LINK 24 The Chemical Structure of Melanin

 

INTRODUCTION

 

One year ago a letter to the Editor entitled ‘’ The Chemical Structure of Melanin ‘’  by Dr. W.L. Cheun and the reply of Drs  J. D. Simon and S. Ito, appeared on  Pigment Cell Research ( 2004 ) 17;  422-424 .  Some new aspects concerning the chemical structure of  melanin and many fundamental papers  ( G.Swan, M.S.Blois, H.Mason, J.Harley-Mason, J.Bu’Lock,  M.G.Peter , H.Wyler, M.Piattelli, E.Fattorusso  V.Hearing, J.Duchon, H.Rorsman, M.R.Chedekel, M.G.Bridelli, R.A.Nicolaus, G.Prota, M.d’Ischia, A.Pezzella, A.Bolognese ) have not been adequately treated. Several important  references, especially for those turning to the study of melanin structure are neglected..The contribute from Italian Research to melanin chemistry starting from Angelo Angeli is ignored.

The main purpose of this paper is  to try to limit the bad scientific information and extend our real knowlrdge  of melanin

 A new classification and recent advances on melanin chemistry and structure ( radical-polarone system of acetylene-black ) are presented . Our experiment will not end the debate about a final melanin structure but it may help to push the research  in a specific and right  direction.

 A list of papers which don’t appear frequently in literature, but  useful for research on melanin, is reported. References for determination of eumelanin  pheomelanin and allomelanin methods are cited. Some points worth of being recalled  to  the  melanin expert are the followings :

1 . Melanoma was found to be constituted  by two different melanins ( M. Bakunin 1904 ) 2 . Simple or complex pyrrole acids were isolated  among the oxidative degradation mixture   of Sepia melanin ( L.Panizzi, R.A.Nicolaus 1952 ). DHI and DHICA were obtained for the first time as degradation products ( R: A: Nicolaus, M.Piattelli 1962 ). 3 . The precursor of pheomelanins and pheochromes  is  the new aminoacid 5-S-Cysteinyldopa   and related isomers  .( R.A.Nicolaus 1968 ). 4 . The chromophores ( dibenzothiazine and dibenzothiazinone ) of pheochromes have been synthetised.  ( B.Kaul  1974 ). 5 .  Melanins are able to bind organic molecules, ions,  liquids, gases ( M.S.Blois 1965), ( H. Rorsman,  1972 )  ( N.G.Lindquist, B.Larsson ,C.Hanson, G.Agrup, E.Rosengren  and the Swedish School since 1972 ).  6 . Melanins exhibit two separate current - voltage characteristics, the on and off. ( J.McGinness,  J.P.Proctor  1974 ) . 7 .  The neuromelanin of the human Substantia nigra was isolated  ( H.Rorsman 1991 ).  8 . Melanins are particles (M.R.Chedekel 1992 ) . 9 . Eumelanin particles (granules covered by a membrane) are called melanosomes. Melanosomes  may be obtained in pure form.  (  V.J.Hearing  1973 ). 10 . The particles are formed by oligomers ( melanochromes ) of low molecular weight  (M.G.Peter  1996) . 11 . All melanins show the radical polarone system of acetylene black ( R.A. Nicolaus 2001 ).  12 . All melanins have about one oxygen atom more than the precursors ( R.A.Nicolaus  2001 ).  13 . The particle may show a certain level of organization ( J.D.Simon  2001 ) . 14 . All black particles, from carbon to sepiomelanin, have similar physical properties.The melanin particle gave  X-ray examination results which may suggest not only   graphite but also  fullerene or helicoidal cages. The helicoidal system may produce a magnetic field.   ( A.Bolognese 2001 ). 15 . The organic black matter in the interstellar clouds is supposed to protect organic material from dangerous rays and to regulate the ion/radical/ molecule balance .  ( B.J.R.Nicolaus,R.A.Nicolaus 1999 ).  All these data and properties suggest important functions of eumelanins in Biology.( 30 )

CLASSIFICATION

The current classification of melanins is not  satisfactory.

The black matter may be organised as followings  :

The black cell matter   ( BCM )

The black synthetic matter  ( BSM )

The  BCM may be  classified as  :

Eumelanin  Mammalian colouring is mainly dependent on the secretory activity of a unique pigmentary system,the melanocyte, which is capable of producing three chemically distinct types of pigments.These are, the dark insoluble, nitrogenous eumelanins, the sulphur-containing alkali-souble pheomelanins which  provide the lighter colours,  the amphoteric pheochromes ( red and red-orange colours ), closely related to pheomelanins  but found only in certain types of reddish hair as well in melanoma urines.

 

 

  eumelanin

 

 Eumelanins are particles generally derived from DOPA, and widespread in the animal kingdom in  skin, hair, furs, eyes, brain, feathers ( 1-6 ).

The particles covered with a membrane are called melanosomes. Melanosomes may be purified with the method of  V.Hearing (7-8) . The particles are formed by olygomers which are derived from precursors of different type like DHI, DHICA,DOPA, Cyclodopa, Decarbossicyclodopa The melanin prepared in vitro by Swan  are different from those of cell origin. ( 4 ), ( 5a ), ( 6 )  Melanins show a typical EPR signal.

  Melanins are sensitive to oxidants.Oxidative degradation products are pyrrole acids. Methods are available for melanin , pheomelanin and allomekanin quantitative determination .

 

. Neuromelanin   particles generally derived from dopamine and cysteinyldopamine. Neuromelanin  from  pigmented neurons..  Substantia nigra  was isolated by H.Rorsman in 1991. Structural studies  by Zecca  in 2001  ( 51-52 )Studies on neuromelanin primates ( 50 ),.Adrenalin-black was studied by J.Bu’Lock in 1960 ( 36d ).

 

 Substantia nigra

As reported by  M.G.Bridelli the  IR of neuromelanin has features similar to those of synthetic dopamine-melanin but different with other catecholamine-black .All that we know about dopamine-black is due to  G.Swan works ( 6 ) Dopamine-melanin have one oxygen more than the precursor

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 Dopamine-melanin found                  :   57.9 C%    5.7 H%     8.0 N%

Calculated for      ( C8 H5 O3 N )x      :   57.2  C%   4.7  H%   8.3 N%

 Calculated for   ( C8 H3 O2 N   )x     :         66.2    C%    2.0 H%   9.6 N%  

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 The pigment was found to be soluble in dilute alkali,to give a lightbrown derivative with benzoyl chloride having one benzoyl group per 8-carbon  unit in the polymer. IR indicate that benzoylation had occurred chiefly at the phenolic hydroxyl groups.Analysis led Swan to suggest that the ‘’ polymer  ‘’ contain uncyclised dopamine units, carboxylated pyrrole units 5,6-dihydroxyindole units.The presence of one oxygen atom more in the polymer was not discussed.As showed by solid state NMR dopamine melanin contains larger proportions of uncyclised units  compared with DOPAmelanin . Black / brown pigmented granules are present in the human central nervous system  The most pigmented regions are two areas: substantia nigra and the locus coeruleus. Histological studies displayed pigmentation in the substantia nigra of other mammals phylogenetically close to humans, including the shimpanzee, and more distant ones, such as horses and sheep. Histochemical studies on human substantia nigra and locus coeruleus found that the pigment had similar properties to the melanins, including  insolubility ,  bleaching by hydrogen peroxide, labelling by silver stains. The pigment was therefore named neuromelanin. Histological studies showed that neuromelanin granules were located in the neuronal perikaryon and were surrounded by a double membrane. In humans and horses, histochemical analyses indicated an association of neuromelanin granules with lipofuscin. In the substantia nigra, neuromelanin accumulates during aging  and is found after the first 2 to 3 years of life. The  substantia nigra neurones are more vulnerable than the non-pigmented ones. However, important questions remain regarding the possible role of neuromelanin in the substantia nigra, both under physiological conditions and in the pathogenesis of Parkinson’s disease. Neuromelanins like others melanins (BCM) show electrical activity.The presence in the brain of  electrical material may be interesting.

Dopamine  may be a precursor of neuromelanin  but like other melanins the polymer has more oxygen than the precursor.

 Dopamine ( C8H11NO2 , mol.weight 153,18 ;  62.7 C% ,  7.2 H%., 9.1 N% ) forms in alkaline medium, by oxidation,  black / brown material..Dopamine is present in bananas being responsible for the blackning of the fruit.Dopamine partecipate to  the biogenesis of some  alkaloids .

Initially, the name neuromelanin was chosen because of its similarity in appearance to skin melanin. However, the electron paramagnetic resonance (EPR) and metal analysis studies indicate that chemically neuromelanin   has a stable free radical structure. Furthermore neuromelanin and synthetic melanin are amorphous semiconductors.

The ability of neuromelanin to bind several inorganic and organic compounds, to chelate metal ions , to store liquids and gases, to show sound and electrical conductivity,  are difficult to understand from a chemical point of view but not from the bionanoscientists.

Degradation analyses using potassium permanganate and hydriodic acid hydrolysis showed that neuromelanin has properties of both pheomelanin and eumelanin. Elemental analyses of neuromelanin revealed a high sulphur content (2.5–2.8%), with a molar C/H ratio lower than that of synthetic melanins, thus indicating the presence of aliphatic groups and benzothiazine rings.

