Zenon J. Kokot

Analysis of the factors that significantly influence the stability of fluoroquinolone–metal complexes

The aim of the present study was to evaluate factors contributing to the differences between the overall stability constants (logbeta(pqr)) of the fluoroquinolone-metal ion complexes. The experiments were performed using potentiometric titration method in wide pH range. The overall stability constants (logbeta(pqr)) were determined using the Hyperquad program. Complexation equilibria of eight different fluoroquinolones with six divalent and trivalent metal ions were investigated in this study. The authors employed a multifactorial ANOVA analysis, fixed effect model to describe the influence of particular variables affecting the stability of the analyzed complex species. Four different variables were set at different levels labeled. The ligand number (LF) was the first factor. LF determined the number of fluorochinolone molecules in the complex structure, and could take the values 1, 2 or 3. The second factor (Me) was connected with the type of the metal ion bonded in the complex. Since six different metal cations were studied, the Me factor was described with six levels. The number of hydrogen or hydroxide groups substituted into the complex molecule was the third variable (HR) with many levels labeled: q, a, s, d, f and g. The last factor FQ described the type of the fluorochinolone used for complex formations. All variables analyzed here were statistically significant (p value lower than 0.01), which indicates that all of them strongly affect the logbeta(pqr) value. Binary interactions (LF-Me, LF-FQ, Me-HR and Me-FQ) between variables were also stated, which suggests that the effects of these variables were higher than we could calculate based on the effect of each variable alone. The ANOVA analysis has shown that the following factors Me, LF and HR were the most important for the stability of the fluoroquinolone-metal ion complexes. It was also found that according to the FQ factor (type of ligand molecule) all analyzed fluoroquinolones formed stable complexes with metals. It was proved that the application of ANOVA for the entire complexation profile of analyzed fluoroquinolones with polyvalent metal ions was a valid technique for detecting the statistically significant differences in the complexation profiles. Such information may be very useful for better understanding and interpretation of differences in bioavailability of fluoroquinolones and their interactions with antacids and other multimineral drugs.

Challenges in biomarker discovery with MALDI-TOF MS

MALDI-TOF MS technique is commonly used in system biology and clinical studies to search for new potential markers associated with pathological conditions. Despite numerous concerns regarding a sample preparation or processing of complex data, this strategy is still recognized as a popular tool and its awareness has risen in the proteomic community over the last decade. In this review, we present comprehensive application of MALDI mass spectrometry with special focus on profiling research. We also discuss major advantages and disadvantages of universal sample preparation methods such as micro-SPE columns, immunodepletion or magnetic beads, and we show the potential of nanostructured materials in capturing low molecular weight subproteomes. Furthermore, as the general protocol considerably affects spectra quality and interpretation, an alternative solution for improved ion detection, including hydrophobic constituents, data processing and statistical analysis is being considered in up-to-date profiling pattern. In conclusion, many reports involving MALDI-TOF MS indicated highly abundant proteins as valuable indicators, and at the same time showed the inaccuracy of available methods in the detection of low abundant proteome that is the most interesting from the clinical perspective. Therefore, the analytical aspects of sample preparation methods should be standardized to provide a reproducible, low sample handling and credible procedure.

Activation of Prodrug Treosulfan at pH 7.4 and 37°C Accompanied by Hydrolysis of Its Active Epoxides: Kinetic Studies with Clinical Relevance

Treosulfan (TREO), originally registered for treatment of ovarian cancer, is currently being investigated for conditioning prior to hematopoietic stem cell transplantation. TREO is a prodrug, which undergoes a pH- and temperature-dependent two-step conversion to active monoepoxide [S,S-EBDM, (2S,3S)-1,2-epoxybutane-3,4-diol-4-methanesulfonate] and diepoxide [S,S-DEB, (2S,3S)-1,2:3,4-diepoxybutane]. In this paper, the kinetics of the nonenzymatic transformation of TREO at pH 7.4 and 37°C were studied for the first time including the effects of the TREO concentration, buffer concentration, ionic strength, and the presence of NaCl. Transformation of TREO was well described by a kinetic model, which included first-order reactions for TREO activation, that is, TREO → S,S-EBDM → S,S-DEB, and pseudo-first-order reactions for the hydrolytic decomposition of S,S-EBDM and S,S-DEB. In contrast to the two-step activation of TREO, the hydrolysis of epoxides was influenced by electrolytes. In phosphate-buffered saline, decomposition of S,S-EBDM and S,S-DEB (mean half-lives 25.7 and 15.4 h) proceeded much slower than their formation (mean half-lives 1.5 and 3.5 h). In conclusion, the kinetics of the nonenzymatic transformation of TREO in the presence of plasma electrolytes cannot contribute to the very low levels of S,S-EBDM and S,S-DEB observed in patient plasma. The results also indicate that elimination of TREO proceeds primarily via conversion to S,S-EBDM.

