Development and validation of a HPLC-MS/MS method for the analysis of fatty acids - in the form of FAME ammonium adducts - in human whole blood and erythrocytes to determine omega-3 index.

Recent scientific studies in the field of health and nutrition have unanimously affirmed the importance of consuming the omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), because of their cardioprotective properties. Fatty acid profiling in erythrocyte membranes allows the omega-3 index, which is a recognized indicator of the risk of developing cardiovascular disease, to be calculated. One consequence of the upward trend in healthy lifestyles and longevity is an increase in the number of studies into the omega-3 index, which requires a reliable method for the quantitative analysis of fatty acids. This article describes the development and validation of a sensitive and reproducible liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the quantitative analysis of 23 fatty acids (in the form of fatty acid methyl esters, FAMEs) in 40 µl of whole blood and erythrocytes. The list of acids includes saturated, omega-9 unsaturated, omega-6 unsaturated and omega-3 unsaturated fatty acids as well as their trans-isomers. The limit of quantitation was 250 ng ml-1 for C12:0, C16:0 and C18:0; and 62.5 ng ml-1 for other FAMEs, including EPA, DHA and trans-isomers of FAME C16:1, C18:1 and C18:2 n-6. Sample preparation for fatty acid (FA) esterification/methylation with boron trifluoride-methanol (BF3) has been optimized. Chromatographic separation has been carried out on a C8 column in gradient mode using a mixture of acetonitrile, isopropanol and water with the addition of 0.1% formic acid and 5 mM ammonium formate. As a result, the problem of separating the cis- and trans-isomers of FAME C16:1, C18:1 and C18:2 n-6 has been solved. The electrospray ionization mass spectrometry (ESI-MS) detection of FAMEs, in the form of ammonium adducts, has been optimized for the first time, which has made the method more sensitive that when the protonated species are used. This method has been applied to 12 samples from healthy subjects that consumed omega-3 supplements and has proven to be a reliable tool for determining the omega-3 index.

[Laparoscopic heminephrureterectomy for duplex kidney in children].

INTRODUCTION: Duplication of the upper urinary tract is one of the most common congenital urological anomalies. In patients with critically decreased or lost function of one of the renal segments, heminephrureterectomy is usually the treatment of choice. Until recently, this was an open surgery; in cases of complete removal of the ureter, an additional incision in the iliac region was required. Currently, heminephrureterectomy is increasingly performed laparoscopically. We report the experience in laparoscopic heminephrureterectomy (LHNUE) in 10 clinics in Russia and Belarus. Some of them have already used this technique for 10 years. AIM: The study aimed to to improve the treatment results in children with urodynamic dysfunction due to duplicated upper urinary tract. MATERIALS AND METHODS: We retrospectively analyzed medical records of 111 children treated from 2007 to 2016. There were 26 (23.4%) boys and 85 (76.6%) girls with mean age 44.6 months (from 2 to 170) at the time of surgery. All children included in the study had complete duplex kidneys, including 51 (45.9%) right-sided and 60 (54.1%) left-sided. All the children underwent LHNUE for a critical decrease or absence of function of the upper or lower segment of the duplex kidney caused by the following pathology: obstruction of the ureterovesical junction with the development of the megaureter of the upper ureteral segment in 57 (51.4%) patients; ureterocele in 28 (25.2%); extra-vesical ectopic ureter with urinary incontinence in 10 (9.0%) girls; high-grade UVR in 16 (14.4%) patients. RESULTS: There were no conversions in this series of patients. The mean operative time was 135 minutes (60-240 min.). All children included in the study were followed for 1 to 9 years after surgery. Complications occurred in 17 (15.3%) patients, of whom 12 (10.8%) required repeat surgery. In one patient with the loss of lower pole function, the treatment result was considered unsatisfactory. CONCLUSION: LHNUE for duplex kidney is performed by a few clinics and is still at the stage of development and accumulation of experience. Nevertheless, LHNUE, though an effective treatment modality, carries the risk of reducing or losing the function of the retained segment.

