Péter Avar

Novel dating method to distinguish between forensic and archeological human skeletal remains by bone mineralization indexes

The fast, high-throughput distinction between paleoanthropological remains and recent forensic/clinical bone samples is of vital importance in the field of medicolegal science. In this paper, a novel screening method has been described, using the crystallinity index (C.I.) and carbonate–phosphate index (C/P) as a means to distinguish between archeological and forensic anthropological skeletal findings. According to the Fourier transform infrared spectroscopy analyses, the archeological bone samples are characterized by a range of C.I. between 2.84 and 3.78 and by low C/P values of 0.10–0.33, while the C.I. and C/P ranges of forensic skeletal remains are 2.55–3.18 and 0.38–0.88, respectively. Significant (p < 0.05) changes were observed in C/P as well as C.I. values between the groups of forensic and archeological skeletal samples. The suggested dating method needs only a few milligramms of bone tissue; thus, it can be extremely useful for distiguishing ancient and recent bone fragments.

Comparative Protein Composition of the Brains of PACAP-Deficient Mice Using Mass Spectrometry-Based Proteomic Analysis

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. The diverse biological actions provide the background for the variety of deficits observed in mice lacking endogenous PACAP. PACAP-deficient mice display several abnormalities, such as sudden infant death syndrome (SIDS)-like phenotype, decreased cell protection, and increased risk of Parkinson’s disease. However, the molecular and proteomic background is still unclear. Therefore, our aim was to investigate the differences in peptide and protein composition in the brains of PACAP-deficient and wild-type mice using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric (MS)-based proteomic analysis. Brains from PACAP-deficient mice were removed, and different brain areas (cortex, hippocampus, diencephalon, mesencephalon, brainstem, and cerebellum) were separated. Brain pieces were weighed, homogenized, and further processed for electrophoretic analysis. Our results revealed several differences in diencephalon and mesencephalon. The protein bands of interest were cut from the gel, samples were digested with trypsin, and the tryptic peptides were measured by matrix-assisted laser desorption ionization time of flight (MALDI TOF) MS. Results were analyzed by MASCOT Search Engine. Among the altered proteins, several are involved in metabolic processes, energy homeostasis, and structural integrity. ATP-synthase and tubulin beta-2A were expressed more strongly in PACAP-knockout mice. In contrast, the expression of more peptides/proteins markedly decreased in knockout mice, like pyruvate kinase, fructose biphosphate aldolase-A, glutathione S-transferase, peptidyl propyl cis-trans isomerase-A, gamma enolase, and aspartate amino transferase. The altered expression of these enzymes might partially account for the decreased antioxidant and detoxifying capacity of PACAP-deficient mice accompanying the increased vulnerability of these animals. Our results provide novel insight into the altered biochemical processes in mice lacking endogenous PACAP.

A validated HPLC-FLD method for analysis of intestinal absorption and metabolism of capsaicin and dihydrocapsaicin in the rat

A sensitive and selective reverse-phase high performance liquid chromatographic method with fluorescence detection has been developed for determination of capsaicin (8-methyl-N-vanillyl-(trans)-6-nonenamid) and dihydrocapsaicin (8-methyl-N-vanillylnonanamide) in samples generated in rat small intestine luminal perfusion experiments. The experiments were designed to study the biotransformation of capsaicinoids in the small intestine in the rat. The chromatographic separation was performed at room temperature on a ZORBAX Eclipse(®) XDB-C8 column using isocratic elution with a mobile phase consisting 0.05M orthophosphoric acid solution and acetonitrile (60:40, v/v; pH 3.0) with a flow rate of 1.5mL/min. Fluorescence detection was performed at excitation and emission wavelengths of 230 and 323nm, respectively. The method was evaluated for a number of validation characteristics (accuracy, repeatability and intermediate precision, limit of detection, limit of quantification and calibration range). The limit of detection (LOD) was 50ng/mL and the limit of quantification (LOQ) was 100ng/mL for both capsaicin and dihydrocapsaicin reference standards dissolved in blank perfusate. The method was successfully applied for investigation of intestinal absorption of capsaicin and dihydrocapsaicin while 30μg/mL standardized Capsicum extract - containing capsaicin and dihydrocapsaicin - was luminally perfused for a 90min period. The structure of the glucuronide metabolites of capsaicin and dihydrocapsaicin appeared in the perfusate was identified by mass spectrometry.

