence of dopamine. We have found that 5-S-cysteinyldopamine (Fig. 7) when hydrolysed by HI yields cysteine and dopamine and thus the demonstration of dopamine after HI treatment of the neuromelanin is evidence that dopamine a n d / o r cysteinyldopamine are included in the polymer in uncyclised form. Significant amounts of PTCA found at oxidation of substantia nigra neuromelanin indicate the presence of indolic monomers. Even though PTCA at KMNO 4 oxidation can arise from oxidation of monomer intermediates, findings of similar amounts at experiments with substantia nigra tissue and with isolated and purified neuromelanin derived therefrom, precludes this possibility. Although PTCA can arise from dopaderived as well as from dopaminederived monomers, the finding of considerable amounts of dopamine, but only traces of dopa in the HI hydrolysates from isolated neuromelanin suggests that the catecholic precursor to this pigment is dopamine [21-24]. This is further supported by the facts that the concentration of dopamine is almost two orders of magnitude higher than that of dopa in the substantia nigra [14] and that dopamine is more easily oxidized than dopa. There was no evidence of inclusion of dopac in the substantia nigra neuromelanin, nor could AHPAc, the product derived from cysteinyldopac, be found after HI-hydrolysis. It can only be a matter of speculation if this fact is due to a lesser tendency for dopac- and cysteinyldopac-derived molecules to be included in the polymer or to a lessened availability of dopac due to some conditon in the cell related to a decrease in oxidative deamination. HI-hydrolysis and permanganate degradation of the frontal cortex did not lead to the formation of any of the melanin products demonstrated after degradation of the substantia nigra neuromelanin. This is not surprising since the concentrations of catecholic compounds are much lower here than in the substantia nigra. However dopamine and dopac are abundant in other non-melanized parts of the brain, i.e. in the
H0 H0-~-CH2-CH2-NHg I S I CH2 5-S-Cysteinyl I dopamine CH /\ H2N COOH Fig. 7. Structure of the main isomer of cysteinyldopamine.
159 putamen and in the caudate nucleus and that oxidation of these substances takes place is evidenced by the occurrence of 5-S-cysteinyldopac and 5-S-cysteinyldopamine in brain, 5-S-cysteinyldopac is also found in normal urine [25]. Thus the mere presence of catecholic compounds and their oxidation products is by itself not sufficient to give rise to pigment formation. Tyrosinase has not been demonstrated in the substantia nigra and therefore oxidation of dopamine and other cathecolic compounds giving cysteinyl addition products and neuromelanin must be induced by other mechanisms. It is well known that autooxidation of cathecolic compounds occur in the body [26]. Thus 5-S-cysteinyldopa, largely produced by oxidation of dopa through tyrosinase in the melanocyte is present in low concentrations also in organs where melanocytes are absent. Moreover, albinos without tyrosinase show urinary excretion of 5-S-cysteinyldopa [27-32]. The oxidation of dopa, dopamine and other catechols in the absence of tyrosinase can be mediated by free radicals. The superoxide anion is a good candidate as an oxidizing agent. Actually oxidation of catecholic substances has been used as a detection system for determination of superoxide anion which is dismutased in their presence [33]. The conditions necessary for pigment formation after oxidation of dopa, dopamine and dopac in the substantia nigra are not known, but the finding that the neuromelanin from the substantia nigra contains not only cysteinyldopamine units, but also indoles, could indicate that the availability of thiols is limited in the neurons, or in the compartments of the neurons where the neuromelanin formation takes place. Since thiols react very rapidly with quinones, cysteine must be virtually absent wherever indole formation occurs. Neuromelanin formation might be a marker for the exhaustion of the glutathione-cysteine protective system. It is now more than 30 years since Bertler and Rosengren [34] first demonstrated that dopamine is localized to the basal ganglia. Since then the catabolism of dopa and dopamine in this part of the brain has ben a subject of intense investigation. The demonstration in the past decade of autooxidation of dopa, dopamine and dopac (for review see Ref. 35) has focussed interest on such events in the pathogenesis of Parkinson's disease and also, more generally, in aging. The present finding that the substantia nigra neuromelanin like most cutaneous melanins [7] contains both indolic and benzothiazinederived monomers - whether in the same polymer or in separate ones can not be deduced from our study - provides us with the information that the substantia nigra neurons oxidize dopamine both in the presence and absence of cysteine. Considerable melanin formation is probably not compatible with further vital functions in the compartment where the
melanin formation occurs, since the function of the tyrosinase of the melanosomes of the melanocytes ceases with increasing melanisation [36]. It seems that the glutathione-cysteine system functions as a protective mechanism in cells in general. In the neurons of the substantia nigra this system is under heavy stress, since the concentration of catechols is high and their quinones consume cysteine and glutathione. Our data are compatible with an exhaustion of the glutathionecysteine system in some parts of the cell since indole formation occurs. If such an exhaustion becomes more generalized in the neurons, it could be responsible for the cell death observed in Parkinson's disease and related conditions.
