Comparative studies on vitamin B1 deficiency inwholeblood of chronically haemodialysed patients: chromatographic, fluorimetricandPCAstudy.

The levels of thiamine diphosphate (ThDP), the most active biologically form of vitamin B1, were assessed in whole blood oflong-term haemodialysed patients (n = 50), by applying chromatographic methods based on RP-HPLC technique with isocratic elution and fluorescence detection. The target analyte, thiochrome diphosphate (ThODP), was obtained by pre-column derivatization of vitamin B1 contained in blood samples, applying deproteination with trichloroacetic acid, following by oxidation with alkaline solution of potassium ferricyanide(III) and stabilization with DTT before assays. A simple and sensitive assay was developed, and the results were referenced to the commercially available test. Steady-state and time-resolved studies on emissive properties of ThODP enabled optimization of the proposed assay. The F-Snedecor test shown no statistically significant differences between both approaches. Assessed parameters of the proposed assay, such as linearity, precision, sensitivity, and recovery, were satisfactory if compared to the reference one. The LOQ value for ThDP in whole blood of studied group of patients was of 0.5 ng/mL and the recovery of88%. The results disclosed high individual variabilities in the interdialytic deficiencies of ThDP among the patients - ranged from afew percent to values close to 100%. A comprehensive clinical data, characterizing patients under study, were processed together, and analysed by employing achemometric discriminative tool, the Principal Components Analysis,to find interdependences among clinical data characterizing patients. The three Principal Components were disclosed, that in sum explained almost 50% of the observed variability of the clinical data set. Among the clinical parameters involved in PCs were dialyzer membrane and type, duration as well as levels of creatinine, haemoglobin, and red blood cells in patients' whole blood.

Neutral endopeptidase (NEP) inhibitors - thiorphan, sialorphin, and its derivatives exert anti-proliferative activity towards colorectal cancer cells in vitro.

Neutral endopeptidase (NEP) is an enzyme implicated in development of different tumors, e.g. colorectal cancer (CRC). In this study, the anti-cancer effects of NEP inhibitors, thiorphan (synthetic compound) and sialorphin (naturally occurring pentapeptide) on CRC cells were investigated. Moreover, we synthesized some derivatives of sialorphin (alanine scan analogues: AHNPR, QANPR, QHAPR, QHNAR; N-acetylated sialorphin; C-amidated sialorphin, and C-amidated alanine scan analogues) to examine the biological activity of these inhibitors on CRC cells. The cytotoxic activity of the NEP inhibitors against CRC cell lines (SW620 and LS180) and normal human fibroblasts (HSF) was evaluated. Additionally, the influence of NEP inhibitors on proliferation, cell cycle progression, induction of apoptosis, and the level of phosphorylation of MAP kinases and mTORC1 signaling pathway proteins in CRC cells were examined. The NEP inhibitors were non-cytotoxic to HSF cells; however, most of them slightly decreased the viability and inhibited proliferation of CRC cells. The N-acetylation or C-amidation of sialorphin or its alanine scan analogues resulted in decreased or abolished anti-proliferative activity of the NEP inhibitors towards the CRC cells. Additionally, thiorphan and sialorphin enhanced the anti-proliferative activity of other CRC-cell growth inhibitors (atrial natriuretic peptide-ANP and melphalan-MEL). The mechanisms involved in the anti-proliferative effects of the tested inhibitors were mediated via NEP and associated with induction of cell cycle arrest in the G0/G1 phase, increased activity of ERK1/2, and a reduced level of phosphorylation of mTOR (Ser2448), 4E-BP1, and p70S6K. However, the NEP inhibitors did not induce apoptosis in the CRC cells. These results have indicated that thiorphan and sialorphin or its derivatives AHNPR, QANPR, QHAPR, and QHNAR have the potential to be used as agents in treatment of patients with CRC.