[Experience of 157 vesikoscopic operations in children].

AIM: Recent advances in the field of minimally invasive surgical technologies in children and adolescents have led to the development of vesicoscopic (transvesical, pneumoscopic) access (VA). Current limitations in using VA emphasize the need for further studies investigating surgical options for the management of various pathological conditions of the bladder and ureterovesical junction, the features of surgical techniques and the course of the early postoperative period when used in pediatric urological practice. MATERIALS AND METHODS: From 2013 to 2017, 157 patients (79 girls and 78 boys) aged between 2 months and 18 years (mean age 4.9-8.7 years) underwent surgery using VD. Unilateral and bilateral vesicoscopic ureterocystoneoimplantation was performed in 110 (70%) and 44 (28%) patients, respectively. A total of 198 ureters was implanted. Three (1.9%) children underwent vesicoscopic excision of the bladder diverticulum. Transvesicoscopic Cohen ureteric reimplantation, pneumovesical Glenn-Anderson procedure, and Chumakov ureterocystoneoimplantation were performed in 151 (96.1%), 2 (1.3%) and 1 (0.6%) patients, respectively. RESULTS: The mean operative time when using VA was 126.8+/-46.7 min. In patients younger than one year, 1-3 years, 4-17 years, it was 136.0+/-43.8 min, 130.1+/-43.5 min and 122.4+/-65.8 min, respectively. The mean length of postoperative hospital stay was 6.2+/-2.3 days. In 3 (1.9%) cases we had to convert to open surgery. Gas migration into the abdominal cavity occurred in 6 (3.8%) patients. Fourteen (9%) patients had early postoperative complications. Transient obstruction of ureterovesical junction occurred in 6 (3.8%) patients. Acute complete obstruction of the distal ureter developed in 3 (1.9%) patients aged three months who did not undergo drainage of the upper urinary tract intraoperatively. A paravesical urine leak occurred in 1 (0.6%) patient. In one (0.6%) of the boys, the distal end of the urinary drainage inserted through the trocar into the ureter migrated in the bladder. The urine leakage from the trocar puncture occurred once (0.6%) and was stopped by indwelling urethral catheterization for seven days. In 2 (1.3%) patients, exacerbation of pyelonephritis required a modification in antibacterial therapy. DISCUSSION: Despite the accumulated experience, vesicoscopic surgery remains a laborious and complicated surgical intervention, requiring long learning curves even for surgeons who have good manual skills in laparoscopic surgery. CONCLUSION: In our opinion, vesicoscopic access allows the entire range of surgical interventions on the vesicoureteral junction and bladder in children to be performed. It is effective, significantly less traumatic than traditional open cystotomy access, and associated with an excellent cosmetic result.

Self-assembled nanocrystals discovered in Chelyabinsk meteorite.

Current interest in nanomaterials is focused mostly on artificial materials fabricated for various applications. However, naturally occurring nanocrystal arrays, like those recently found in the meteorite that fell near Chelyabinsk in Russia on 15 February, 2013, can provide some new insights into the nature of nanomaterials, including the conditions for their natural occurrence. Here we report the results of our spectroscopic investigation of a fragment of the Chelyabinsk meteorite. The atomic structure of a fragment of the Chelyabinsk meteorite was studied using high-resolution transmission electron microscopy. Elemental and phase analysis of the object that was carried out by energy dispersive X-ray spectroscopy and electron diffraction revealed the presence of crystalline phases of different chemical compounds specific to meteorites of the LL group. In addition to single-crystal inclusions, extensive areas with ferropericlase nanocrystals having characteristic sizes from 3 to 15 nm were found in the structure of the meteorite. The study of the meteorite employing combination scattering of light (Raman) and photoluminescence spectroscopy methods has revealed quantum effects of ferropericlase nanoparticles and related photoluminescence with a maximum in the range of 675-800 nm.

Preparation and characterisation of anion exchangers with dihydroxy-containing alkyl substitutes in the quaternary ammonium functional groups.