Acknowledgements We thank Mrs Gunilla Westerdahl for skillful assistance with LC-MS. This investigation was supported with grants from the Swedish Cancer Society (Project 626-890-18XC), The Swedish Medical Research Council, The Walter, Ellen and Lennart Hesselman Foundation for Scientific Research, The Edward Welander Foundation for Scientific Research, The Thure Carlsson Foundation for Scientific Research, The Crafoord Foundation for Scientific Research, the donation funds of the Lund University Hospital and the donation funds of the Faculty of Medicine, University of Lund.
References 1 Van Woert, M.H., Prasad, K.N. and Borg, D.C. (1967) J. Neurochem. 14, 707-716. 2 Nordgren, L., Rorsman, H., Rosengren, A.-M. and Rosengren, E. (1971) Experientia 27, 1178-1179. 3 Das, K.C., Abramson, M.B. and Katzman, R. (1978) J. Neurochem. 30, 601-605. 4 Barden, H. (1981) in Age Pigments (Sohal, R.S., ed.), pp. 155-180, Elsevier, Amsterdam. 5 Nicolaus, R.A. (1968) Mel'anins, Hermann, Paris. 6 Prota, G. (1972) in Pigmentation: Its genesis and biologic control (Riley, V., ed.), pp. 615-630, Appleton Century Crofts, New York. 7 Prota, G., Rorsman, H., Rosengren, A.-M. and Rosengren E. (1976) Experientia 32, 970-971. 8 Rorsman, H., Agrup, G., Rosengren, A.-M. and Rosengren E. (1979) in Pigment Cell (Klaus, S.N., ed.), Vol 4, pp. 244-252, Karger, Basel. 9 Lerner, A.B., Fitzpatrick, T.B., Calkins, E. and Summerson, W.H. (1949) J. Biol. Chem. 178, 185-195. 10 Barden, H. (1969) J. Neuropathol. Exp. Neurol. 28, 419-441. 11 Bertler, ,~., Falck, B., Gonfries, C.-G., Ljunggren, L. and Rosengren, E. (1964) Acta Pharm. et Toxicol. 21,283-289. 12 Barden, H. and Martin E. (1972) in Pigmentation: Its genesis and biologic control (Riley, V., ed.), pp. 631-638, Appleton Century Crofts, New York. 13 Rosengren, E., Linder-Eliasson, E. and Carlsson, A. (1985) J. Neural. Transm. 63, 247-253. 14 Fornstedt, B., Rosengren, E. and Carlsson, A. (1986) Neuropharmacology 25, 451-454. 15 Ito, S. and Jimbow, K. (1983) J. Invest. Dermatol. 80, 268-272.
160 16 Ito, S., Fujita, K. (1984) Analytical Biochemistry 144, 527-536. 17 Agrup, G., Edholm, L.E., Rorsman, H. and Rosengren, E. (1983) Acta Derm. Venereol. (Stockh.) 63, 59-61. 18 Karg, E., Rosengren, E. and Rorsman H. (1990) J. Invest. Dermatol., in press. 19 Ito, S. (1989) Pigment Cell Research 2, 53-56. 20 Patil, D.G. and Chedekel, M.R. (1984) J. Organ. Chem. 49, 997-1000. 21 Piatelli, M., Faturosso, E., Magno, S. and Nicolaus, R.A. (1962) Tetrahedron 18, 941-949. 22 Palumbo, A., d'Ischia, M., Misuraca, G. and Prota, G. (1987) Biochim. Biophys. Acta 925, 203-209. 23 Palumbo, A., d'Ischia, M., Misuraca, G., Prota, G. and Schultz TM. (1988) Biochim. Biophys. Acta 964, 193-199. 24 Corradini, M.G., Napolitano, A. and Prota, G. (1986) Tetrahedron 42, 2083-2088. 25 Carstam, R., Brinck, C., Fornstedt, B., Rorsman, H. and Rosengren, E. (1990) Acta Derm. Venereol. (Stockh.) 70, 373-377. 26 Fehling, C., Hansson, C., Poulsen, J., Rorsman, H. and Rosengren, E. (1981) Acta Derm. Venereol. (Stockh.) 61,339-342. 27 Aquaron, R.R., Rouge, F. and Aubert, C. (1981) in Pigment Cell, Phenotypic expressions in pigment cells. (Seiji, M., ed.), pp. 97-103, University of Tokyo Press, Tokyo. 28 Carstam, R., Hansson, C., Krook, G., Rorsman, H. and Rosengren, E. (1985) Acta Derm. Venereol. (Stockh.) 65, 435-437.