Amino Acid Profiles of Serum and Urine in Search for Prostate Cancer Biomarkers: a Pilot Study

There is a great interest in searching for diagnostic biomarkers in prostate cancer patients. The aim of the pilot study was to evaluate free amino acid profiles in their serum and urine. The presented paper shows the first comprehensive analysis of a wide panel of amino acids in two different physiological fluids obtained from the same groups of prostate cancer patients (n = 49) and healthy men (n = 40). The potential of free amino acids, both proteinogenic and non-proteinogenic, as prostate cancer biomarkers and their utility in classification of study participants have been assessed. Several metabolites, which deserve special attention in the further metabolomic investigations on searching for prostate cancer markers, were indicated. Moreover, free amino acid profiles enabled to classify samples to one of the studied groups with high sensitivity and specificity. The presented research provides a strong evidence that ethanolamine, arginine and branched-chain amino acids metabolic pathways can be a valuable source of markers for prostate cancer. The altered concentrations of the above-mentioned metabolites suggest their role in pathogenesis of prostate cancer and they should be further evaluated as clinically useful markers of prostate cancer.

Complexes of Fe(III) ions with mefenamic acid.

Mononuclear and binuclear complexes of mefenamic acid with Fe(III) ions of formulae Na[Fe(C15H14O2N)4] and Fe2Cl(C15N14O2N)5 were obtained in the solid phase. Characterization of the complexes was performed by elemental analysis and visible, IR, 1H and 13C NMR spectroscopy and possible structures are proposed.

Evaluation of serum amino acid profiles’ utility in non-small cell lung cancer detection in Polish population

OBJECTIVES Data from studies performed in Japanese and Korean populations suggest that free amino acid profiles have the potential to aid in non-small cell lung cancer (NSCLC) detection. However, there is still no data regarding abnormalities of free amino acids and their usefulness in NSCLC detection in European populations. The aim of the study was an evaluation of utility of amino acid profiles in NSCLC detection in Polish patients. MATERIALS AND METHODS Levels of 31 free amino acids were determined in 153 serum samples applying a liquid chromatography-tandem mass spectrometry-based methodology. Patients with I stage lung cancer represented a significant part of the studied group (46.7%). The obtained metabolite profiles along with clinical data were subjected to multivariate statistical tests. RESULTS The presented study indicated that the increased serum level of phenylalanine and decreased level of citrulline are among the most robust cancer signatures in blood of NSCLC group. In addition, increased levels of aspartic acid and β-alanine were also recognized as important features of NSCLC. Amino acid selected based on studies of Asian patients were found to have insufficient specificity in NSCLC detection in the studied population. Therefore, we proposed a new set of 6 amino acids (aspartic acid, β-alanine, histidine, asparagine, phenylalanine and serine), which ensured higher accuracy in sample classification (from 90.3% to 77.1% depending of histological type). CONCLUSION We indicated that some of the free amino acid alterations occur in serum of NSCLC patients in early stage of disease and thus they can be valuable components of a blood multi-marker panel for NSCLC detection.

A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus

The aim of this pilot study was to apply a novel combined metabolomic and proteomic approach in analysis of gestational diabetes mellitus. The investigation was performed with plasma samples derived from pregnant women with diagnosed gestational diabetes mellitus (n = 18) and a matched control group (n = 13). The mass spectrometry-based analyses allowed to determine 42 free amino acids and low molecular-weight peptide profiles. Different expressions of several peptides and altered amino acid profiles were observed in the analyzed groups. The combination of proteomic and metabolomic data allowed obtaining the model with a high discriminatory power, where amino acids ethanolamine, l-citrulline, l-asparagine, and peptide ions with m/z 1488.59; 4111.89 and 2913.15 had the highest contribution to the model. The sensitivity (94.44%) and specificity (84.62%), as well as the total group membership classification value (90.32%) calculated from the post hoc classification matrix of a joint model were the highest when compared with a single analysis of either amino acid levels or peptide ion intensities. The obtained results indicated a high potential of integration of proteomic and metabolomics analysis regardless the sample size. This promising approach together with clinical evaluation of the subjects can also be used in the study of other diseases.

