New approach in determination of urinary diagnostic markers for prostate cancer by MALDI-TOF/MS.

In our study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is proposed as a novel tool, which can be applied to analyze lipids in urine samples. For this reason, the main aim of the study was to develop and optimize the preparation protocol for urine samples in lipidomics, using urine samples obtained from patients with diagnosed cancer and non-cancer controls. Several conditions like extraction method and types of matrices were evaluated. For this purpose, two methods for the extraction of lipids, namely modified Folch and Bligh & Dyer were employed. Furthermore, two types of matrices (α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxybenzoic acid (DHB)) for the separation of lipids into individual components was tested. The results of this study can serve as an essential source for the selection of appropriate extraction methods and the appropriate choice of a matrix for the purification and identification of a particular class of lipid in human biological fluids. Based on it, Bligh & Dyer method associated with the usage of HCCA matrix was found to be the most effective for lipidomics using MALDI-TOF/MS. The optimized method was applied to compare the lipid profile of 139 urine samples collected from both healthy individuals and patients with prostate cancer. The tandem spectroscopic analysis allowed to identify lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and triacylglycerols in urine samples. Finally, MALDI-TOF/MS analysis enabled to discriminate between the two tested groups (healthy individuals and patients with prostate cancer). A preliminary statistical model suggested that classification accuracy ranging from 83.3 to100.0% may be achieved by using pre-selected MS signals.

GC-MS and LC-MS approaches for determination of tocopherols and tocotrienols in biological and food matrices.

Tocopherols and tocotrienols, widely described as vitamin E derivatives, have been proven to take part in a number of important biological functions. Among them, antioxidant properties had been investigated and documented in the literature. Since tocochromanols have revealed their plausible beneficial impact on several pathological processes, such as cancerogenesis or cognitive impairment diseases, there is a growing interest in quantitative determination of these compounds in biological fluids, tissues and plant organs. However, due to vitamin E chemical features, such as lipophilic and non-polar characteristics, quantitative determination of the compounds seems to be problematic. In this paper we present current analytical approaches in tocopherols and tocotrienols determination in biological and food matrices with the use of chromatographic techniques, especially gas chromatography (GC) and high performance liquid chromatography (HPLC) coupled with mass spectrometry. Derivatization techniques applied for GC-MS analysis in the case of tocol derivatives, especially silylation and acylation, are described. Significant attention is paid to ionization process of tocopherols and tocotrienols.

Studies of the effect of grasshopper abdominal secretion on wound healing with the use of murine model.

ETHNOPHARMACOLOGICAL RELEVANCE: Grasshopper, belonging to Chorthippus sp., is a widespread insect inhabiting Polish territory. According to folk knowledge and folk tales, the grasshopper abdominal secretion was used by villagers of Central and South-West Poland as a natural drug accelerating the wound healing process. AIM OF THE STUDY: In the reported study the hypothesis about beneficial properties of grasshopper abdominal secretion on hard to heal wounds was verified. MATERIALS AND METHODS: The study was carried out with the use of a murine model (mice C57BL/6). In order to verify the beneficial properties of grasshopper abdominal secretion, the wounds of 8mm in diameter were formed on one side of each tested mouse. The influence of ethanolic extract of insects' secretion on healing process was evaluated in comparison to ethanolic solution of allantoin and 30% aqueous solution of ethanol (medium). The observation was carried out over a 14 day period. Finally the statistical analysis (ANOVA) was carried out to highlight the differences in wound healing rate between applied preparations. Moreover, qualitative composition of grasshoppers' secretion was studied with the use of GC/MS technique. RESULTS: During the first three days of observation, wounds treated with allantoin were healed with higher efficiency in comparison to ethanol and insect secretion preparations. The trend of healing changed from the 4th day of observation. Wounds treated with grasshoppers' abdominal secretion were closuring faster than wounds treated with allantoin or ethanol. In this part of observation, in the case of allantoin and ethanol application, the wound healing efficiency was similar. Since the 9th day of experiment the measurement of wounds size was problematic, due to crust formation. Finally at the 14th day of the study, wounds were totally healed. Morphological study enabled to observe all the phases of healing. In the 5th and 8th day, the infiltration of neutrophils and mononuclear cells in dermis was observed, which is characteristic for inflammatory phase of wound healing. On the 8th day of experiments, granulation of the tissue was clearly observed in the tested groups. Reepithelialization phase was observed from the 5th to 14th day, when the wound was totally healed. The analytical approach enabled to identify 38 compounds of hydrophobic or hydrophilic character. Among them, 6 amino acids, 14 organic acids and their derivatives, one sterol, 4 hydrocarbons, 5 carbohydrates, 2 inorganic acids, 4 alcohols, one diamine and one nucleoside were identified. CONCLUSION: The obtained results enabled to recognize the composition of grasshopper abdominal secretion. Some of the identified compounds possess therapeutic properties described in the literature. The performed in vivo study proved that application of insects secretion accelerates the healing process. The obtained results positively verified the scientific hypothesis based on ethnopharmacological premises about the beneficial properties of grasshopper abdominal secretion on wound healing process.