29 Ekelund, M.C., Carstam, R., Hansson. C., Rorsman, H. and Rosengren, E. (1985) Acta Derm. Venereol. (Stockh.) 65, 437439. 30 Fehling, C., Hansson, C., Poulsen, J., Rorsman, H. and Rosengren, E. (1981) J. Neurol. Transm. 52, 251-257. 31 lto, S., Kato, T. and Fujita, K. (1986) Chromatographia 21, 645-647. 32 Nimmo, J.E., Hunter, J.A., Percy-Robb, I.W., Jay, B., Philips, C.I. and Taylor, O.G. (1985) Acta Derm. Venereol. (Stockh.) 65, 169-171. 33 Sawyer, T.D. Richens, T.D., Nanni, E.J. and Stallings, M.D. (1980) in Chemical and biochemical aspects of superoxide and superoxide dismutase. (Bannister, J.V. and Hill, H.A.O., eds.). pp. 1-26, Elsevier, Amsterdam 34 Bertler, ,~. and Rosengren, E. (1959) Acta Physiol. Scand. 47, 350-361. 35 Fornstedt. B. (1990) Cysteinyl adducts as putative indices of catechol autooxidation in dopaminergic brain regions. Thesis, University of Gothenburg, Gothenburg. 36 Seiji, M. (1981) in Pigment Cell, Phenotypic expressions in pigment cells. (Seiji, M., ed.) pp. 97-103, University of Tokyo Press. Tokyo.
Biochimica et Biophysica Acta, 1097 (1991) 152-160 © 1991 Elsevier Science Publishers B.V. All rights reserved 0925-4439/91/$03.50 ADONIS 092544399100110B
BBADIS 61068
The neuromelanin of the human substantia nigra Ragnar Carstam 1, Carita Brinck 2, Annika Hindemith-Augustsson 3, Hans Rorsman l and Evald Rosengren 3 Department of Dermatology, University of Lund, Lund (Sweden), 2 Department of Ecology, Unit~ersityofLund, Lund (Sweden) and "~Department of Pharmacology, University ofLund, Lund (Sweden) (Received 7 January 1991)
Key words: Cysteinyldopamine; C'ysteinyldopa; Dopamine; Dopa; Parkinson's disease; (Aging)
The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with K M N O 4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyidopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.
Introduction
The nature of the dark brown pigment in the substantia nigra is still unknown. The pigment has been assumed to be an oxidation product of catechol derivatives and thus to be similar to the cutaneous melanins [1-4]. The melanin of the epidermis and the hair has dopa as a common precursor and is traditionally divided into two main classes: eumelanin which is black, insoluble and composed mainly of indole monomers; and pheomelanin which is brown, alkali-soluble and formed from oxidized cysteinyldopa products [5,6]. However, most of the melanins of the skin are co-polymers of the two main classes [7,8]. In the skin the formation of melanin is catalyzed by tyrosinase, which is a bi-functional enzyme oxygenating tyrosine to dopa and oxidizing dopa to dopaquinone [9], but the enzyme does not seem to be present in the substantia nigra [10]. Large amounts of dopa are formed in this brain
Abbreviations: AHPEA, 4-amino-3-hydroxyphenylethylamine; AHP, 4-amino-3-hydroxyphenylalanine; AHPAc, 4-amino-3-bydroxyphenylacetic acid; PTCA, pyrrole-2,3,5-tricarboxylic acid. Correspondence: H. Rorsman, Department of Dermatology, University of Lund, Lasarettet, S-221 85, Lund, Sweden.
nucleus by another enzyme, tyrosine hydroxylase, but the dopa produced is rapidly decarboxylated to dopamine. The quantities of dopa are consequently low, but large amounts of dopamine are present in the perikarya of the neurons of the pars compacta and the pars reticulata with the axons running to the nuclei of the corpus striatum [11]. It is known that the pigment of the substantia nigra contains much sulphur [12] and cysteine-containing catechol derivatives in the brain indicate that neuromelanin may contain oxidation products of catechols such as cysteinyldopamine [13,14]. Hydrolysis techniques make it possible to identify different types of melanin formed from catechol derivatives [15,16]. We have used such techniques for the investigation of the neuromelanin from the substantia nigra. We have synthetized 4-amino-3-hydroxyphenyletylamine (AHPEA) which is the product of reductive hydrolysis of cysteinyldopamine-melanin and also applied techniques for identification of pyrrole2,3,5-tricarboxylic acid (PTCA), the compound obtained from dopa- and dopamine-melanin at oxidative degradation. Certain characteristics of synthetic melanins formed from dopamine in the presence of cysteine will also be described. This investigation was in part presented at a workshop at the XIVth International Pigment Cell Conference in Kobe, on November 3rd 1990.
153 Materials and Methods
Materials The chemicals used were 3-hydroxytyramine (Sigma), cysteine (Merck), mushroom tyrosinase (3430 units/mg solid) (Sigma), catalase (11000 unit/mg protein) (Sigma), Catroniazid (Pharma-Stern, Hamburg, F.R.G.), H3PO 2 and HI (BDH Chemicals, England), 5-hydroxyindole-2-carboxylic acid (Aldrich Chemical, F.R.G.), u-3,4-dihydroxyphenyl-[3-14C]-alanine (10 tzCi) (Amersham, England). 5-S-Cysteinyldopa was prepared according to Agrup et al. [17], 4-amino-3-hydroxyphenylacetic acid (AHPAc) was prepared according to Karg et al. [18]. All other chemicals were of analytical grade.