Infrared spectroscopy of neuromelanin ( and other melanins BCM ) revealed the presence of aliphatic groups and a low intensity aromatic component, whereas in synthetic melanins the aliphatic groups were absent. Chemical degradation studies showed that neuromelanin  is a mixture of melanin (BCM)  and pheomelanin  .

Neuromelanin  shows a peptide component of about 15%. The amino acids could be derived from a direct reaction between the melanic polymer and proteins, or dopamine molecules bound to cysteinic residues of polypeptidic chains. Indeed, the precursor of neuromelanin synthesis has been suggested to be cysteinyl-dopamine, although a study using hydriodic acid hydrolysis failed to identify the corresponding degradation products. Moreover, nuclear magnetic resonance spectroscopy indicates that the presence of both aliphatic and aromatic hydrogens, and the ratio of aliphatic to aromatic hydrogens is again higher in neuromelanin than in synthetic melanins, suggesting that dopamine cannot be the only precursor in neuromelanin synthesis.

x Ray difraction studies have shown that neuromelanin has a multilayer (graphite-like) three dimensional structure similar to synthetic and naturally occurring melanins. The three dimensional structure is derived from planar overlapped sheets consisting of cyclic molecules of indolebenzothiazine rings. However,these sheets are stacked much higher in neuromelanin than in any other synthetic and naturally occurring melanins.

The process of neuromelanin formation is difficult to understand.  It has long been debated whether the synthesis of neuromelanin is enzymatically mediated or whether it is a pure autooxidation process of dopamine derivatives. For eumelanin synthesis, the enzyme tyrosinase   catalyses the conversion of tyrosine to L-dopa and then to dopa-quinone. Some authors proposed that tyrosinase could also be involved in neuromelanin biosynthesis because tyrosinase mRNA and promoter activity have been detected in the substantia nigra.

Moreover, albinos who lack tyrosinase display normally pigmented substantia nigra. Alternative enzymatic actions have been suggested, including tyrosine hydroxylase mediated oxidation of dopamine. In another study, peroxidase catalysed the oxidation of tyrosine to dopa and then dopamine, and further oxidisation to the respective quinones that are possible precursors of neuromelanin.

Alternatively, neuromelanin could derive from non-enzymatic oxidation. The autooxidation  of catechols to quinones with the addition of a thiol has been demonstrated in the brain.

A dopamine-melanin can be synthesised by the autooxidation of dopamine, although there are several structural differences between synthetic melanins and the natural one isolated from the substantia nigra. Recently, neuromelanin synthesis was induced in rat substantia nigra neurones and PC cell cultures by exposure to L-dopa. The pigment produced in this model contains a stable free radical; in addition, both light and electron microscopy have shown that the pigment synthesised in these cells appears to be identical to human neuromelanin, and the granules are surroundedby a double membrane, similar to the naturally occurring neuromelanin of the substantia nigra. In those experiments, treatment with the iron chelator desferrioxamine inhibited neuromelanin synthesis stimulated by L-dopa; therefore, it seems that iron ( or an iron catalyst ) is involved in neuromelanin formation. In this model, neuromelanin synthesis was shown to be driven by an excess of cytosolic catecholamines not accumulated in synaptic vesicles.

The herbicide paraquat has a molecular structure similar to that of MPTP, and has been proposed as a Parkinson’s disease inducing agent. The pesticide is accumulated in neuromelanin containing nerve cells, where it appeared that the neuromelanin adsorbed intraneuronal paraquat, protecting the neurones from consequent damage.

Neuromelanin like others BCM can also accumulate chlorpromazine, haloperidol, and imipramine, thereby contributing to the control of the intraneuronal concentration of these molecules.

 Because higher intraneuronal concentrations of dopaminergic drugs might be toxic to substantia nigra neurones, neuromelanin can influence this toxicity. The association of neuromelanin with lipids has been described in several studies.

Although previous studies proposed that lipids were part of the neuromelanin molecule, recent work has shown that neuromelanin contains about 20% adsorbed lipids. Cholesterol is a minor component in this lipid mixture, with the major component being a new class of polyunsaturated lipid with a high molecular weight, low volatility, and low oxygen content.

High concentrations of iron and other  metals are present in several brain nuclei. Neuromelanin from the substantia nigra can interact with many heavy metal ions such as zinc, copper, manganese, chromium, cobalt, mercury, lead, and cadmium; in addition, it binds iron particularly EPR studies showed that in the substantia nigra the ferric iron is bound to     neuromelanin as a high spin complex with an octahedral configuration. Mössbauer spectroscopy demonstrates that ferric iron is  chelated by the neuromelanin polymer and that the iron sites are arranged in a ferritin-like iron-oxy-hydroxide cluster form. The cluster conductivity is unknown.

X rays absorption fine structure spectroscopy and infrared spectroscopy studies confirmed thatiron in neuromelanin was bound by oxygen derived phenolic groups in an octahedral configuration. In substantia nigra tissue, neuromelanin is only about 50% saturated withFe(III), therefore maintaining an important residual chelating capability, which can protect against iron toxicity.Values of iron complex conductivity are unknown.

Neuromelanin can sequester redox active iron ions, reducing the formation of free hydroxyl radicals. Thus, in normal subject s, neuromelanin may play a protective role b    inactivating the iron ions that induce oxidative   stress. The ability of neuromelanin to chelate other redox active metals such as copper, manganese, chromium, and toxic metals including cadmium, mercury, and lead  strengthens the hypothesis that neuromelanin may have a high capacity storage trapping system for metal ions which  may prevent neuronal damage.

Electrical conductivity of the different chelates has not determined until now. Neuromelanin accumulates normally with age in human substantia nigra neurones. A neuronal pigment has also been observed in the ubstantia nigra of adult rats and dogs, and its concentration seems to depend upon age. In very old (23 months) rats, but not in younger animals, neuromelanin granules were detected by electron microscopy; similar results were observed in aged dogs. Neuromelanin granules were also detected in catecholaminergic cerebellar cells of monkeys  and their presence correlated with age. In human substantia nigra, the first small, brown neuromelanin granules were clearly discernable at approxim tely 3 to 5 years of age. The neuromelanin content of neurones is highest in individuals in th ir 60s, after which it decreases; this phenomenon may reflect the neuronal loss observed in these anatomical structures during aging. However, there is no significant loss of catecholaminergic neurones in the substantia nigra of normal subjects until very old ages.

In patients with Parkinson’s disease, neuromelanin values were 1.2–1.5 mg/g of substantia nigra , which is less than 50% of that seen in age matched controls . The absolute number of pigmented neurones in the substantia nigra of normal subjects may be dependent upon ethnicity—an Indian population was found to have fewer pigmented neurones than an agematched Western population.. Because the neuromelanin concentration in substantia nigra neurones increases, and the number of pigmented neurones appears to be constant over the life span, it seems that neuromelanin accumulates only in a subpopulation of nigral neurones, whereas other dopaminergic neurones remain nonpigmented. The observed decrease in the neuromelanin concentration occurring in the substantia nigra of patients with Parkinson’s disease confirms the loss of pigmented neurones occurring in the substantia nigra of these patients, as has been reported in neuropathological studies. Other studies indicate that neuromelanin values decrease in the surviving neurones of the substantia nigra during Parkinson’s disease. This could be the result of reduced neuromelanin synthesis, neuromelanin degradation, or higher vulnerability of the pigmented neurones.

Although in idiopathic Parkinson’s disease the neurones are depleted in both the substantia nigra and locus coeruleus, in MPTP intoxicated subjects, locus coeruleus neurones are spared. Such a different neuronal vulnerability might eventually be explained by structural differences in the neuromelanin of the substantia nigra and locus coeruleus.

Although neuromelanin may play a cytoprotective role by sequestering redox active metals, toxic metals, and organic toxic compounds, neuromelanin might also become a source of free radicals by reaction with hydrogen peroxide. When free neuronal iron increases to the point where neuromelanin becomes saturated and it starts to catalyse the production of free radicals, neuromelanin would become cytotoxic with an increasing of the conductivity.

 Moreover, because hydrogen peroxide can degrade neuromelanin, the pigmented neurones could loose this putatively protective agent. The consequence may be a release of iron and other cytotoxic metals or compounds from neuromelanin that could accelerate neuronal death.

The pigmented neurones of the substantia nigra are typically lost in Parkinson’s disease; however, the possible relation between neuronal vulnerability and thepresence of neuromelanin has not been elucidated. Early histological studies revealedthe presence of increasing amounts of neuromelanin in the substantia nigra with aging in higher mammals, showed that the neuromelanin granules are surrounded by a membrane, and comparatively evaluated the pigmentation of the substantia nigra in different animal species. Histochemical studies showed the association of neuromelanin with lipofuscins. However, systematic investigations of the structure, synthesis, and molecular interactions of neuromelanin have been undertaken only during the past decade. In these later studies, neuromelanin was identified as a genuine melanin with a strong chelating ability for iron and an affinity for compounds such as lipids, pesticides, MPP+  (methylphenylpyridine ion ). The affinity of neuromelaninfor a variety of inorganic and organic toxins is consistent with a postulated protective function for neuromelanin. Moreover, the neuronal accumulation of neuromelanin during aging and the link between its synthesis and a high cytosolic concentration of catechols suggest a protective role. However, its putative neuroprotective effects could be quenched in conditions of toxin overload.