Novel poly(styrene-divinylbenzene) (PS-DVB) based anion exchangers having one and two (2,3-dihydroxypropyl) substitutes in the quaternary ammonium functional groups are prepared and characterised by ion chromatography (IC). The introduction of bulky and hydrophilic substitutes to the anion-exchange groups allows the elimination of non-ionic interactions between the polarisable anions and the aromatic rings from the matrix and the improvement of separation selectivity as compared with the traditional trimethylammonium (TMA) functionalised ion exchangers. The synthesis of the ion exchangers includes acylation of PS-DVB particles with acetic anhydride followed by reductive amination either with methylamine hydrochloride or dimethylamine and further alkylation with oxiranes under varied conditions. The ion exchange selectivity and separation efficiency of nine adsorbents having different structure of bonded groups, ion exchange capacity (9-98µequiv.g(-1)) or particle size is studied for model mixture of inorganic anions (F(-), Cl(-), NO2(-), Br(-), NO3(-), HPO4(2-) and SO4(2-)) using carbonate and hydroxide eluents. The adsorbents with more hydrophilic substitutes provided superior columns efficiencies and better peak symmetry as compared with analogues having hydrophobic functional groups. The calculated values of column efficiencies for polarisable NO3(-) and HPO4(2-) are 18,500 and 29,000N/m, respectively, for anion-exchanger, having N-methyl-N',N″-di-(2,3-dihydroxypropyl)ammonium groups which is significantly higher than 1800 and 12,000N/m obtained for these anions with anion exchanger bearing TMA functional groups.

[Experience in surgery of diastatic ruptures of the colon].

An experience with treatment of 58 patients operated upon for carcinoma of the colon complicated by acute intestinal obstruction and diastatic perforation has been analyzed, 13 patients had incomplete diastatic ruptures (tears) of the colon. In 23 patients the IV stage of the tumor process (remote metastases) were found and the other 35 patients had the III stage. In early terms after operation 37 (63.8%) patients out of 58 patients died. Lethality after incomplete diastatic ruptures of the colon made up 53.8%, and after diastatic perforation - 67.7%. The main cause of lethal outcomes was polyorganic insufficiency against the background of intoxication of mixed genesis (cancerous + endogenous). Ablation of colonic tumor is justified in cases of local peritonitis. Hemicolectomy with ablation of the diastatic rupture is indicated for the right-side localization of the tumor Obstructive resection is expedient for left-side localization of the tumor and rupture, the diastatic ruptures should be sutured or extraperitonized with application of cecostoma. Subtotal colectomy is possible for left-side localization of the tumor and diastatic ruptures of the right half of the colon. Symptomatic operations are indicated for canceromatosis of the peritoneum, terminal phase of peritonitis or infectious-toxic shock.

[Comparative characteristic of the methods of immunocytochemistry and immunomagnetic separation for detection of cytokeratin-positive cells in bone marrow and lymph nodes].

Comparative study of cytokeratin-positive (CK+) cells, isolated from bone marrow and lymph node micrometastases in patients with lung and esophageal cancer, was performed using the methods of immunocytochemistry and immunomagnetic separation. It was found that immunocytochemical analysis permitted the detection of CK+ cells in the smears of bone marrow of oncologic patients. All the samples of bone marrow with immunocytochemically demonstrated CK+ cells, contained also the cellular complexes marked by magnetic spheres. The method of positive immunomagnetic separation has some advantages for the intraoperative diagnosis. However, when either method was applied, among CK+ cells in bone marrow and lymph nodes some false-positive, questionable cellular forms and hemopoietic cells expressing epithelial markers were found. Theses results suggest that for verification of micrometastases in these organs, an additional study using the standard staining of the slides, is required.

Investigation of ionene adsorption on quartz surfaces by thermal-lens spectrometry.

Thermal-lens spectrometry was used for the investigation of the adsorption of ionene to quartz surfaces. The thermooptical analysis of the surface makes it possible to distinguish the modified surface from a clean quartz surface and to provide sensitive direct concentration measurements of the light absorbing co-adsorbed substance. The co-adsorption of chromate ions and 2,10-ionene from aqueous solutions to quartz surfaces was investigated and the desorption procedure proposed.