Chromatography Condition A. In the preparation of AHPEA we used semipreparative chromatography. We employed an LKB pump (LKB Bromma, Sweden) with a UV-detector Varichrom (Varian, CA, U.S.A.) set at 275 nm. A Bondapak column, C~s (Waters Associates, MA, U.S.A.), 5 /x, 300 × 7.8 mm i.d. was used. Mobile phase: 40 mg sodium octanesulphonic acid, 11 ml H3PO4/1 water (pH 1.75). The flow rate was 1.2 ml/min. Condition B1. The tissue contents of AHPEA were determined by high-performance liquid chromatography (HPLC) with UV-detection or with electrochemical detection. An LKB pump was employed with a UV-detector set at 275 nm. A Nucleosil column, C ls (Machery, Nagel & Co., Duren, S.F.R.), 5 ~, 250 x 5 mm i.d. was used. Mobile phase: 0.15% trifluoracetic acid. The flow rate was 1.2 ml/min. Condition B2. An LKB pump was employed with an electrochemical detector set at +0.67 mV vs. an Ag/AgC1 reference electrode. A Supelcosil column, OH
OH
H2NI
H2N/
Benzothiazolyl unit
AHPEA
KMn04
HO dihydroxyindole unit
H00C
HOOC
COOH
tt PTCA
Fig. 1. Degradation products from melanin containing cysteinyldopamine-derived benzothiazolyl units and dopamine-derived indole units.
LC 18 DB (Supelco, Bellafonte, PA, U.S.A.), 3 Ix, 150 x 4 . 6 mm i.d. was used. Mobile phase: 30 mg sodium octanesulphonic acid/l water, pH adjusted to 2.25 with H3PO 4. The flow rate was 1.2 ml/min. Condition C. Identification of AHPEA: the effluent from the HPLC column was introduced into a VG Trio 3 mass quadropole mass spectrometer. A Nucleosil column, SA 5 Iz 150 × 4.6 mm i.d. was used. Mobile phase: 5% MeOH in water, 0.3 M ammonium acetate (pH 5.5). The flow rate was 1 ml/min. Postcolumn addition of ammonium acetate buffer was employed to give a final buffer concentration of 0.3 M. The vaporizer capillary had a temperature of 210°C. A standard VG 11/253 data system configuration was employed to aquire mass spectral data. Condition D. The contents of PTCA were determined by HPLC. An LKB pump was employed with a UV-detector set at 270 nm. A Supelcosil column, LC 18 DB, 3 Iz, 150 × 4.6 mm i.d. was used. Mobile phase: 0.1 M KI-I2PO4,20% Methanol (pH 2.1). The flow rate was 1.2 ml/min.
HI-hydrolysis Reductive degradation in HI was performed according to Ito [16].
Potassium permanganate oxidation Oxidative degradation in potassium permanganate was performed according to Ito [16].
Preparation of 4-amino-3-hydroxyphenylethylamine (AHPEA) AHPEA was obtained by HI-hydrolysis of a melanin produced by enzymatic oxidation of dopamine in the presence of cysteine in the ratio 1.0/1.5, according to Ito [19]. 100/zg of the insoluble melanin was degraded by HI-hydrolysis and the resulting solution was put onto a semipreparative HPLC column (condition A). The elution was monitored by a UV-detector and the material having an UV maximum peak at 275 nm was collected [20]. A sample of the compound with the absorbance maximum at 275 nm was injected into a mass spectrometer by direct inlet and CI ionization (ammonia). The spectrum showed one major peak which correponded to (M + H) + m / z 153 for 4-amino3-hydroxyphenylethylamine. When the material from the semipreparative column corresponding to the peak was analyzed by LC-MS, in addition to a large peak at m / z 153 another one at m / z 149 was observed, probably representing the indole derivative of the original substance. Another sample of the compound isolated was treated with a monoamineoxidase preparation from kidney cortex of the guinea pig. Both kidneys from one animal were homogenized in 20 ml 0.5 M KH2PO 4 buffer (pH 7.4) with a Polytron homogenizer (Kine-
154 matics PT 10-35) setting 7 for 15 s. The suspension was centrifuged for 60 rain at 100000 ×g. The pellet was suspended in 5 ml of the phosphate buffer. 0.5 ml of the suspension containing high activity of monoamineoxidase was incubated in 1 ml 0.5 M KH2PO 4 buffer (pH 7.5) together with 3-4 /~g of the substance with UV-maximum at 275 nm obtained from semipreparative HPLC chromatography. The reaction was followed by HPLC (condition B2). During the incubation the peak corresponding to AHPEA gradually decreased and a peak corresponding to AHPAc [18] appeared. In a separate experiment the MAO inhibitor Catroniazid was added to a similar incubation mixture and the reaction was followed. In this case the AHPEA peak had a constant height and no peak could be observed at the place of AHPAc in the chromatogram.