Black / brown pigmented granules in the human central nervous system has been observed since the half past century  (1) The most pigmented regions are two mesencephalic areas: Sömmering’s substantia nigra and the locus coeruleus. Histological studies displayed pigmentation in the substantia nigra of other mammals phylogenetically close to humans, including the shimpanzee, gibbon, and baboon, and more distant ones, such as horses and sheep. Histochemical studies on human substantia nigra and locus coeruleus found that the pigment had similar properties to the melanins, including being insoluble in organic solvents, being bleached by hydrogen peroxide, and being labelled by silver stains. The pigment was therefore named neuromelanin.Histological studies showed that neuromelanin granules were located in the neuronal perikaryon and were surrounded by a double membrane. In humans and horses, histochemical analyses indicated an association of neuromelanin granules with lipofuscin.In the substantia nigra, neuromelanin accumulates during aging and is found after thefirst 2 to 3 years of life.

Parkinson’s disease is a neurodegenerative disorder caused by the selective death of pigmented substantia nigra neurones, giving rise to dopamine depletion in the neostriatum, and resulting in clinical syndrome characterised by tremor, rigidity, and severely impaired motility. The pigmented substantia nigra neurones are more vulnerable than the non-pigmented ones. However, important questions remain regarding the possible role of neuromelanin in the substantia nigra, both under physiological conditions and in the pathogenesis of Parkinson’s disease. Here, we review those studies undertaken during the past 10 years on the molecular aspects of neuromelanin, and attempt to integrate these structural aspects with morphological findings.

Initially, the name neuromelanin was chosen because of its similarity in appearance to cutaneous melanin. However, recent electron paramagnetic resonance (EPR) and metal analysis studies indicate that chemically neuromelanin is indeed a genuine melanin because it has a stable free radical structure and avidly chelates metals. The ability of neuromelanin to interact with several inorganic and organic compounds, including metal ions and lipids, complicates studies of the structure of this pigment. Degradation analyses using potassium permanganate and hydriodic acid hydrolysis showed that neuromelanin has properties of both pheomelanins and eumelanins. Elemental analyses of neuromelanin revealed a high sulphur content (2.5–2.8%), with a molar C/H ratio lower than that of synthetic melanins, thus indicating the presence of aliphatic groups and benzothiazine rings. Infrared spectroscopy of neuromelanin revealed the presence of aliphatic groups and a low intensity aromatic component, whereas in synthetic melanins the aliphatic groups were absent. Chemical degradation studies showed that neuromelanin contains equal amounts of indole and benzothiazine molecules. Neuromelanin consistently shows a peptide component of about 15%. The amino acids could be derived from a direct reaction between the melanic polymer and proteins, or dopamine molecules bound to cysteinic residues of polypeptidic chains. Indeed, the precursor of neuromelanin synthesis has been suggested to be cysteinyl-dopamine, although a study using hydriodic acid hydrolysis failed to identify the corresponding degradation products. Moreover, nuclear magnetic resonance spectroscopy indicates that the presence of both aliphatic and aromatic hydrogens, and the ratio of aliphatic to aromatic hydrogens is again higher in neuromelanin than in synthetic melanins, suggesting that dopamine cannot be the only precursor in neuromelanin synthesis. x Ray difraction studies have shown that neuromelanin has a multilayer (graphite-like) three dimensional structure similar to synthetic and naturally occurring melanins. The three dimensional structure is derived from planar overlapped sheets consisting of cyclic molecules of indolebenzothiazine rings. However,these sheets are stacked much higher in neuromelanin than in any other synthetic and naturally occurring melanins. The process of neuromelanin formation is obscure, although a recent in vitro study has clearly established some steps of this complex process. It has long been debated whether the synthesis of neuromelanin is enzymatically mediated or whether it is a pure autooxidation process of dopamine derivatives. For eumelanin synthesis, the enzyme tyrosinase (also known as monophenol monoxygenase) catalyses the conversion of tyrosine to L-dopa and then to dopa-quinone. Some authors proposed that tyrosinase could also be involved in neuromelanin biosynthesis because tyrosinase mRNA and promoter activity have been detected in the substantia nigra. However, tyrosinase has not been detected in the substantia nigra by immunohistochemistry. Moreover, albinos who lack tyrosinase display normally pigmented substantia nigra. Alternative enzymatic actions have been suggested, including tyrosine hydroxylase mediated oxidation of dopamine. In another study, peroxidase catalysed the oxidation of tyrosine to dopa and then dopamine, and further oxidisation to the respective quinones that are possible precursors of neuromelanin. It was proposed that prostaglandin H synthase, which has peroxidase activity and is located on the mitochondrial membrane, could mediate the oxidation of dopamine to dopaminequinone, which can internally cyclise to form a pseudo indole derivative called dopachrome. In addition, enzymatic activity of macrophage migration inhibitory factor was suggested for neuromelanin synthesis, because it converts catecholamines intodihydroxyindole derivatives, which are potential precursors of neuromelanin. Alternatively, neuromelanin could derive from non-enzymatic oxidation. The autooxidation of catechols to quinones with H2O2 formation and addition of a thiol has been demonstrated in the brain. A dopamine-melanin can be synthesised by the autooxidation of dopamine, although there are several structural differences between synthetic melanins and the natural one isolated from the substantia nigra. Recently, neuromelanin synthesis was induced in rat substantia nigra neurones and PC cell cultures by exposure to L-dopa. The pigment produced in this model contains a stable free radical; in addition, both light and electron microscopy have shown that the pigment synthesised in these cells appears to be identical to humanneuromelanin, and the granules are surrounded by a double membrane, similar to the naturally occurring neuromelanin of the substantia nigra. In those experiments, treatment with the iron chelator desferrioxamine inhibited neuromelanin synthesis stimulated by L-dopa; therefore, it seems that iron is involved in neuromelanin formation. In this model, neuromelanin synthesis was shown to be driven by an excess of cytosolic catecholamines not accumulated in synaptic vesicles. Neuromelanin interacts with numerous organic molecules including lipids, pesticides, and toxic compounds. MPTP (1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin that after systemic administration selectively kills substantia nigra neurones by conversion through monoamino-oxidase type B activity to methylphenylpyridine (MPP+), which in turn stops the respiratory chain at the NADH-CoQ1 reductase stage, leading in humans and several other animal species to an irreversible parkinsonian syndrome. Neuromelanin might reduce the toxicity of MPTP by accumulating its toxic metabolite MPP+ in vivo. The herbicide paraquat has a molecular structure similar to that of MPTP, and has been proposed as a Parkinson’s disease inducing agent. The pesticide is accumulated in neuromelanin containing nerve cells, where it appeared that the neuromelanin adsorbed intraneuronal paraquat, protecting the neurones from consequent damage. Neuromelanin can also accumulate chlorpromazine, haloperidol, and imipramine, thereby contributing to the control of the intraneuronal concentration of these molecules. Because higher intraneuronal concentrations of dopaminergic drugs might be toxic to substantia nigra neurones, neuromelanin can influence this toxicity. The association of neuromelanin with lipids has been described in several studies. Although previous studies proposed that lipids were part of the neuromelanin molecule, recent work has shown that neuromelanin contains about 20% adsorbed lipids. Cholesterol is a minor component in this lipid mixture, with the major component being a new class of polyunsaturated lipid with a high molecular mass, low volatility, and low oxygen content.31 It may be that neuromelanin itself catalyses the synthesis of this type of lipid. Alternatively, neuromelanin could originate from lipofuscin by an enzymatic reaction occurring in lysosomes, although this hypothesis is not supported by recent observations. In this case, high molecular mass lipids could be derived from a lysosomal metabolic pathway and might interact with neuromelanin within these organelles. High concentrations of iron and other nonalkaline metals are present in several brain nuclei. Neuromelanin from the substantia nigra can interact with many heavy metal ions such as zinc, copper, manganese, chromium, cobalt, mercury, lead, and cadmium; in addition, it binds iron particularly strongly. In the course of Parkinson’s disease and related syndromes, the concentration of iron in the substantia nigra increases by 30–35%. This accumulation of nigral iron seems to occur within the neuromelanin granules: the concentration of iron in these granules is higher in patients with Parkinson’s disease than in normal subjects. Although a neuromelanin–glycolipid complex was proposed as a good chelating and insolubilising system to bind iron ions, it now appears that iron is bound to catecholic groups and not to lipids. EPR studies showed that in the substantia nigra the ferric iron is bound to neuromelanin as a high spin complex with an octahedral configuration. Mössbauer spectroscopy demonstrates that ferric iron is chelated by the neuromelanin polymer and that the iron sites are arranged in a ferritin-like ironoxyhydroxide cluster form. x Ray absorption fine structure spectroscopy and infrared spectroscopy studies confirmed that iron in neuromelanin was bound by oxygen derived phenolic groups in an octahedral con-figuration. In substantia nigra tissue, neuromelanin is only about 50% saturated with Fe(III), therefore maintaining an important residual chelating capability, which can protect against iron toxicity. Neuromelanin can sequester redox active iron ions, reducing the formation of free hydroxyl radicals. Thus, in normal subjects, neuromelanin may play a protective role by inactivating the iron ions that induce oxidative stress. The ability of neuromelanin to chelate other redox active metals such as copper, manganese, chromium, and toxic metals including cadmium, mercury, and lead  strengthens the hypothesis that neuromelanin may be a high capacity storage trapping system for metal ions and, as such, may prevent neuronal damage. Neuromelanin accumulates normally with age in human substantia nigra neurones. A neuronal pigment has also been observed in the substantia nigra of adult rats and dogs, and its concentration seems to depend upon age. In very old (23 months) rats, but not in younger animals, neuromelanin granules were detected by electron microscopy; similar results were observed in aged dogs. Neuromelanin granules were also detected in catecholaminergic cerebellar cells of monkeys (Macaca mulatta and Macaca nemertina) and their presence correlated with age. In human substantia nigra, the first small, brown neuromelanin granules were clearly discernable at approximately 3 to 5 years of age. The neuromelanin content of neurones is highest in individuals in their 60s, after which it decreases; this phenomenon may reflect the neuronal loss observed in these anatomical structures during aging. However, there is no significant loss of catecholaminergic neurones in the substantia nigra of normal subjects until very old ages. A new spectrophotometric method indicates that neuromelanin is not detectable during the 1st year of life, but starts to accumulate thereafter, with a continuous linear trend, and reaches a concentration of 2.3–3.7 mg/g of substantia nigra pars compacta in 50–90 year old individuals. Male and female subjects showed the same age trend of neuromelanin concentration. In patients with Parkinson’s disease, neuromelanin values were 1.2–1.5 mg/g of substantia nigra pars compacta, which is less than 50% of that seen in age matched controls . The absolute number of pigmented neurones in the substantia nigra of normal subjects may be dependent upon ethnicity­an Indian population was found to have fewer pigmented neurones than an age matched Western population. These observations suggest that neurodegenerative disorders characterised by nigral neurone loss, best typified by Parkinson’s disease and other parkinsonian syndromes, are not the result of early aging, as hypothesised in the past. Because the neuromelanin concentration in substantia nigra neurones increases, and the number of pigmented neurones appears to be constant over the life span, it seems that neuromelanin accumulates only in a subpopulation of nigral neurones, whereas other dopaminergic neurones remain nonpigmented. The observed decrease in the neuromelanin concentration occurring in the substantia nigra of patients with Parkinson’s disease confirms the loss of pigmented neurones occurring in the substantia nigra of these patients, as has been reported in neuropathological studies. Other studies indicate that neuromelanin values decrease in the surviving neurones of the substantia nigra during Parkinson’s disease. This could be the result of reduced neuromelanin synthesis, neuromelanin degradation, or higher vulnerability of the pigmented neurones. Neuropathological investigations have examined the presence of extraneuronal neuromelanin in subjects with idiopathic Parkinson’s disease and MPTP intoxication. Most of this extraneuronal neuromelanin is phagocytosed by microglia and is associated with astrocytic and microglial activation. It may be that neuromelanin could be the eVector of a chronic inflammation process in the substantia nigra. Although in idiopathic Parkinson’s disease the neurones are depleted in both the substantia nigra and locus coeruleus, in MPTP intoxicated subjects, locus coeruleus neurones are spared. Such a different neuronal vulnerability might eventually be explained by structural differences in the neuromelanin of the substantia nigra and locus coeruleus. Although neuromelanin may play a cytoprotective role by sequestering redox active metals, toxic metals, and organic toxic compounds,81 neuromelanin might also become a source of free radicals by reaction with hydrogen peroxide. When free neuronal iron increases to the point where neuromelanin becomes saturated and it starts to catalyse the production of free radicals, neuromelanin would become cytotoxic. Moreover, because hydrogen peroxide can degrade neuromelanin, the pigmented neurones could loose this putatively protective agent. The consequence may be a release of iron and other cytotoxic metals or compounds from neuromelanin that could accelerate neuronal death. A critical review of the function of neuromelanin and an attempt to provide a unified theory was recently reported ( Bruno J.R. Nicolaus Medical Hypotheses 2005 65, 791-796 ). This paper  provides a critical review of the numerous and various biological functions so far attributed to neuromelanin and an attempt to provide a unified theory based on the peculiar physical and chemical properties of the black particle (the neuromelanin cage).It is stressed that neuromelanin is not homogeneous, as is commonlyaccepted, but is made up of different substrate specific black pigments formed by the oxidation of o.diphenols or other oxygenated precursors (substantia nigra melanin, locus coeruleus melanin, retinal pigmented epithelium or ocular melanin, inner ear melanin, and so on). Ocular melanin is believed to protect the eye by trapping metals and free radicals. The paper shows that this unconfirmed mechanism is a rather fortuitous irreversible molecular accident, which a times may prove itself deleterious.Albinism often leads to deafness in animals, indicating a geneticcorrelation. These two conditions appear to be correlated at a molecular level to eye/ ear pigmentation and suggest verifying this hypothesis in normal and albino human individuals.Skin and ocular melanin are chemically different. However, they are both involved in lightabsorption/dissipation. The black particle structure (melanin cage) is believed to be fundamental to this process because there is a common bioelectric mechanism. The latter isworth of further investigation. It is also proposed checking how ocular melanin dissipates the excessive absorbed light (as heat or as current?). It has been claimed that inner ear melanin mutes acoustic waves.This paper suggests investigating the underlying mechanism and also studying whether this pigment is bio-electrically involved in audio logy. According to numerous authors, Substantia nigra melanin is only biological garbage. This view is rejected, and it is stressed that intracellular melanogenesis is a fundamental and genetically controlled physiological process. It has been repeatedly claimed that the binding of iron, heavy metals, free radicals and harmful chemicals by substantia nigra melanin is fundamental to body detoxification/protection. Presumably, such irreversible and generic binding mechanisms have no physiological foundation; it is suggested the alternative that, substantia nigra melanin acts as semiconductor, transmitting and modulating nervous impulses, in a reversible way. In fact, substantia nigra melanin is absent or significantly scarce in two conditions of life in which the coordination of movement is either inefficient (newborn babies) or strongly compromised (Parkinson). To check this assumption, further investigation of nucleus caudatus, putamen, globus pallidus,substantia nigra pars compacta and reticulata, nucleus hypothalamicus is recommended .