HPLC-MS/MS analysis of steroid hormones in environmental water samples

Today, freshwaters, such as lakes and rivers, are subject to controlled pollution. Steroid hormones are chemically very stable highly lipophilic molecules. Their biological properties have a strong impact on the endocrine regulation of species. Steroids have estrogenic, androgenic, thyroidogenic or progestogenic effects and based on them, they could disturb the physiological mechanisms of freshwater species. We focused on progestins as they are the main active ingredients of contraceptive pharmaceuticals. Progestins have been shown to impair reproduction in fish, amphibians, and mollusks at low ng/L concentrations. Certain progestins, such as levonorgestrel (LNG) have androgenic properties also. We selected the most used active substances drospirenone (DRO), LNG, and progesterone (PRG) and then developed and optimized a liquid chromatographic-mass spectrometric method with solid-phase extraction to measure them. Using our sensitive method (LOQ 0.03-0.11 ng/L) we could measure steroids even between 0.1 and 1 ng/L. Analyzing freshwater samples from the Lake Balaton catchment area, we found influents where the concentration of these hormones was 0.26-4.30 (DRO), 0.85-3.40 (LNG), and 0.23-13.67 (PRG) ng/L. Out of 53 collecting places, 21 contained measurable progestin levels, which clearly demonstrates the applicability of our method, legitimates toxicology experiments with effected species, and indicates monitoring efforts.

Bone tuberculosis in Roman Period Pannonia (western Hungary)

The purpose of this study was to analyse a skeleton (adult female, 25-30 years) that presented evidence of tuberculous spondylitis. The skeleton, dated from the Roman Period (III-VI centuries), was excavated near the town of Győr, in western Hungary. The skeleton was examined by gross observation supplemented with mycolic acid and proteomic analyses using MALDI-TOF/TOF tandem mass spectrometry. The biomolecular analyses supported the morphological diagnosis.

Oxidation of Hydroxy- and Dihydroxybenzoic Acids Under the Udenfriend's Conditions. An HPLC Study

Background: Non-enzymatic hydroxylation of aromatic compounds to the respective phenolic derivatives is a possible metabolic pathway of xenobiotics. The formed metabolites can undergo consecutive oxidative reactions with free radicals to form potential toxic molecules. Objective: Development of HPLC methods to separate, identify and quantitate the main products formed from salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid under in vitro hydroxylation conditions (Udenfriends system). Method: An RP-HPLC-UV-Vis method was developed to separate salicylic acid and isomeric dihydroxybenzoic acids formed in the Udenfriends system. Confirmation of structures of the oxidized products of salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was performed by HPLC-ESI-MS/MS method. Results: The HPLC-UV-Vis method was evaluated for a number of validation characteristics (selectivity, repeatability and intermediate precision, LOD, LOQ and calibration range). It was found that oxidation of salicylic acid resulted in the formation of 2,3- and 2,5-dihydroxybenzoic acids. Furthermore, the hydroxylated metabolites can be further metabolized under the Udenfriend’s conditions. Conclusion: The results give evidence for possible involvement of the oxidized metabolites of salicylic acid in the development of biological action of salicylates at the site of inflammation, where high hydroxyl radical level can be detected.

Investigation of estrogen activity in the raw and treated waters of riverbank infiltration using a yeast estrogen screen and chemical analysis

Exposure to various endocrine disrupting chemicals (EDCs) can lead to adverse effects on reproductive physiology and behavior in both animals and humans. An adequate strategy for the prevention of environmental contamination and eliminating the effects of them must be established. Chemicals with estrogenic activity were selected, and the effectiveness of their removal during the purification processes in two drinking water treatment plants (DWTPs) using riverbank infiltrated water was determined. Thirty-five water samples in two sampling campaigns throughout different seasons were collected and screened with a yeast estrogen test; furthermore, bisphenol A (BPA), 17ß-estradiol (E2) and ethinyl-estradiol (EE2) content were measured using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Our results confirm that estrogenic compounds are present in sewage effluents and raw surface river water of DWTPs. Very low estrogen activity and pg/L concentrations of BPA and E2 were detected during drinking water processing and occasionally in drinking water. Based on this study, applied riverbank filtration and water treatment procedures do not seem to be suitable for the total removal of estrogenic chemicals. Local contamination could play an important role in increasing the BPA content of the drinking water at the consumer endpoint.

Automated SPE and nanoLC–MS analysis of somatostatin

ABSTRACT Plasma peptides are widely used in clinical diagnosis and therapy monitoring. Our aim was an efficient system development for the enrichment of small, low abundant peptides from plasma by robotized sample preparation. The automation of SPE yields additional time saving and improves system robustness and repeatability. Automation is based on combining a Waters SPE kit with Oasis HLB sorbent and a multichannel liquid handling workstation with cheap commercially available electronic devices such as a programmable logic controller (PLC) and an AVR controller. Reversed phase nano liquid chromatography coupled with a sensitive quadrupole time of flight mass spectrometer (QTOF MS) was used for the quantitative determination of somatostatin (SST). We quantified SST from mouse plasma, where lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 2.5 and 8.3 fmol/ml, respectively. We investigated linearity of response, accuracy, precision, recovery, reproducibility and stability of SST during both short-term sample processing and long-term storage. This method allows reliable quantification of plasma peptides. The developed automated sample preparation solid phase extraction method can be easily and conveniently adopted for different volumes and amounts of sample in routine analysis using controlled vacuum. The highest advantage is enhanced reproducibility, which makes it suitable for the investigations of large sample cohorts in clinical studies. GRAPHICAL ABSTRACT