TABLE 1
Analytical data of synthetic melanin formed from dopamine Melanin was formed by incubation of dopamine with tyrosinase at different cysteine concentrations and in the presence of catalase. The molar ratio of cysteine (C) and dopamine (DA) in the incubate and the ratio of sulphur to nitrogen in the formed insoluble melanin are given. The values for products obtained after HI-hydrolysis (AHPEA, DA) and after degradation by KMNO 4 (PTCA), are also given. 1 mg melanin was used for analysis. Data represent mean values-+ S.D. for 10 incubations. Values for PTCA, DA and AHPEA are in p~g. C/DA
S/N
PTCA
DA
AHPEA
0 0.25 0.50 1.00 1.5
0.04 0.23 0.30 0.35 0.42
0.27 _+0.018 0.31 _+0.019 0.27 _+0.014 0.017_+0.004 0.011 + 0.001
67_+2 86_+7 72_+7 27_+2 25 _+3
122_+ 8 123_+10 211_+11 235 + 24
Preparation of pyrroIe-2,3,5-tricarboxylic acid (PTCA) As a standard for determination of PTCA we prepared the compound by permanganate oxidation of 5-hydroxyindole-2-carboxylic acid in 1 M H2SO 4 according to the method of Ito and Fujita [16]. The main lipid soluble product obtained after permanganate degradation was analyzed by HPLC-diode array. The substance was found to have an absorption maximum at 270 nm. On analysis with LC-MS and CI (ammonia) a major peak was obtained at m / z 200, (M + H) +, and a smaller one at m/z 217, (M + NH4) +, as could be expected for a pyrrole-tricarboxylic acid (Fig. 4). For quantification of PTCA the compound was prepared by KMNO 4 degradation of a melanin prepared from [14C]dopa. L-3,4-Dihydroxyphenyl-[3-14C]-alanine (10 /~Ci) was incubated together with 0.5 mmol unlabelled L-dopa, 1 mg mushroom tyrosinase and 0.25 mg catalase in 20 ml 0.05 M phosphate buffer (pH 6.8), for 4 days. The black melanin formed was spun down and dried. It was degraded to give PTCA according to the method of Ito and Fujita [16] and the pyrrole further purified with HPLC (condition D). The amount of pyrrole-2,3,5-tricarboxylic acid isolated was determined by measuring its radioactivity and the sample was then used as a standard in the quantification of PTCA
Preparation of melanins from dopamine and dopamine plus cysteine Since we suspected that the pigment of the substantia nigra was a polymer formed by the oxidation of dopamine in the presence of cysteine, we performed some model experiments on such melanins. Preparations were made according to the method of Ito [19] and the incubation conditions are presented in Table I. The samples were freeze-dried. Sulphur and nitrogen contents of the dried melanins were determined. The solubility of the different melanins were tested using buffers with different pH values. Melanins from dopa,
dopa plus cysteine, dopa plus glutathione and dopamine plus glutathione were prepared in an analogous way. The yields of PTCA and AHPEA from different melanins were determined after degradation by the methods of Ito and Fujita [16]. Table I gives the results of oxidative degradation and HI-hydrolysis of melanins formed by oxidation of dopamine alone and from dopamine with various amount of cysteine.
Preparation and analysis of the substantia nigra The substantia nigra and pieces of the frontal cortex were obtained at autopsies performed at the department of Pathology, Lund University Hospital, Lund. The patients had no history of Parkinsons disease, nor of medication known to interfere with dopaminergic neurons. The tissues were excised and then frozen. Crude samples of 1-2 mg of the frozen tissue from the substantia nigra and the frontal cortex were analyzed for PTCA and AHPEA according to the method of Ito and Fujita. Neuromelanin was also isolated, as follows, from the substantia nigra for further analysis of the compounds with the chromatographic properties of PTCA and AHPEA. Frozen tissue (approx. 1 g) was homogenized with twelve strokes in a Dounce glass-glass homogenizer with 10 ml of a solution containing 2% sodium cholate in 30% ethanol. The homogenate was centrifuged for 60 rain at 100000×g and the colorless supernatant was discarded. The pellet was suspended in 10 ml of a solution containing 10% glycerol and 2% sodium dodecyl sulphate (SDS) in water and homogenized with five strokes in a Dounce glass-glass homogenizer. The suspension was heated to 60°C for 30 min and centrifuged for 60 min at 3000 × g and the colorless supernatant was then discarded. The pellet was not uniformly colored, a black substance was concentrated at the bottom
155 m/z:149
m/z:153
8:54
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100
50
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'
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I
u
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~
U
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I
'
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'
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m / z : 149 I00
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iI
6:40
l
I
I
I
8:20 10:00
rain
1:40
3:20
5:00
6:40
8:20 10:00
rain
Fig. 2. Mass fragmentograms of: A, 3-amino-4-hydroxyphenylethylamine(7.45) and 4-amino-3-hydroxyphenylethylamine(8.54); and B, HI-hydrolysate of human substantia nigra melanin.
100
100
8O
8O
6o
=
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;
A1
~" 4O
IL
2O
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;
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u
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.t ..... I u
*
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¸
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..~
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f
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.] L, 1
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149 i'"l"
i
~
m/z
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100
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i
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Fig. 3. Mass spectra of: 3-amino-4-hydroxy-phenylethylamine, (AI) and 4-amino-3-hydroxyphenylethylamine, (A2)" HI-hydrolysate of human substantia nigra melanin, peak at 7.44-7.46 min, (B1) and peak at 8.55 minutes, (B2).
