The 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts

The 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts. June 4-7, 2019, Szczyrk, Polan

Secretion of proinflammatory cytokines by normal human melanocytes in response to lipopolysaccharide

A large body of evidence suggests that epidermal melanocytes are an integral part of the skin immune system and can be considered immunocompetent cells. Recently, it has been reported that human melanocytes constitutively express Toll-like receptors and may be involved in the induction of several inflammatory cytokines. In the study the secretion of IL-1β, IL-6 and TNF-α by cultured normal melanocytes was investigated after stimulation with lipopolysaccharide. LPS increased the secretion of IL-1β in a dose-dependent manner. IL-1β stimulated release of IL-6 and TNF-α by melanocytes, whereas LPS activated production of TNF-α, but not of IL-6. These observations indicate that LPS can participate in the regulation of cytokine activity in normal human melanocytes and suggest that cytokines released by melanocytes could affect melanocytes themselves or/and other cells of the epidermis

GC/MS determination of non-steroidal anti-infl ammatory drugs in human serum: an optimization of the drug extraction procedure

Determination of non-steroidal anti-infl ammatory drugs (NSAIDs) in human and animal physiological fl uids requires the use of appropriate pro-cedures permitting rapid and effi cient recovery of the drugs from biological matrices. The aim of this study was to compare various methods of NSAIDs extraction from human serum that is essential for GC/MS analysis of the drugs. Commercially available biochemistry control human serum supplemented with ibuprofen, paracetamol, fl urbiprofen, nabumeton, naproxen, ketoprofen, mefenamic acid and diclofenac sodium salt was used. Solid phase extraction, HybridSPE- -PPT technology and liquid-liquid extraction were applied for NSAIDs isolation from the serum. GC/MS analysis of the drugs was preceded by their derivatization to trimethylsilyl esters with BSTFA. The highest extraction effi ciency and the best separation of the drugs from endogenous components of the serum were achieved using the solid phase extraction on octadecyl-bonded silica.Oznaczanie niesteroidowych leków przeciwzapalnych (NLPZ) w płynach fizjologicznych ludzi i zwierząt wymaga zastosowania szybkich i wydajnych procedur izolacji tych leków z biologicznej matrycy. Celem pracy było porównanie różnych metod ekstrakcji NLPZ z surowicy ludzkiej, jako etapu poprzedzającego analizę tych leków techniką GC/MS. W badaniach wykorzystano dostępną handlowo surowicę stosowaną jako materiał kontrolny w oznaczeniach biochemicznych, do której dodano ibuprofen, paracetamol, flurbiprofen, nabumeton, naproksen, ketoprofen, kwas mefenamowy oraz sól sodową diklofenaku. W celu wyizolowania NLPZ z surowicy zastosowano ekstrakcję do fazy stałej, technikę HybridSPE- -PPT oraz ekstrakcję ciecz-ciecz. Leki poddano derywatyzacji do pochodnych trimetylosililowych za pomocą BSTFA, a następnie analizowano techniką GC/MS. Najwyższą wydajność ekstrakcji oraz najlepszy stopień oddzielenia NLPZ od endogennych składników surowicy uzyskano metodą ekstrakcji do fazy stałej w postaci żelu krzemionkowego z aktywnymi grupami oktadecylowymi

Melanin from Epidermal Human Melanocytes: Study by Pyrolytic GC/MS

Pigmentation of human skin is determined by the presence of melanin, the polymeric pigment that is produced in melanocytes and transferred to adjacent keratinocytes. Epidermal melanocytes produce two distinct types of melanin pigments: eumelanin, composed mainly of indole-type monomers, and pheomelanin that contains benzothiazine-type backbone. Eumelanin protects skin against UV-induced damages, whereas pheomelanin is believed to act as a potent UV photosensitizer and promote carcinogenesis. In this study, pyrolysis in combination with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural studies of the epidermal pigment isolated from the cultured human melanocytes. The analysis was preceded by investigations of DOPA-originated synthetic eumelanin and pheomelanin standards. This allowed determination of pyrolytic markers for both types of melanin pigments. To obtain additional information on the natural pigment structure, the samples were thermally degraded in the presence of tetramethylammonium hydroxide as the derivatizing agent. It was shown that the analyzed pigment from normal human epidermal melanocytes derived from moderately pigmented skin is of eumelanin type with little incorporation of a pheomelanin component. The results indicate that Py-GC/MS is a rapid and efficient technique for the differentiation of epidermal melanin types and may be an alternative to commonly used methods based on chemical degradation

Peroxynitrite mediated linoleic acid oxidation and tyrosine nitration in the presence of synthetic neuromelanins.

