Slawomir Kurkiewicz

Pyrolytic GC-MS analysis of melanin from black, gray and yellow strains of Drosophila melanogaster

Abstract Properties of melanins depend strongly on their chemical constitution. Melanins from differently colored strains of Drosophila melanogaster demonstrate various proprieties, and biological activity in the presence of toxic ions or radiation. In the presented work, we analyzed chemical constitution of the melanins derived from black, gray and yellow D. melanogaster strains. Analysis of the products forming during thermal degradation of the biopolymers by pyrolitic gas chromatography followed by mass spectrometry allowed us to determine chemical composition of these biopolymers and classified melanins derived from black and gray strains as eumelanins and derived from yellow strains as pheomelanins. These findings allow us to determine chemical and biological properties of the melanins in vivo, and can explain susceptibility of differently colored strains of D. melanogaster to metal ions.

GC/MS determination of fatty acid picolinyl esters by direct Curie-point pyrolysis of whole bacterial cells

A single-step method suitable for cellular fatty acid derivatization to picolinyl esters with the use of a pyrolyzer as a thermochemical micro-reactor was developed for whole bacterial cells. This reduced the preparation time from several hours to less than two minutes. In addition, the minimal bacterial mass required for analysis was reduced from several milligrams to micrograms. The profiling of cellular fatty acids of Gram-positive and Gram-negative bacteria was achieved using three derivatization methods: preparation of methyl esters, β-picolinyl esters by Harvey’s method and a new method based on pyrolytic derivatization to β-picolinyl esters. It was shown that there are great similarities between profiles of bacterial fatty acids determined by the pyrolytic derivatization method and traditional preparation methods of picolinyl and methyl esters prior to GC analysis. Results obtained by application of the new technique have immense diagnostic value due to vast similarities between profiles of fatty acids derivatized to either picolinyl and methyl esters. Although the latter are referred to in the literature most often, mass spectra of picolinyl esters contain fragment ions that provide structural information about the chain branching, position of unsaturation, and other substituents.

Structural investigations of neuromelanin by pyrolysis-gas chromatography/mass spectrometry

Summary.Pyrolysis combined with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural investigations of the human substantia nigra neuromelanin. Using synthetic neuromelanins, we have demonstrated that Py-GC/MS is suitable for identification and differentiation of both eumelanin (dopamine-derived) and pheomelanin (cysteinyldopamine-derived) component of the pigment. Structural information on melanin monomers was inferred from their pyrolytic markers. When the human neuromelanin was subjected to pyrolysis, none of the heterocyclic, sulfur-containing markers of pheomelanin component was detected among the thermal degradation products. We have concluded that nigral pigment isolated from normal brain tissue does not contain benzothiazine-type monomers, and that cysteinyldopamine-originated units may be incorporated into the polymer in uncyclized form. The most abundant pyrolysis product was identified as limonene, which indicates that nigral pigment is tightly associated with an isoprenoid-type compound. Pyrolysis in the presence of the methylating reagent allowed identification of high levels of saturated and monounsaturated straight-chain C14–C18 fatty acid species chemically bound to the pigment macromolecule.

Detection and quantitation of a pheomelanin component in melanin pigments using pyrolysis-gas chromatography/tandem mass spectrometry system with multiple reaction monitoring mode.

Here, we describe the reliable method for the detection and quantitation of a pheomelanin component in melanin pigments. Synthetic melanins with various contents of pheomelanin-type structural units were thermally degraded, and the multiple reaction monitoring mode was applied to detect the pheomelanin markers in the pyrolysates by GC/MS/MS. The method allowed the specific detection and quantitation of a pheomelanin component in melanin with the incorporation of pheomelanin-type units as low as 0.05%. Considering highly universal character of the pheomelanin markers, the method could be applied for structural studies of natural melanin pigments being mixtures of eumelanin and pheomelanin.