156 of the tube, covered by a light brown layer, in turn covered by a colorless gelatinous substance. This substance was carefully sucked off using a Pasteur pipette connected to a water suction device. The residue was then washed twice with 97% ethanol and twice with water to get rid of the light brown layer. The black pellet remaining was analysed for solubility and for degradation products. For investigation of the presence of pheomelanin units in the isolated pigment the pellet was hydrolysed in HI according to the method of Ito and Fujita [16]. After hydrolysing and evaporation the residue was dissolved in water and analysed on reverse phase HPLC with two mobile phases. Samples were injected onto a HPLC column (conditions B1). The peaks corresponding to the standard AHPEA were collected, usually in 3-400 /xl. A sample of 100 /xl was injected onto another HPLC column (conditions B2) to establish that the peak corresponded with AHPEA. In another set of experiments four hydrolysates were combined and placed in a round-bottomed bottle connected to a Buchi rotary evaporator and evaporated to almost dryness. The residue was dissolved in 0.5 ml water and evaporated to dryness, again dissolved in 0.5 ml water and injected onto a HPLC column (condition B1) and the eluate was followed with a UV-detector set at 275 nm. The material with absorption at 275 nm was collected and the combined samples evaporated to dryness. The samples were anlaysed by means of LCMS (condition C). The sample was dissolved in the eluent. Mass fragmentography using the m / z 153 and m / z 149 was carried out. Melanin synthesized from cysteine and dopamine in ratio 1 : 1 was treated in the same way as the substantia nigra melanin sample and the hydrolysation products were used as references. (Figs. 2 and 3) In six experiments the pellet was treated with KMNO 4 and analysed according to Ito and Fujita [16]. Results
Observation on the synthetic melanins In the course of our work with isolation of the pigment of the substantia nigra it became evident that the pigment had properties different from the melanins of the skin, hair and eyes. Therefore a series of model experiments with synthetic melanins and their degradation products were performed and the result of this work provides a basis for our investigation on the pigment of the substantia nigra. Incubation of dopamine with tyrosinase gave an insoluble totally black pigment that could easily be spun down. At incubation of dopamine together with low concentrations of cysteine, black precipitates were obtained. At high concentrations of cysteine only small amounts of insoluble material were formed and the
supernatants after centrifugation were intensely colored. The precipitates with high concentrations of cysteine were brownish black. The ratio S / N in the insoluble melanin increased with the ratio of cysteine/ dopamine in the incubation mixture. When the ratio of cysteine/dopamine in the incubate was higher than 1.5 no melanin could be precipitated. Dopamine-cysteine melanin precipitated at neutral pH, but was soluble in alkali like dopa-cysteine melanin [6]. Melanin obtained by enzymatic oxidation of dopamine was degraded with KMNO 4. The product formed from dopamine-melanin had the same chromatographic properties as and could not be separated from, the corresponding compound obtained from 5hydroxyindole-2-carboxylic acid, at LC-MS, in several systems, indicating that PTCA prepared from dopamine has its three carboxyl groups in positions 2, 3 and 5. Table I shows that the yields of PTCA after KMNO 4 degradation of the melanin formed from dopamine was 0.03% which is somewhat lower than that observed by Ito and Fujita. Only small amounts of PTCA were obtained from melanin formed in the presence of large quantities of cysteine. In spite of the low yield, the method was found to be adequate for quantification of the eumelanin. The yields of AHPEA formed from dopamine oxidized in the presence of cysteine were much higher than those of PTCA from eumelanin. The original amount of pheo- and eumelanin was estimated by multiplying with a factor of 4 and 3000, respectively, for the two compounds. HI-hydrolysis of all dopamine melanins gave considerable amounts of dopamine. Thus the yield of dopamine from HI-hydrolysis of the melanin formed from dopamine was 7 percent and the yields from the sulphur containing melanins were also high. The dopamine found after HI-hydrolysis could not be explained by absorption of the amine onto the polymer, since careful washing of the melanin with 6 M HCI did not decrease the amounts obtained. The finding indicates instead that the melanins formed from dopamine and cysteinyldopamine contain a substantial amount of uncyclized monomers. The yield of dopa from dopamelanin prepared and analyzed in the same way was only 2 percent. Dopamine-melanin thus contains more uncyclized units of catechol derivatives than dopamelanin, which may be related to the fact that the amino group in dopamine is ionized at physiological pH, a fact which is less favorable for the formation of indoles. Dopamelanin, like dopaminemelanin was not soluble in buffers. Pigment formed from dopa plus glutathione or dopamine plus glutathione were soluble in all buffers used, pH 1-13.
Observations on the substantia nigra neuromelanin The results of K / V I N O 4 degradation and of HI-hydrolysis of the substantia nigra tissues without prior
157 2O0
TABLE II
100'
(M+H) +
Composition of substantia nigra neuromelanin as reflected in yields of degradation products
217
80.
Samples of substanta nigra tissue were hydrolysed in HI and oxidized by K/VINO4 (Materials and Methods). PTCA is a degradation product from eumelanin, yield approx 0.03% of original amount eumelanin. AHPEA, AHP and AHPAc from pheomelanin, yield approx 25%. Data from nine samples. All values in /zg/g.