 

 

Pheomelanin  particles generally derived from the aminoacid 5-S-cysteinyldopa ( R.A.Nicolaus  1965) . Light brown, or reddish , free or as protein complex alkali soluble animal pigments found in hair, fur, feather . The pigments of New Hampshire chicken feathers ( gallopheomelanins ) are polycondensed structure showing the presence of Benzothiazole,Benzothiazine,Quinoline rings.

pheomelanin

 

 

 

Typical degradation products are thiazole acids, 3-amino-4-hydroxy-benzoic acid , 3-hydroxy-4-aminobenzoic acid,  3-amino-4-hydroxytoluene, 3-hydroxy-4-aminotoluene, 1,6-(4-hydroxybenzothiazolyl)-alanine,  beta-6-(2-methyl-4-hydroxybenzothiazolyl)-alanine,  beta-7-(4-hydroxybenzothiazolyl)-alanine, beta-7-(3-oxo-5-hydroxy-3,4-dihydro-2H-1,4-benzothiazinyl)-alanine, 3-hydroxy-4-aminophenylalanine.After methylation of the pigments the followings degradation products were obtained  :  2,3,4,6,-tetracarbomethoxypyridine,   3,4,6-tricarbomethoxypyridine-2-carboxamide,  2-(  2’-(4’,5’-dicarbomethoxythiazolyl)  )-3,4,6-tricarbomethoxypyridine , 3-Carbomethoxy-6-methoxy-7-aminoisoquinoline, 1-methyl-3-carbomethoxy-6-methoxy-7-aminoisoquinoline, 1-methyl-3-carbomethoxy-6,7-dimethoxy-isoquinoline. Some of these products are utilized for pheomelanin identification.

 Eumelanins and Pheomelanins may coexist (copolymerized ?  ) in Nature ( 1a ), ( 4 )  ( 9 ).

 Some of these degradative compounds have been used for pheomelanin identification.

Pheochromes   red or  yellow crystallisable, low molecular weight, compounds derived from 5-S-Cysteinyldopa ( Cysdopa ) and isomers . The only   pigment structure elucidated (see formula below) is that  isolated for the first time from the  New Hampshire chicken feathers ( R.A.Nicolaus 1967 ) which also occurs in red hair.  Different confusing names are trichosiderins, tricochromes, pyrrotricholes  ( 1a ), ( 4 ) . Pheomelanins and pheochromes have many similar degradation products. Chromophores (  II, III, IV ) have been synthetised by  B.L.Kaul ( 10 ) and G.Prota  ( 4 )

 

 

 

 

 

     The Pigment of red hair

The cromophores

Allomelanin  ( 5 )  derived from nitrogen-free precursors like catechol and 1,8-dihydroxynaphtalene have been  found in plants,  mould, yeast, bacteria, humic acid, fulvic acid  etc. Benzenepolycarboxylic acid  and  catechol derivatives were obtained as oxidative degradation products. A typical allomelanin is the graphite-like pigment of the Aspergillus niger discovered by A.Quilico.   ( 11 ),  ( 4- 6 ) ( 1a, 1b,1m ). Mellitic acid in high yield is produced  by oxidation of the pigment.

The photo was a gift of Professor A.Quilico ( Milan 1960 ).Allomelanin are difficult to obtain in pure form.Chemistry of allomelanins is little known

Some well known and famous  BSM are  : Acetylene-black ( 12 ),( 13), Pyrrole-black, Aniline-black.

 

 

Sepiomelanin  chemistry    

 Melanin chemistry ( 1-4 ), ( 6) has produced poor results so far, due to the use of  raw materials in chemical and physical analyses and to scarce knowledge of solid state science by the researchers involved. The discovery of the particle ( Chedekel 1992 ) is expected, in our view, to change the strategy of future structural and functional melanin studies.  Mass spectrometry  is expected  to be  the most relevant fragmentation tool, replacing the old oxidative and reductive degradation techniques used in the chemistry of organic natural products.