Influence of Time of Day on Propofol Pharmacokinetics and Pharmacodynamics in Rabbits

This study evaluates the administration time-of-day effects on propofol pharmacokinetics and sedative response in rabbits. Nine rabbits were sedated with 5 mg/kg propofol at three local clock times: 10:00, 16:00, and 22:00 h. Each rabbit served as its own control by being given a single infusion at the three different times of day on three separate occasions. Ten arterial blood samples were collected during each clock-time experiment for propofol assay. A two-compartment model was used to describe propofol pharmacokinetics, and the pedal withdrawal reflex was used as the sedation pharmacodynamic response. The categorical data comprising the presence or absence of pedal withdrawal reflex was described by a logistic model. The typical volume of the central compartment equaled 7.67 L and depended on rabbit body weight. The elimination rate constant depended on drug administration time; it was lowest at 10:00 h, highest at 16:00 h, and intermediate at 22:00 h. Delay of the anesthetic effect, with respect to plasma concentrations, was described by the effect compartment, with the rate constant for the distribution to the effector compartment equal to 0.335 min−1. Drug concentration had a large effect on the probability of anesthesia. The degree of anesthesia was largest at 10:00 h, lowest at 16:00 h, and intermediate at 22:00 h. In summary, both the pharmacokinetics and pharmacodynamics of propofol in rabbits depended on administration time. The developed population approach may be used to assess chronopharmacokinetics and chronopharmacodynamics of medications in animals and humans. (Author correspondence: agnbienert@op.pl)

Inductively coupled plasma mass spectrometry determination of metals in honeybee venom.

Inductively coupled plasma mass spectrometry (ICP-MS) technique was used to analyze the contamination of selected 20 metals in 32 samples of honeybee venom and to demonstrate differences in the content of these elements. Among the analyzed metal microelements (Al, Co, Cu, Zn, Mn, Mo, B, V, Sr and Ni), macro-elements (Ca, Mg, K and Na) and toxic metals (As, Ba, Pb, Cd, Sb and Cr) were identified. The presented results showed that the metal levels in honeybee venom are much lower than the tolerable upper intake levels for the elements. Also the toxic metal contamination is much lower than the permissible levels for drugs established by the United States Pharmacopeia and the European Pharmacopeia. As opposed to the pharmacopeial tests for metals, a multi-element ICP-MS method has been developed. In order to confirm data obtained, the following steps and parameters were taken into account for the validation of the method: calibration verification, recovery, accuracy, precision, detection limit (LOD), quantitation limit (LOQ), spectral and matrix interference and comparison between ICP-MS and GFAAS (graphite furnace atomic absorption spectrometry) for Mn. All steps of validation proved the accuracy of the results. This is most likely the first study in which the metal content in honeybee venom was evaluated by ICP-MS.

Identification of Serum Peptidome Signatures of Non-Small Cell Lung Cancer.

Due to high mortality rates of lung cancer, there is a need for identification of new, clinically useful markers, which improve detection of this tumor in early stage of disease. In the current study, serum peptide profiling was evaluated as a diagnostic tool for non-small cell lung cancer patients. The combination of the ZipTip technology with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the analysis of peptide pattern of cancer patients (n = 153) and control subjects (n = 63) was presented for the first time. Based on the observed significant differences between cancer patients and control subjects, the classification model was created, which allowed for accurate group discrimination. The model turned out to be robust enough to discriminate a new validation set of samples with satisfactory sensitivity and specificity. Two peptides from the diagnostic pattern for non-small cell lung cancer (NSCLC) were identified as fragments of C3 and fibrinogen α chain. Since ELISA test did not confirm significant differences in the expression of complement component C3, further study will involve a quantitative approach to prove clinical utility of the other proteins from the proposed multi-peptide cancer signature.