(M+NH4)+ 60 ¸
40 ¸ HOOC
Compound
PTCA
AHPEA
DA
AHP
0.15+_0.06
200+6
2 2 + _ 4 Trace
AHPAc
_•COOH COOH
H
20
n.d.
0
i
i
i
~9o
,
L
2O0
zi0
i
z~0
2~o
zi0
m/z
isolation of the pigment are summarised in Table II. Significant amounts of PTCA, the degradation product of indolic melanins, were found when the tissues obtained from the substantia nigra was treated with K_MNO4. AHPEA, the product at HI-hydrolysis of melanin formed from dopamine in the presence of cysteine, was also found in the hydrolysates from the substantia nigra. The amounts of PTCA and AHPEA roughly corresponds to those of synthetic melanins prepared from cysteine and dopamine in molar ratios between 1/2 and 1/1, see Table I. Trace quantities of AHP indicating the presence of units formed by oxidation of cysteinyldopa were also detected. No products from cysteinyldopac-melanin were found in the HI-hydrolysate. In samples of the frontal cortex treated in the same way as the substantia nigra, none of the degradation products could be found. The isolation of neuromelanin from substantia nigra met with several obstacles since it was difficult to separate from brain lipids. However neuromelanin could be cleared from contaminating substances if organic solvents were used together with a detergent. We found the following procedure for the isolation of the neuromelanin most effective: Extraction of lipids and proteins by 2% sodium cholate in 30% ethanol followed by extraction with 2% SDS in 10% glycerol. With this extraction procedure the neuromelanin was obtained as a fine black pellet. At HI-hydrolysis of the pellet and following HPLCanalyses with UV-detection or electrochemical detection, a predominant peak corresponding to AHPEA was found (Fig. 5). Small quantities of AHP, but not AHPAc, was observed indicating that the pigment polymer contained small amounts of cysteinyldopa derived units, but no cysteinyldopae derived units. A large peak corresponding to dopamine was seen. The quantity of dopa was 1% of that of AHPEA in the substantia nigra neuromelanin. The mass-fragmentogram during the LC run after loading the sample from the isolated substantia nigra pigment onto LC-mass apparatus, exhibited fragments at m/z 153 and m/z
Fig. 4. Ammonium CI mass spectrum of pyrrole-2,3,5-tricarboxylic acid (PTCA).
149. The retention time of the substance responsible for these ions were identical to that of authentic AHPEA. After degradation with IQV[NO4 of the isolated substantia nigra neuromelanin we obtained a substance with the same optical absorption as PTCA and with the same properties as PTCA in several chromatographic systems (Fig. 6).
B
A
i
0
I
I
5
10
rain
I
I
I
0
5
10
rain
Fig. 5. HPLC-chromatogram of HI-hydrolysis products of: A, authentic AHPEA; and B, neuromelanin isolated from the substantia nigra. Arrows indicate AHPEA.
158
B
A
~TCA
5
1'0
1'5 min
0
5
f0
1'5 rain
Fig. 6. HPLC-chromatogram of KMNO4 dcgradation products of: A,
melanin prepared from dopamine and cysteine (1/1); and B, neuromelanin isolated from the substantia nigra.
Solubility of the substantia nigra neuromelanin In three experiments the solubility of the neuromeianin in the pellet was tested. The pellets were dissolved in 0.1 M NaOH. After centrifugation of the sample for 5 rain at 3000 x g, a yellow-brown supernatant was obtained. The supernatant was transferred to another test-tube and acidified to pH 1 with 1 M HC1 and the test tube was left for 10 min. At that time a yellow-brown precipitate had appeared. It could be pelleted by centrifugation at 3000 x g for 10 min. The yellow-brown sediment was again dissolved in 1 M NaOH.