Melanin generally does not dissolve in water, acids, alkalies or organic solvents. Sonication may produce a fine dispersion in alkali. IR and NMR spectra show the presence of OH, COOH,  NH2, NH, CH2 groups.

 Alkali fusion of eumelanin gave 5,6-dihydroxyindole ( DHI ) together with 5,6-dihydroxyindole-2-carboxylic acid ( DHICA ) ( Table I ,  14, 15 ) Typical pyrrolic acids ( TABLE I ) are formed by oxidation of eumelanin ( 16-21, 22-25 ).Pyrrole-tricarboxylic acid is also formed by boiling sepiomelanin with dilute NaOH 4% . Although degradation products are formed in low yield they gave interesting information about melanin structure.

 

 

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TABLE  I

:Sepiomelanin degradation products  : alkali fusion .

                                       DHICA

5,6-dihydroxyindole                                             5,6-dihydroxyindole-2-carboxylic acid

 

Sepiomelanin degradation products  : KMnO4  oxidation

 

Pyrrole acids ( 1 – 23, 44- 45 )

 

 

                                     

 

 

 

Complex pyrrole acids obtained as degradation products of sepiomelanin  (  26a, 26b, 42  )  :

 

                                        

 


 

 

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 On heating at 200° CO2 is evolved. Melanin reacts with diazomethane ( Table II ) and CH3OH+HCl giving an ether and an ester respectively. Melanin is oxidised by H2O2 ( a process which occurs also in vivo), KMnO4  , chlorine and bromine water, but is difficult to be reduced. Oxidation produces not only simple but also   complex pyrrole acids ( isolated as Ba salt ) ( 26a ). MALDI spectra  (26b)  show degradation products useful in   the study  of the chemical structure of melanin), ( 27 ), . It is possible that all eumelanin samples obtained until now are artefacts ( breakdown of the benzenoid part ) produced by  cellular H2O2

 

.  

 

TABLE II

Percentage of nitrogen , methoxyl goups in different methylated pigments.

CO2 evolved during heating at 190-200°.

 (Piattelli M.,Fattorusso E, Magno S.,Nicolaus R.A. The structure of melanins and melanogenesis II.Sepiomelanin and synthetic pigments. Tetrahedron 1962, 18, 941-949 ).

The methylated pigments are light brown in colour,infusible and insoluble.

  Methylated samples                                                                               Free samples

                                                                       % N            % OCH3       % N         % CO2         

 Sepiomelanin                                                 7.4                 18.8              8.5              9.1

DOPA-melanin (tyrosinase)                            7.4               20.6           7.3           7.5

DOPA-melanin ( tyrosinase+catalase )            6.5                 19.6              6.3             5.2

DHI-melanin ( autoxidation )                           7.5                  19.1              7.6             9.3

DHI-melanin   ( tyrosinase )                             7.7                   21.6             8.0              9.6

DHI-melanin ( tyrosinase + catalase )                7.7                  21.5           8.2              4.7

Pyrrole black                                                     14.0                   8.6             13.8             4.9

____________________________________________________________________

BCM  are particles derived from polyphenols .The black particles discovered by M.R. Chedekel in  1992 ( 28  ) show  an interesting  internal organisation as reported by M.G.Bridelli and J.D.Simon   ( 29-30 )  but results are  still uncertain because the use of raw material in the atomic force microscopy experiments. The particles are formed  from radical-polarone oligomers  of acetylene-black type, as established by theorethical calculations ( 12 ), ( 13 ) . They show high binding power towards organic and inorganic compounds, to liquids and  gases.These properties may influence or distort the results  of chemical and physical analysis. The black particles are conductors  showing the McGinness-Proctor ( 31 ) effect ( melanin exhibits the unusual characteristics of an amorphous semiconductor with threshold switching ). Melanin  may act as an innovative sensor and energy generator, with the capacity of growing and shrinking in length and volume, by electric stimulation.  It is interesting to note that the particle may exhibit non-invasive control of the shape and growth of mammalian cells, as showed by D.E.Ingber (  32 ). Melanins show typical broad EPR, IR, solid state   NMR (  spectra show an aliphatic part  ) spectra (1a), ( 2 ), ( 4 ), ( 5a,5c ), ( 6a,6b ) ( 49). The MALDI  procedures are not useful for molecular weight determination as in the case of proteins. It is obvious that for a particle the term “ molecular weight “, frequently reported in literature  cannot be used in melanin chemistry. ( 33-34 ).

Particles are formed by oligomers . It is interesting to note that in those oligomers the system indicated by the red line in the formulae corresponds to the structure of the acetylene-black  and is present in many BCM ( Black Cell Matter ) and BSM ( Black Synthetic Matter ) . Formation of indolequinone hydrate was observed in the polymerization process of dopamine or DHI ( 5,6-dihydroxyindole )    ( 35 ) .   The oligomeric indole units   may form a graphite-like stack with spacing of 3.4 Å,  a fullerene structure or, according to recent molecular mechanics  calculations, they may have a helical structure ( 33 -34 ) .

It seems that indole oligomers may form particles of different size and shape due to the preparation method.The size and shape, type of oligomers  may influence some physical properties, like conductivity.

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TABLE III

DHI-melanin and neuromelanin oligomers

 

DHI-melanin

 

 

 

Dopamine-black  ( neuromelanin ? ) in vitro

 

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The structures show ( TABLE III )   neuromelanin oligomers compared with acetylene black system   . Dopamine units predominate in neuromelanin  in vitro ( 5-6, 11,  36 ). The structure of neuromelanin in vivo is unknown but is probably a  mixture of dopamine-melanin , DHI-melanin, cysteinyldopamine-melanin . Also  to remember is  that  adrenalin forms in presence or absence of oxygen a melanin. ( 4-5, 36  )  Adrenalin-melanin is different from neuromelanin .  Generally melanins have about one oxygen more than the corresponding precursors.

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DHI ( .5,6-Dihydroxyindole  ), whih may obtained also by melanin degradation,  is generally considered the precursor of eumelanin . Differences between  DHI-melanin  and the natural pigment   are observed in IR, 13C  NMR ,   MALDI spectra, and   AFM figures . As regards MALDI ( matrix assisted laser ionization/desorption )  and MALDI-TOF  ( TOF=time of flight ) spectra, no high mass peaks have been detected,whereas a lot of ,low colourless fragments arising fron melanin breakdown have been recorded TABLE  IV

TABLE IV

MALDI spectra

Sepiomelanin

273, 313, 335, 349, 363, 369, 373, 391, 450, 526, 552, 685.

DHI-melanin ( autoxidation)

497, 516, 542, 572, 579, 692, 722, 744, 778, 874, 930, 983, 1047, 1080, 1136.

DHI-melanin ( peroxidase )

497, 524, 540, 552, 572, 703, 714, 731, 868

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  This fragmentation, which occurs both in vitro and in vivo was discovered by D.I.McAuliffe ( 37 ) and   resembles the LASER graphite fission . Sepiomelanin and DHI-melanin methylated with diazomethane have similar percentages of -OCH3 groups as reported by R.A.Nicolaus ( 26-27 )   The methoxy group value  is different from the theoretical values. An  interpretation given by A.Bolognese ( 34 )  suggest that about half of the hydroxyls present are masked and not methylable.  The black particles have a  structure (internal) organisation which  may explain the binding effect of solid, liquid and gases observed by H.Rorsman ( 38 ) . Natural and synthetic melanins are still today considered polyindolequinones, justifying the difference between the found  (three oxygen for every indole unit) and  calculated values of polymer analysis by the presence of molecules of water  ( 39 ). Thermogravimetric analysis performed on freshly prepared sepiomelanin samples recorded two transition temperatures of about 100° and 150° according to the loss of two possibly different kinds of water ( 40 ).In conclusion chemistry only plays a small  role in  the determination of chemical structure of melanins. Degradation products and analysis show sepiomelanin oligomers to be chiefly formed   by DHI and DHICA units. Pyrrole units are present.The study of  the solid state seems more promising. It is  necessary to work on purified and analytically tested melanin samples ( recommended elemental analysis for  C , H,  N, S, Cu, Fe, Ni, Zn ).

THE INK GLAND

This pigment, contained in the ink gland of  Sepia officinalis  was well studied from a chemical point of view. The ink gland described by A.Palumbo ( 41 - 42 )  is a highly specialized organ with  immature cells in the inner portion , from where the cells gradually mature, migrate towards the outer portion of the gland and become competent to produce melanin giving rise to mature particles  (melanosomes ). When cell maturation  is complete, melanin is secreted into  the lumen of the gland, accumulated into the ink sac and ejected on demand . The pigment samples were found to differ in properties and chemical composition depending to the mode of extraction  and storage time. Fresh ejected ink differs from the ink remaining into the sack.  Simple extraction can induce some transformations, like morphology, hydroxylation processes, further oxidation, benzenoid ring  breakdown.