Occupational Exposure to Aromatic Hydrocarbons at a Coke Plant : Part II. Exposure Assessment of Volatile Organic Compounds

Occupational Exposure to Aromatic Hydrocarbons at a Coke Plant: Part II. Exposure Assessment of Volatile Organic Compounds: Grażyna Bieniek, et al. Department of Molecular Biology, Biochemistry and Biopharmacy, Faculty of Pharmacy Medical University of Silesia, Poland— The objective of the study is to assess the external and internal exposures to aromatic hydrocarbons in the tar and oil naphthalene distillation processes at a coke plant. 69 workers engaged as operators in tar and oil naphthalene distillation processes and 25 non‐exposed subjects were examined. Personal analyses of the benzene, toluene, xylene isomers, ethylbenzene, naphthalene, indan, indene and acenaphthene in the breathing zone air allowed us to determine the time weighted average exposure levels to the aromatic hydrocarbons listed above. The internal exposure was investigated by measurement of the urinary excretion of naphthols, 2‐methylphenol and dimethylphenol isomers by means of gas chromatography with a flame ionization detection (GC/FID). Urine metabolites were extracted after enzymatic hydrolysis by solid‐phase extraction with styrene‐divinylbenzene resin. The time‐weighted average concentrations of the hydrocarbons detected in the breathing zone air shows that the exposure levels of the workers are relatively low in comparison to the exposure limits. Statistically significant differences between average concentrations of aromatic hydrocarbons (benzene, toluene, xylene isomers) determined at the workplaces in the tar distillation department have been found. Concentrations of the naphthalene and acenaphthene detected in workers from the oil distillation department are higher that those from the tar distillation department. Concentrations of naphthols, 2‐methoxyphenol and dimethylphenol isomers in the urine of occupationally exposed workers were significantly higher than those of non‐exposed subjects. Concentrations of the 2methoxyphenol and dimethylphenol isomers in urine were significantly higher for the tar distillation workers, whereas concentrations of naphthols were higher for the oil naphthalene distillation workers. Operators at the tar and naphthalene oil distillation processes are simultaneously exposed to a mixture of different hydrocarbons, mainly benzene and naphthalene homologues.

Occupational Exposure to Aromatic Hydrocarbons at a Coke Plant: Part I. Identification of Hydrocarbons in Air and their Metabolites in Urine by a Gas Chromatography-Mass Spectrometry Method

Occupational Exposure to Aromatic Hydrocarbons at a Coke Plant: Part I. Identification of Hydrocarbons in Air and their Metabolites in Urine by a Gas Chromatography‐Mass Spectrometry Method: Grażyna Bieniek, et al. Department of Molecular Biology, Biochemistry and Biopharmacy, Faculty of Pharmacy, Medical University of Silesia, Poland—A method for the qualitative analysis of aromatic hydrocarbons in air and their various urinary metabolites is presented. The air was sampled in charcoal tubes and extracted with carbon disulfide. The hydrocarbons were identified as being aliphatic hydrocarbons (C9–C19), aromatic hydrocarbons and heterocyclic compounds. The urinary metabolites after enzymatic hydrolysis were analyzed by solid‐phase extraction with a styrene‐divinylbenzene resin, silylation with N,Obis(trimethylsilyl)acetamide and GC/MS for separation and detection. Satisfactory separation of all compounds investigated was achieved without interference due to matrix peaks. The following compounds were identified in the urine of workers: dimethylphenol isomers, 4‐ethyl‐1,3‐benzenediol, 2ethoxybenzoic acid and methoxyphenols. Trimethylsilyl derivatives of aromatic hydroxyacids and hydroxymethoxyacids were found in the urine of occupationally exposed workers by means of a silylation procedure.

Effect of Cu2+ and Zn2+ ions on DOPA-melanin structure as analyzed by pyrolysis-gas chromatography-mass spectrometry and EPR spectroscopy

Abstract Synthetic polymers, obtained by oxidative polymerization of 3,4-dihydroxyphenylalanine (DOPA) in the presence and in the absence of copper or zinc ions, were analyzed by pyrolysis–gas chromatography–mass spectrometry and electron paramagnetic resonance (EPR) methods. Pyrolytic profiles of DOPA–Cu 2+ - and DOPA–Zn 2+ -melanins were characterized by the abundance of pyridine- and pyrazine-derivatives and by considerably reduced levels of pyrrole and phenols that were predominant pyrolysis products of metal-free DOPA-melanin. It was found that incorporation of paramagnetic Cu 2+ ions and diamagnetic Zn 2+ ions into DOPA-melanin effects its free radical properties. It was concluded that the presence of Cu 2+ and Zn 2+ ions during DOPA-melanin synthesis modifies the structure of the biopolymer formed.