Quantification of the neuromelanin from the substantia nigra Absorption measurement showed a general absorption curve without any distinct maxima. A similar spectrum was obtained if synthetic melanin prepared from dopamine in the presence of cysteine ( 1 / 1 ) was dissolved in alkali. The amounts of neuromelanin from 1 g substantia nigra were estimated to be 1.2, 1.5 and 2.0 mg from the A determined in three cases, these numbers are however rough estimates due to the unfavourable spectrophotometric properties of the materials. Discussion
The finding of A H P E A after HI-hydrolysis of the pigment of the substantia nigra shows that 5-Scysteinyldopamine, a compound previously demonstrated in this part of the brain can further be oxidized to form benzothiazines which are polymerized to neu-
romelanin. Results from mass spectometry with pyrolytic degradation seems to be in accordance with our results (L. Zecca, personal communication). Analysis of the HI-hydrolysates of the neuromelanin from the substantia nigra also showed the presence of dopamine. We have found that 5-S-cysteinyldopamine (Fig. 7) when hydrolysed by HI yields cysteine and dopamine and thus the demonstration of dopamine after HI treatment of the neuromelanin is evidence that dopamine a n d / o r cysteinyldopamine are included in the polymer in uncyclised form. Significant amounts of PTCA found at oxidation of substantia nigra neuromelanin indicate the presence of indolic monomers. Even though PTCA at KMNO 4 oxidation can arise from oxidation of monomer intermediates, findings of similar amounts at experiments with substantia nigra tissue and with isolated and purified neuromelanin derived therefrom, precludes this possibility. Although PTCA can arise from dopaderived as well as from dopaminederived monomers, the finding of considerable amounts of dopamine, but only traces of dopa in the HI hydrolysates from isolated neuromelanin suggests that the catecholic precursor to this pigment is dopamine [21-24]. This is further supported by the facts that the concentration of dopamine is almost two orders of magnitude higher than that of dopa in the substantia nigra [14] and that dopamine is more easily oxidized than dopa. There was no evidence of inclusion of dopac in the substantia nigra neuromelanin, nor could AHPAc, the product derived from cysteinyldopac, be found after HI-hydrolysis. It can only be a matter of speculation if this fact is due to a lesser tendency for dopac- and cysteinyldopac-derived molecules to be included in the polymer or to a lessened availability of dopac due to some conditon in the cell related to a decrease in oxidative deamination. HI-hydrolysis and permanganate degradation of the frontal cortex did not lead to the formation of any of the melanin products demonstrated after degradation of the substantia nigra neuromelanin. This is not surprising since the concentrations of catecholic compounds are much lower here than in the substantia nigra. However dopamine and dopac are abundant in other non-melanized parts of the brain, i.e. in the
H0 H0-~-CH2-CH2-NHg I S I CH2 5-S-Cysteinyl I dopamine CH /\ H2N COOH Fig. 7. Structure of the main isomer of cysteinyldopamine.
159 putamen and in the caudate nucleus and that oxidation of these substances takes place is evidenced by the occurrence of 5-S-cysteinyldopac and 5-S-cysteinyldopamine in brain, 5-S-cysteinyldopac is also found in normal urine [25]. Thus the mere presence of catecholic compounds and their oxidation products is by itself not sufficient to give rise to pigment formation. Tyrosinase has not been demonstrated in the substantia nigra and therefore oxidation of dopamine and other cathecolic compounds giving cysteinyl addition products and neuromelanin must be induced by other mechanisms. It is well known that autooxidation of cathecolic compounds occur in the body [26]. Thus 5-S-cysteinyldopa, largely produced by oxidation of dopa through tyrosinase in the melanocyte is present in low concentrations also in organs where melanocytes are absent. Moreover, albinos without tyrosinase show urinary excretion of 5-S-cysteinyldopa [27-32]. The oxidation of dopa, dopamine and other catechols in the absence of tyrosinase can be mediated by free radicals. The superoxide anion is a good candidate as an oxidizing agent. Actually oxidation of catecholic substances has been used as a detection system for determination of superoxide anion which is dismutased in their presence [33]. The conditions necessary for pigment formation after oxidation of dopa, dopamine and dopac in the substantia nigra are not known, but the finding that the neuromelanin from the substantia nigra contains not only cysteinyldopamine units, but also indoles, could indicate that the availability of thiols is limited in the neurons, or in the compartments of the neurons where the neuromelanin formation takes place. Since thiols react very rapidly with quinones, cysteine must be virtually absent wherever indole formation occurs. Neuromelanin formation might be a marker for the exhaustion of the glutathione-cysteine protective system. It is now more than 30 years since Bertler and Rosengren [34] first demonstrated that dopamine is localized to the basal ganglia. Since then the catabolism of dopa and dopamine in this part of the brain has ben a subject of intense investigation. The demonstration in the past decade of autooxidation of dopa, dopamine and dopac (for review see Ref. 35) has focussed interest on such events in the pathogenesis of Parkinson's disease and also, more generally, in aging. The present finding that the substantia nigra neuromelanin like most cutaneous melanins [7] contains both indolic and benzothiazinederived monomers - whether in the same polymer or in separate ones can not be deduced from our study - provides us with the information that the substantia nigra neurons oxidize dopamine both in the presence and absence of cysteine. Considerable melanin formation is probably not compatible with further vital functions in the compartment where the
melanin formation occurs, since the function of the tyrosinase of the melanosomes of the melanocytes ceases with increasing melanisation [36]. It seems that the glutathione-cysteine system functions as a protective mechanism in cells in general. In the neurons of the substantia nigra this system is under heavy stress, since the concentration of catechols is high and their quinones consume cysteine and glutathione. Our data are compatible with an exhaustion of the glutathionecysteine system in some parts of the cell since indole formation occurs. If such an exhaustion becomes more generalized in the neurons, it could be responsible for the cell death observed in Parkinson's disease and related conditions.
Acknowledgements We thank Mrs Gunilla Westerdahl for skillful assistance with LC-MS. This investigation was supported with grants from the Swedish Cancer Society (Project 626-890-18XC), The Swedish Medical Research Council, The Walter, Ellen and Lennart Hesselman Foundation for Scientific Research, The Edward Welander Foundation for Scientific Research, The Thure Carlsson Foundation for Scientific Research, The Crafoord Foundation for Scientific Research, the donation funds of the Lund University Hospital and the donation funds of the Faculty of Medicine, University of Lund.
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