COLOURATION

The methylether obtained treating the pigment with diazomethane was in the form of an infusible light brown powder The change of  “colour” indicates that a physical “colour” contributes to the black “colouration”  of the particle. In 1987  a  unified  physical model of  pigment “colour” was presented   The model  is based on  measurements of the optical constants of  eumelanin and pheomelanin. Using the results of exact Mie calculations of the scattering  and absorption cross- sections for individual  pigment granules, it was shown  that the  colours produced by dispersions  of  eumelanin or  pheomelanin granules are strongly dependent  on the pigment granule  size  as reported by L.Wolfram ( 46 ), ( 47 ).In conclusion chemistry only plays a small  role in  the determination of chemical structure of melanins.  The study of  the solid state seems more promising. It is  necessary to work on purified and analytically tested melanin samples ( recommended elemental analysis for  C , H,  N, S, Cu, Fe, Ni, Zn ).

HELICOIDAL STRUCTURE OF OLIGOMERS

Molecular modelling studies on the 5,6-indole-dione monohydrate at 5 position showed that linear dimeric, trimeric, tetrameric, pentameric, etc. forms may assume two low energy conformations sheets and helixes . The first conformation,  sheet,  shows  two areas with different polarity, the second one is like a helix was observed by A.Bolognese   (  36,  40 ) .Constrained by steric hindrance, the alternated, pleated sheet and the helix are held in their shapes by hydrogen bonds between a hydroxyl group and the heterocyclic nitrogen. The hydroxyl group over (or under) the indolic plane and the heterocyclic nitrogen generate the helix, whereas a bond between the hydroxyl group, alternatively, over and under the indole plane and the heterocyclic nitrogen determines the alternated pleated sheet. Whenever the helix behaves as an organic conductor, according to reported acquisitions on acetylene-black, it could constitute a very long solenoid able to generate a magnetic field at the far end of the molecule with the long axis of the helix coiling around the backbone formed by double bond linking 4 and 7 positions of starting 5,6-indol-di-one monohydrate.Even if the unsaturated carbon backbone is not completely planar as reported on the acetylene black, nevertheless partial orbital overlapping is possible, as the bond length between the two junction carbons of the dienic unit demonstrates 1.49 Å. This distance is very close to the length of an aromatic conjugate C-C bond. Both proposed conformations can satisfy the  properties and different roles of melanin in the animal kingdom. It is to note that reactions involving the radical or the cation site of the melanin radical-polarone produce a local carbon hybrid change and determine variation of the strand orientation in  space. This response to chemical radical attack could determinate abrupt conductivity changes and  indicate a possible chemical switching function of this pigments. On this topic, it was reported that melanins respond to a critical applied field by changing their conductivity and that the nature of response depends on hydration and temperature of sample and on external circuitry   Melanin  exhibits two separate current-voltage characteristics, the on and off state. Experiments have demonstrated that their switching depends on hydratation (gem-diol formation).

REFERENCES

1) . a   R.A.Nicolaus (1978)   Melanins     in  METHODICUM CHIMICUM  A critical survey of

proven methods and their application in chemistry, natural science and medicine,  Vol.11, Part 3,   Ed.  Friedheim Korte, Academic Press New York, Georg Thieme Stuttgart, Maruzen Co. Tokyo, pp.190-199.

b.  Nicolaus R.A.  La Chimica delle Melanine  Accademia Nazionale  dei Lincei, Fondazione Donegani,III° Corso Estivo di Chimica, Varenna 23 settembre-7 ottobre 1958, 261-275.

 Nicolaus R.A., Piattelli M. Narni G. The structure of sepiomelanin  Tetrahedron Letters 1959, 22, 14-17.

c. Nicolaus RA, Piattelli M., Narni G. Sulla struttura della sepiomelanina Rend.acc.sci.fis.mat. 1960 Vol XXVII, 1-12

d. Nicolaus R.A. Piattelli M.  Structure of melanins and melanogenesis  J.Polym.Science 1962, 158, 1133-1140.

e . NicolausR.A.  Biogenesi delle melanine   Accademia Nazionale dei Lincei , Fondazione Donegani, VII Corso estivo di Chimica, Milano 21 Settembre-3 Ottobre 1962 pp.291-319.

Fattorusso E. Piattelli M. Nicolaus R.A.  Su alcuni neri naturali  Rend.Acc.Sci.Fis.Mat. 1965, Vol.XXXII, 3-4 .

 f. Nicolaus R.A. Piattelli M.  Progress in the Chemistry of Natural Black Pigments  Rend.Acc.Sci.Fis.Mat. 1965, Vol.XXXII, 3-17.

g.  Fattorusso E. Piattelli M. Nicolaus R.A.  Su alcune melanine naturali  Rend.Acc.Sci.Fis.Mat. 1965, Vol.XXXII, 200-20

h.  Prota G.Piattelli M. Nicolaus R.A.  Preliminary results in the study of phaeomelanins  Rend.Acc.Sci.Fis.Mat. 1966, Vol.XXXIII, 146-150.

i.  Fattorusso E. Cimino G.  Ulteriori ricerche sulla sepiomelanina  Rend.Acc.Sci.Fis.Mat. 1966,Vol.XXXIII, 378-380.

l. Nicolaus R.A. Comments on Howard S.Mason Paper.The structure of Melanin  in Advances in Biology of Skin-Volume VIII The Pigmentary System. Proceedings of a Symposium held at The University of Oregon Medical School 1966. Pergamon Press 1967.

m.  Fattorusso E. Minale L. Sodano G. Feomelanine ed eumelanine da nuove fonti naturali  Gazz.Chim.Ital. 1970, 100, 452-460.

n.   Nicolaus R.A.  The Nature of Mammalian Colors  La Chimica e l’Industria  1972, 54, 427-433.

 o . Piattelli M. Nicolaus R.A.  Struttura e biogenesi delle melanine  Rend.Acc.Sci.Fis.Mat. 1960, Vol.XXVII, 499-502 .

2 ) . ) . a,_________________________________________________________________________________________________________________________ Benathan M.  Contibution à l’analyse quantitative des melanines. Application comparèe de methodes de caractèrisation et de dègradation oxydative à la melanine de l’encre de seiche,à la mèlanine de l’iris de bœuf et à la dopa-melanine   Universitè de Lausanne, Facultè des Sciences, Thèse de Doctorat , Imprivite S.A.,Lausanne 1980 .

3 ) . Benathan M. Wyler H. Contribution a l’analyse quantitative des melanines  Yale J. Biol.med.1980, 53, 389 (ABS) ;

4 ) . Prota.G.  (1992 )  Melanins and melanogenesis   AP   San Diego pp 1-279

5 ). a  Nicolaus R. A. (1968) Melanins  in Chemistry of Natural Products, Series edited by Edgar Lederer, Hermann,  Paris pp.1-305. .See also (1), (2), (9), (12), (19), (20).

b  . Look at  INTERNET the voices allomelanin,  fungal melanin, microbial melanin, humin, humic acids, fulvic acid.

  c  . Thomson R.H.  Melanins   in Comparative Biochemistry.Vol.III.Costituents of Life,-Part A, pp. 727-753. Ed. Florkin M. , Mason H.S.  AP, 1962 

6 ) . a.  Swan G.A. Structure chemistry and biosynthesis of the melanins  in Fort.Chem.Org.Nat., Vol 31 , 522-528 , SV Wien 1974

 b. Nicolaus R.A.(1984) Melanine  in Quaderni della Accademia Pontaniana n°4, Ed.Giannini Napoli pp. 1-59. The most important papers of G.A.Swan are reported.

7 ). Hearing VJ. Lutzner MA.  Mammalian melanosomal proteins : Characterization by Polyacrylamide Gel Electrophoresis.  Yale Journal of Biology and Medicine 1973,46, 553-559 ; Hearing V.G., Ekel T.M., Montague P.M., Nicholson J.M., BBA 1980, 611, 251-268

8 ) . Kushimoto T. Basur V. Valencia J. Matsunaga J. Vieira WD Ferrarsi VJ. Muller J. Appella E. Hearing VJ. A model for melanosome biogenesis based on the purification and analysis of early melanosomes  2001 PNAS 98, 10698-10703

9 ) . a  Santacroce C. Sica D. Prota G. Nicolaus R. A.  Delta2,2 –BI (5 hydroxy-7-methyl-8-(2-hydroxy-4-methylphenoxy)-2H-1,4-benzothiazine) : an interesting model compound for the study of chicken feather  pigments  Rend.Acc.Sci.Fis.Mat 1968,Vol.XXXV, 3-5.

b.   Thomson R.H. The pigments of reddish hair and feathers Angew. Chim. Int. Ed. English 1974; 13 ; 305 -312

10 ) . Kaul  B.L.  Studies on Heterocyclic Colouring Matters. Part II: ?2,2'-Bi(2H-1,4-benzothiazines), Helv. Chem. Acta 1974;  57; 2664-2678  The chromophore of pheomelanin was synthesed  ; Prota G. Ponsiglione E. Ruggiero R. Synthesis and photochromism of Bi-(2H-1,4-benzothiazine) Tetrahedron 1974, 30, 2781-2784. 

11 ) .  See  (1), (41), (47).

12 ) . Medrano J. Dudis D. (1990) Quasi-Particles in Polymeric conductors in organic superconductivity, Ed. Vladimir Z. Kresin and William A. Little, Plenum Press  pp 275-285

13 ) . Blumstein A.  S. Subramanyam S.   (1990) Conjugated ionic polyacetylenes : novel structures and model for a polymeric high Tc  superconductor  in Organic Superconductivity, edited by Vladimir Z. Kresin and William A. Little, Plenum Press pp.335-341.