Evaluation of Melanogenesis in A-375 Cells in the Presence of DMSO and Analysis of Pyrolytic Profile of Isolated Melanin

The increase of a skin malignant melanoma (melanoma malignum) incidence in the world has been observed in recent years. The tumour, especially in advanced stadium with metastases, is highly resistant to conventional treatment. One of the strategies is to modulate melanogenesis using chemical compounds. In this study, the processes of differentiation and melanogenesis induced by dimethylsulfoxide (DMSO) in human melanoma cells (A-375) were investigated. Natural melanin isolated from A-375 melanoma cell line treated with 0.3% DMSO was analyzed by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) method. The products derived from pheomelanin have not been stated in the pyrolytic profile of analyzed melanin. Within all products derived from eumelanins, 1,2-benzenediol has been predominated. It has been shown that in the melanoma cells stimulated with 0.3% and 1% DMSO, the increase of transcriptional activity of the tyrosinase gene took place. It was accompanied by the rise of tyrosinase activity and an accumulation of melanin in the cells. The better knowledge about the structure of melanins can contribute to establish the uniform criteria of malignant melanoma morbidity risk.

Evaluation of melanogenesis in A-375 melanoma cells treated with 5,7-dimethoxycoumarin and valproic acid

Malignant melanoma (melanoma malignum) is one of the most dangerous types of tumor. It is very difficult to cure. In recent years, a lot of attention has been given to chemoprevention. This method uses natural and synthetic compounds to interfere with and inhibit the process of carcinogenesis. In this study, a new treatment strategy was proposed consisting of a combination of 5,7-dimethoxycoumarin (DMC), an activator of melanogenesis, and valproic acid (VPA), a well-known drug that is one of the histone deacetylase inhibitors (HDACis). In conjunction with 1 mM VPA, all of the tested concentrations of DMC (10–150 μM) significantly decreased the proliferation of A-375 cells. VPA and DMC also induced the synthesis of melanin and the formation of dendrite and star-shaped cells. Tyrosinase gene expression and tyrosinase activity significantly increased in response to VPA treatment. Pyrolysis with gas chromatography and mass spectrometry (Py-GC/MS) was used to investigate the structure of the isolated melanin. This showed that the quantitative and qualitative components of melanin degradation products are dependent on the type of applied melanogenesis inductor. Products derived from eumelanin were detected in the pyrolytic profile of melanin isolated from A-375 cells stimulated with DMC. Thermal degradation of melanin isolated from melanoma cells after exposure to VPA or a mixture of VPA and DMC revealed the additional presence of products derived from pheomelanin.

Chemical composition of Desulfovibrio desulfuricans lipid A

Lipopolysaccharides also called endotoxins are an integral component of the outer membrane of Gram-negative bacteria. When released from the bacterial surface, they interact with a host immune system, triggering excessive inflammatory response. Lipid A is the biologically most active part of endotoxin, and its activity is modulated by the quantity, quality and arrangement of its fatty acids. Desulfovibrio desulfuricans is sulfate-reducing, Gram-negative bacterium that is supposed to be opportunistic pathogens of humans and animals. In the present study, chemical composition of lipid A from various strains of D. desulfuricans was analyzed by gas chromatography/mass spectrometry. It was found that the fatty acid component of the lipid A contains dodecanoic, tetradecanoic, 3-hydroxytetradecanoic and hexadecanoic acids, and its carbohydrate core is composed of glucosamine. The analysis of 3-acyloxyacyl residue of the lipid A revealed the presence of amide-bound 3-(dodecanoyloxy)tetradecanoic and 3-(hexadecanoyloxy)tetradecanoic acids and ester-bound 3-(tetradecanoyloxy)tetradecanoic acid. It was concluded that both fatty acid and 3-acyloxyacyl residue profiles of the lipid A from the studied bacteria were similar to those of E. coli and S.enterica.