Peroxynitrite-mediated linoleic acid oxidation and tyrosine nitration were analysed in the presence of synthetic model neuromelanins: dopamine (DA) -melanin, cysteinyldopamine (CysDA) -melanin and various DA/CysDA copolymers. The presence of melanin significantly decreased the amount of 3-nitrotyrosine formed. This inhibitory effect depended on the type and concentration of melanin polymer. It was found that incorporation of CysDA-derived units into melanin attenuated its protective effect on tyrosine nitration induced by peroxynitrite. In the presence of bicarbonate, the melanins also inhibited 3-nitrotyrosine formation in a concentration dependent manner, although the extent of inhibition was lower than in the absence of bicarbonate. The tested melanins inhibited peroxynitrite-induced formation of linoleic acid hydroperoxides, both in the absence and in the presence of bicarbonate. In the presence of bicarbonate, among the oxidation products appeared 4-hydroxynonenal (HNE). CysDA-melanin inhibited the formation of HNE, while DA-melanin did not affect the aldehyde level. The results of the presented study suggest that neuromelanin can act as a natural scavenger of peroxynitrite

Zastosowanie pirolitycznej metylacji w oznaczaniu profi lu krótkołańcuchowych kwasów tłuszczowych w wybranych preparatach farmaceutycznych techniką GC/MS

Lactic Acid Bacteria (LAB) belong to the normal fl ora of human alimentary tract and vaginal epithelium. The main products of LAB metabolism are short chain fatty acids (SCFA), which are known to have signifi cant infl uence on human health. The aim of this study was to examine the suitability of direct pyrolytic methylation in GC/MS profi ling of SCFA in the selected pharmaceutical preparations of LAB. For method optimization, standard SCFA samples consisting of acetic, propionic, butyric and lactic acids were pyrolyzed under various temperature conditions in the presence of methanolic solution of tetramethylammonium hydroxide (TMAH) as a derivatizing reagent. It was demonstrated that pyrolytic derivatization of standard SCFA to methyl esters was the most effi cient with the use 10% TMAH and the pyrolytic fi laments with Curie temperature of 480°C, when the pyrolysis cell was kept at 150°C. It was shown that the method is suitable for GC/MS qualitative analysis of SCFA profi le in the pharmaceutical preparations that contain LAB in lyophilized form.Bakterie kwasu mlekowego (Lactic Acid Bacteria – LAB) należą do naturalnej flory bakteryjnej zasiedlającej przewód pokarmowy oraz nabłonek pochwy człowieka. Głównym produktem metabolizmu LAB są krótkołańcuchowe kwasy tłuszczowe (short-chain fatty acids – SCFA) wywierające istotny wpływ na zdrowie człowieka. W pracy poddano ocenie przydatność techniki bezpośredniej metylacji pirolitycznej w oznaczaniu profi lu SCFA w wybranych preparatach farmaceutycznych LAB techniką GC/MS. W celu optymalizacji metody, wzorcowe próbki SCFA zawierające kwas octowy, propionowy, masłowy i mlekowy poddano pirolizie w obecności metanolowego roztworu wodorotlenku tetrametyloamoniowego (TMAH) w różnych warunkach temperaturowych. Stwierdzono, że derywatyzacja analizowanych kwasów tłuszczowych do estrów metylowych zachodziła z największą wydajnością pod wpływem 10% TMAH, gdy zastosowano druty pirolityczne o temperaturze Curie 480°C, a komorę pirolizera utrzymywano w temperaturze 150°C. Wykazano, że zoptymalizowana metoda jest przydatna w prowadzonej techniką GC/MS analizie jakościowej profi lu SCFA w wybranych preparatach farmaceutycznych zawierających LAB w formie liofi lizatu

GRAŻYNA KLUPIŃSKA Nocturnal Secretion of Melatonin in Patients with Duodenal Ulcer Disease and Ulcer−Like Dyspepsia* Nocne wydzielanie melatoniny u osób z chorobą wrzodową dwunastnicy i z dyspepsją wrzodopodobną

Abstract Background. Many recent experimental investigations have shown that melatonin has gastroprotective properties. However, its role in the pathogenesis of upper digestive tract diseases is still unclear. Objectives. Investigating the nocturnal secretion of melatonin in patients with duodenal ulcer or ulcer−like dyspepsia. Material and Methods. The investigations were carried out in 34 patients with recent duodenal ulcer (DUD), 36 patients with functional ulcer−like dyspepsia (ULD, according to the Rome Criteria II), and in 30 healthy control sub− jects. Blood samples were taken for examination in a red−lit room at 10 p.m. and then at 2 and 6 a.m. The serum con− centration of melatonin was measured with ELISA before and after a six−week treatment with omeprazole (40 mg daily). Patients with H. pylori infection additionally took amoxicyllin (2.0 g) and clarithromycin (1.0 g) for seven days. Results. The average melatonin concentration in duodenal ulcer patients was lower than in the healthy subjects (25.50 ± 6.22 pg/ml and 34.71 ± 4.72 pg/ml, respectively; p < 0.05). The highest concentration of melatonin was found in patients with ulcer−like dyspepsia (42.42 ± 9.93 pg/ml, p < 0.05). After six weeks, the duodenal ulcers healed and dyspeptic symptoms improved in all patients, but nocturnal secretion had not changed significantly. Conclusions. The findings suggest that melatonin may play an important role in the pathogenesis of duodenal ulcer disease. In patients with ulcer−like dyspepsia, the influence of melatonin on nocturnal and fasting abdominal pains is also possible (Adv Clin Exp Med 2006, 15, 5, 811-815)

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