14 ) a.  Piattelli M. Fattorusso E . Magno S. Nicolaus R.A.  Alkali fusion of sepiomelanin : isolation of 5,6-dihydroxyindole  Rend.Acc.Sci.Fis.Mat. 1962, Vol.XXIX,  3-4 .

b . Piattelli M. Fattorusso E. Magno S. Alkali fusion of sepiomelanin : identification of 5,6-dihydroxyindole-2-carboxylic acid   Rend.Acc.Sci.Fis.Mat. 1962, Vol.XXIX, 1-2.

15 ) .  Fattorusso E. Nicolaus R.A. Sussman H. Kertesz D. Sul processo di trasformazione del 5,6-diossindolo in melanina  Rend.Acc.Sci.Fis.Mat. 1966, Vol.XXXIII, 372-377.

16 ).a Nicolaus R.  Sugli acidi pirrolcarbonici .Nota I. Acido 2,3,5-pirroltricarbonico Gazz.Chim.Ital. 1953, 83, 239-251.

b.  Binns F. Swan G.A. Oxidation of some synthetic melanins  Chem.and Ind.1957, 396-404.

17 ) . Nicolaus R. Sugli acidi pirrolcarbonici  La ricerca scientifica 1953, 23,1836-1638

18 ) . Nicolaus R., Oriente G.  Sugli acidi pirrolcarbonici : acido 2,3,4,5-pirroltetracarbonico  Gazz.Chim.Ital.1954, 84, 230-241

19 ) . Nicolaus R.A.  Cromatografia su carta dei prodotti di demolizione con H2O2     .di melanine naturali e della tirosina melanina  Gazz.Chim.Ital. 1955, 85, 659-664 .

20 ) . Nicolaus R.A., Mangoni L., Sugli acidi pirrolcarbonici.Nota IV. Cromatografia su carta degli acidi pirrolcarbonici e applicazioni allo studio dei pigmenti neri  Gazz.Chim.Ital. 1955, 85, 1397-1404.

21 ) . Nicolaus R.A., Mangoni L. Sugli acidi pirrolcarbonici.Nota V.Acido 2,3,4-pirroltricarbonico

Gazz.Chim.Ital. 1956, 86, 358-370.  1957, Vol.XXII, 311-317.

22 ) .a. Vitale A. Piattelli M. Nicolaus R.A.  Sugli acidi pirrolcarbonici.Nota XIII.Sulla preparazione dell’acido 2,3,4,5-pirroltetracarbonico  Rend.Acc.Sci.Fis.Mat. 1959, Vol XXVI, 267-271.

b .  Nicolaus R.A. Scarpati R. Forino C. Sugli acidi pirrolcarbonici.Nota X. Ossidabilità degli acidi metilpirrolici  Rend.Acc.Sci.Fis.Mat. 1959, Vol.XXVI, 51-66.

23 ) . Scarpati R. Nicolaus R.A.  Sugli acidi pirrolcarbonici.Nota XI.Ossidabilità dell’acido 2,3,4-pirroltricarbonico in relazione a sistemi naturali a scheletro pirrolico  Rend.Acc.Sci.Fis.Mat.  1959,  Vol.XXVI,  3-11.

 24 )  Piattelli M. Fattorusso E. Magno S.  Isolation of pyrrole-2,3,4-tricarboxylic acid and pyrrole-2,3,4,5-tetracarboxylic acid from sepiomelanin oxidation products  Tetrahedron Letters 1961, 20,718-719.

25 ) .Piattelli M. Fattorusso E. Magno S. Nicolaus R.A. Identificazione degli acidi 2,4-pirroldicarbonico e 2,5-pirroldicarbonico tra i prodotti di ossidazione della sepiomelanina decarbossilata  Rend.Acc.Sci.Fis. Mat. 1961, Vol. XXVIII, 165-167.

26 ) a. Panizzi L.Nicolaus R.A:  Ricerche sulle melanine  Gazz.Chim.Ital. 1952, 82, 435-460  ; Piattelli M. Nicolaus R.A. The structure of melanins and melanogenesis-I-The structure of melanin in Sepia  Tetrahedron 1961;  15; 66-75 ; 1962; 18; 941-949 ; 1964 ;  20 ; 1163-1172

 b.  Nicolaus R.A. MALDI mass spectrometry and melanins   Rend.Acc.Sci.Fis.Mat, 1997, Vol. LXIV, 315-324

27 ) .Piattelli M. Fattorusso E. Magno S. Nicolaus  R.A. The structure of melanins and melanogenesis-III-The structure of sepiomelanin  Tetrahedron 1963; 19; 2061-2072.

28 ) . Zeisel L. Addison R.B. Chedekel   M.R.  Bioanlytical studies of eumelanin.I.Characterization of melanin  : the particle  Pigment Cell Res Suppl. 2,  1992,  48-53 .

29 ) . Bridelli  M.G.  Self-assembly of melanin studied by Laser light scattering   Biophys.Chem. 1998;  73; 227-239 .

30 ) .  Clancy C.M.  Simon  J.D. Ultrastructural organization of eumelanin from Sepia officinalis measured by atomic force microscopy   Biochemistry 2001 , 40, 13353-17360  ; Clancy C.M.R , NofsingerJ.B. Hanks R.K. Simon J.D.   A Hierarchical Self-Assembly of Eumelanin  J.Phys.Chem B 2000,104,7871-7873.

31 ) . McGinness J.Corry J.  Proctor  J. P.   Amorphous semicoductor switching in melanin  Science 1974; 183;  853-858

32 ) . Wong J.Y. Langer R. Ingber  D.E.   Electrically conducting polymers can non invasively control the shape and the growth of mammalian cells     Proc. Natl. Acad. Sci. USA , 1994;  91; 3201-3205.

33 ) .  Nicolaus R.A. Bolognese A.  Nicolaus B. The Pigment Cell and its Biogenesis  Atti della Accademia Pontaniana 2002; Vol L  225-243 .

 34 ) . Nicolaus R.A. Bolognese A. La Vecchia A.  Mazzoni O. B.Nicolaus, I.Romano Nicolaus G.  Perspectives in melanin chemistry  Atti Accademia Pontaniana  2004; Vol. LIII ; 415-441

35 ) .  Kroesche C. Peter M.G.   Detection of   melanochromes by MALDI-TOF Mass Spectrometry    Tetrahedron 1996 ;  52; 3947-3954 ; Peter M.G. Chemical modification of biopolymers by quinines and quinones methides  Angew.Chemie Int.Ed.Engl. 1989, 28, 555-570  ; Peter M.G.,Forster H.,  The structure of eumelanins.Identification of compositions patterns by solid-state NMR spectroscopy  Angew,Chemie Int.Ed.Engl., 1989, 28, 741-743.

36 ) . Short Communications of the Atti della Accademia Pontaniana :  a. Nicolaus R:A: The chemistry of interstellar black matter 1999,Vol.XLVIII. b. Nicolaus R.A. Soluzione dell’enigma chimico : prospettive in Biologia  2000,Vol.XLIX. c. Bolognese A., Nicolaus R.A. About the structure of sepiomelanin  2000, Vol. XLIX.  d . Bolognese A., Nicolaus R.A. Nero di Adrenalina  2002, Vol. L. e. Bolognese A., Nicolaus R.A.  Conduttori biologici neri  2002, Vol. L  6)  Nicolaus R.A.  Revisione dello schema di Raper  2002, Vol. L .

37 ) .  Jacques SL.McAuliffe DJ.   The melanosome : treshold temperature for explosive vaporization and internal absorption coefficient during pulses Laser irradiation   Photochemistry and photobiology 1991 ; 73; 769-764

38 ) .  A vast literature on pheomelanin  and  the binding effect of melanin is disponable thanks to the Rorsman Swedish school. ( Rorsman, Agrup, Rosengren Larsson, Lindquist,Tjaslve, Mars )

39 ) . Beer  R.J.S. Broadhurst T.  Robertson A.  The chemistry of melanins. Part V . The autoxidation of 5,6-dihydroxyindoles     J Chem Soc  1954; 1947-1953 .

40 ) . Nicolaus RA. Bolognese A. Lavecchia A. Mazzoni O. Novellino E. Light on black.Melanins :opening doors into biological conductors Atti del IV Convegno Nazionale sulla Scienza e Tecnologia dei Materiali, B31,Ischia Porto (NA) 29 giugno-2 luglio 2003.

41 ) . Palumbo  A.  Melanogenesis  in the ink gland of  Sepia officinalis  Pigment Cell Res  2003 ; 16; 517-522

42 ) . Nicolaus RA.  Nicolaus G.   The ink in Sepia   Atti   Accademia  Pontaniana   2005 ; Vol. LIV ; 1-26

43 ) . Nicolaus R.A., Caglioti L.  Ricerche di acidi pirrolici nelle miscele di ossidazione  La Ricerca Scientifica  1957, 27, 113-116.

44 ) . Piattelli M. Fattorusso E. Magno S. Nicolaus R.A.  Sepiomelanina e pigmenti sintetici  Rend Acc.Sci.Fis.Mat. 1961, Vol. XXVIII, 337-339.

45 ) . Piattelli M. Fattorusso E. Nicolaus R.A.  Formazione di acido 2,3,5-pirroltricarbonico dalla sepiomelanina per azione degli alcali diluiti  Rend.Acc.Sci.Fis.Mat 1964,Vol.XXXI. 333-334

46 ) . Kurtz S.K. Albrect L. Schultz T. Wolfram L. ( 1987 )  The physical origin of colour in melanin pigment dispersion Communication presented at the first meeting of  The European Society for Pigment Cell Research held in Sorrento October 11-14.

47 ) . Nicolaus RA. Perspectives in Chemistry and Biology of the Melanin Particle   Late-breaking abstracts PP008A  XIX  International Pigment Cell Conference, Reston,Virginia,USA, September  18-23 , 2005

48.) a . Nicolaus G. Nicolaus RA. Melanins,Cosmoids,Fullerenes Rend Acc Sci Fis Mat  1999 ; Vol. LXVI ; 131-158.b. Nicolaus R.A.,Scherillo G., La Melanina.Un riesame su struttura,proprietà e sistemi  Atti Accademia Pontaniana 1995, Vol. XLIV, 265-287. c. Nicolaus R.A. Divagazioni sulla struttura a banda del colore in Natura : il nero  Rend.Acc.Sci.Fis.Mat.,1997, Vol. LXIV, 145-216. d. Nicolaus B.J.R., Nicolaus R.A. Speculating on the band colours in Nature  Atti Accademia Pontaniana, 1997, Vol., XLV, 365-385. e. Nicolaus R.A. MALDI mass spectrometry and melanins  Rend.Acc.Sci.Fis.Mat, 1997, Vol. LXIV, 315-324. f.Nicolaus R.A.Coloured organic semiconductors  Rend.Acc.Sci.Fis.Mat. 1997, Vol LXIV, 325-360 g. Olivieri M.,Nicolaus R.A.  Sulla DHI-melanina  Rend.Acc.Sci.Fis.Mat. 1999, Vol.LXVI, 85-96. h.  Nicolaus B.J.R., Nicolaus R.A., Olivieri M.  Riflessioni sulla chimica della materia nera interstellare   Rend.Acc.Sci.Fis.Mat., 1999 , Vol.LXVI, 113-129. i.  Nicolaus B.J.R., Nicolaus R.A., Lo scrigno oscuro della vita  Atti Accademia Pontaniana , 2000, Vol.XLVIII, 355-380. m. Nicolaus,RA The chemical structure of melanin Atti Accademia Pontaniana,2005,Vol.LIV,1-22.

49) . Adhyaru B.B. Akhmedov N.G. Katritzky A.R. Bowers C.R.  ‘’  Solid-state cross-polirazation magic angle spinning 13C and 15N   NMR characterization of Sepia melanin,Sepia melanin free acid and Human hair melanin in comparison with several model compounds  ‘’  Magnetic Resonance in Chemistry, 2003, 41, 466-474

 

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133 ) Ikemoto K, Nagatsu I, Ito S, King RA, Nishimura A, Nagatsu T.
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134 ) Bisceglia M, Nirchio V, Attino V, Di Cerbo A, Mantovani W, Pasquinelli G

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135 ) Nishio T, Furukawa S, Akiguchi I, Sunohara N.

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146 ) Sulzer D, Zecca L

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The effects of nitric oxide on the oxidations of l-dopa and dopamine mediated by tyrosinase and peroxidase. J Biol Chem. 2001, 276,11214-11222. 

 

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CHEMICAL METHODS IN EUMELANIN , PHEOMELANIN, ALLOMELANIN DETERMINATION .

A ) . Nicolaus R.A.  The Chromatographic study of pyrrolic acids arising from oxidative degradation of natural pigments  Rassegna di Medicina Sperimentale,Anno VII, Supplemento N° 2Ed.Idelson ,Napoli 1960 B ). Nicolaus R.A., Piattelli M., Fattorusso E.,  The structure of melanins and melanogenesis-IV-On some natural melanins  Tetrahedron  1964, 20, 1163-1172.  C ) . Sealy R.C. , Hyde J.S., Felix C.C., Menon I.A., Prota G.  Eumelanins and Pheomelanins : Characterization by Electron Spin Resonance Spectroscopy  Science 1982, 217, 545-547 .D ) . Ito S., Jimbow K.   Quantitative Analysis of Eumelanin  and Pheomelanin in Hair and     Melanomas  J.Invest.Derm. 1983, 80, 268-272. E ) . Wakamatsu K., Ito S., Rees J.L.  The usefulness of 4-Amino-3-hydroxyphenylalanine as a Specific Marker of Pheomelanin  Pigment Cell Res. 2002, 15,225-232  F )  . Wakamatsu K., Ito S.  Advanced Chemical Methods in Melanin Determination  Pigment Cell Res.2002, 15, 174-182  G ) . Ito S., Fujita K.  Microanalysis of eumelanin and pheomelanin in hair and melanoma by chemical degradation and liquid chromatography  Anal.Biochem., 1985, 144, 527-536. H ) . Ito S., Wakamatsu K., Ozeki H.,  Spectrophotometric assay of eumelanin in tissue samples  Anal.Biochem. 1993, 215, 273-277. I ) . Ozeki H., Ito S., Wakamatsu K., Thody H.J.   Spectrophotometric characterization of eumelanin and pheomelanin in hair  Pigment Cell Res. 1996, 9 , 265-270. L ) . Kolb A.M., Lentjes E., Smit N., Schothorst A., Vermeer B.J., Pavel  S. Determination of Pheomelanin by measurement of aminohydroxyphenylalanine isomers by high-performance liquid chromatography  Anal.Biochem. 1997, 252, 293-298.  M ) . Takasaki A., Nezirevi D., Johanson M., Wakamatsu K., Ito S.,Kagedal B.   Analysis of pheomelanin degradation products in biological samples  Pigment Cell.Res. 2001, 14, 408-409.    N ).  .Scrocco M. Nicolaus R.  Studio spettrofotometrico nell’infrarosso di alcuni derivati del 4-metil-3,5-dicarbetossi- pirrolo  Rendiconti  Accademia Nazionale dei Lincei,  1956, Vol XXI, 103-109. O ) . Scrocco M. NicolausR. Ricerche spettrofotometriche IR ed UV su una serie di esteri pirrolici  Rendiconti della Accademia Nazionale dei Lincei, 1957, Vol.XXII, 500-503. P ) Scrocco M. Nicolaus R. Costanti di ionizzazione acida e comportamento spettrofotometrico nell’IR e nell’ UV di acidi C-metil-pirrolici  Rendiconti della Accademia Nazionale dei Lincei ,

Riassunto in lingua italiana

Circa un anno fa è apparsa sulla rivista Pigment Cell Research 2004,17,422-424 una lettera all’editore dal titolo   ‘’ The Chemical Structure of Melanin  ‘’ del Dr. W.L.Cheun ( College of Pharmacy St.John’s University,USA )  e una replica del Dr.J.D.Simon ( Duke University, USA ) e del Dr.S.Ito ( Fujita Health University, Japan ).

Nella lettera e la replica  vi è poco per quanto è stato fatto sulla  struttura chimica della melanina come indicato nel titolo, mentre vengono omessi importanti riferimenti scientifici e bibliografici .Il contributo fondamentale dato dalla ricerca italiana a partire da Angelo Angeli è quasi completamente ignorato. Lo studio fondamentale sulla struttura della melanina di M.G.Peter viene negletto. Gli studi ai raggi-X, la spettrometria di massa,   la microscopia a forza atomica,  lo studio analitico sulle eumelanine e feomelanine ai quali gli autori fanno  riferimento in modo confuso sono ancora incerti a causa   del materiale grezzo usato negli esperimenti di laboratorio. Va inoltre considerato che gli studi chimici  hanno dato  risultati poco soddisfacenti anche  per la  scarsa conoscenza  della scienza dello stato solido.

 La attuale nota presentata per gli Atti  della Accademia Pontaniana ha lo scopo di cercare di colmare le lacune bibliografiche e scientifiche presenti nelle lettere all’editore dei  dottori  Cheun, Simon ed Ito.

Le melanine posseggono interessanti proprietà quali quelle di sequestrare molecole organiche, ioni,  liquidi e gas nonché quella di condurre la corrente elettrica.Tutte le particelle nere a partire dal carbone fino alla melanina del cervello o dello spazio hanno proprietà molto simili e possono  presentare gabbie grafitiche, fullereniche o elicoidali.Le forme elicoidali potrebbero creare,al passaggio della corrente, campi magnetici di interesse biologico.

Lo studio con AFM della particella scoperta di recente  e l’uso della spettrometria di massa quale tecnica degradtiva  sembrano oggi   poter migliorare le nostre conoscenze della struttura chimica della melanina . La particella melaninica mostra una particolare organizzazione che può , fra l’altro, spiegare la sua capacità di legare molecole, ioni, liquidi, gas.

 E’ necessario , nello studio del pigmento,  usare campioni di melanina ( melanosomi ) purificati, ad esempio  secondo il procedimento di V.J.Hearing,  e corredati dalla analisi elementare per C, H, N, S, Cu, Fe, Ni, Cd.

 

 

 

Prof. Rodolfo  Alessandro Nicolaus, Accademia Pontaniana, Presidente della Classe di Scienze Naturali ,Via Mezzocannone 8, I-80134, Napoli. accponta@tin.it       www.pontaniana.unina.it

rnicolaus@tightrope.it       www.tightrope.it/nicolaus/index.htmScritto in Napoli il 25 Settembre 2005. . Approvato per la pubblicazione sugli Atti della AccademiaPontaniana  il 15 Dicembre 2005. Rispetto al lavoro presentato alla Accademia  Pontaniana , il testo è stato revisionato ed illustrato nel Gennaio 2006.