Optical photothermal detection in HPLC.

A mode-mismatched parallel dual-beam thermal lens spectrometer with a far-field single-channel detector system was used as a detector in HPLC. An expert estimation of the measurement results was applied to optimize the optical-scheme configuration of the spectrometer to achieve the longest linear calibration range and highest repeatability under chromatographic flow conditions. Chelates with 4-(2-pyridylazo)resorcinol were separated and determined with the limits of detection of n x 10(-8)- n x 10(-7) mol L(-1); the relative standard deviation of measurements was 46%. Xylenol Orange, 4-(2-thiazolylazo)resorcinol, and dithizone were studied as post-column reagents in thermal lens detection in ion chromatography. The limits of detection were n x 10(-8)- n x 10(-7) mol L(-1); the linear calibration ranges were about three orders; the relative standard deviation of measurements was 3-7%. A combined photothermal-refractometric detector for HPLC based on a polarization interferometer is proposed. Metal complexes as 4-(pyridylazo)resorcinol chelates (limits of detection of n x 10(-8)- n x 10(-7) mol L(-1)) and sugars (limits of detection of 10-20 ng L(-1)) were investigated as model substances. Obtained results were compared with results for traditional detectors, which show that photothermal detection has higher sensitivity than photometric and other absorption detectors.

Application of polyelectrolyte complexes as novel pseudo-stationary phases in MEKC.

Polyacrylic acid (PAA) and polymethacrylic acid (PMAA) with carboxyl groups partially blocked by dodecyltrimethylammonium bromide (DTAB) and tetrabutylammonium bromide (TBAB) were tested as new pseudo-stationary phases in micellar electrokinetic chromatography (MEKC). The separation of was examined using PAA and PMAA. Excellent resolution of the substituted phenols and derivatized amino acids was demonstrated using additives of PAA-DTAB polyelectrolyte complex in the running phosphate buffer. It was found that the capacity factors were proportional to the concentration of the complex PAA/DTAB. Critical micelle concentration was effectively zero. It was found that the migration times and efficiency of separation of phenols and derivatives of amino acids depended on the type of polymers and alkyltrimethylammonium salts used.

Separation and enantioseparation of derivatized amino acids and biogenic amines by high-performance liquid chromatography with reversed and chiral stationary phases.

This study demonstrates that an amino-beta-cyclodextrin-bonded phase column exhibits enantioselectivity for various amino acid derivatives. Mixtures of methanol, acetonitrile, tetrahydrofuran or dioxan and triethylamine buffers (pH 4.0-7.0) were used as mobile phases. The effect of the mobile phase on the resolution process was studied by varying the mobile phase composition (type and percentage of organic modifiers, pH, and ionic strength of the buffer solution). The 1-octanol-water partition coefficients are calculated and tabulated for 16 derivatized amino acids. The chromatographic data for 42 pairs of derivatized amino acids resolved on the amino-beta-cyclodextrin-bonded phase are summarised. The separation of adrenaline, noradrenaline and amphetamine on a novel vancomycin stationary phase is demonstrated.

Changes in the balance between membrane-bound and soluble forms of CD95 (Fas) during selection of tumor cells in vivo.

Studies concerning the expression of the receptor CD95 (Fas) by tumor cells and the role of this protein in apoptosis induced by the effector host cells that bear Fas-ligand are mainly focused on the membrane-bound form of Fas. There are only a few data about the production of the soluble form of Fas by the tumor cells, its role in the interaction with the effector host cells, and the possible changes in the synthesis of this protein during tumor progression. In the present work, three in vitro transformed parental cell lines of different origin and 24 of their variants isolated after a short cycle of natural selection in vivo were studied. It was demonstrated for the first time that: 1) production of the soluble Fas by all selected in vivo variant tumor cell lines increased significantly (2-10-fold) in comparison to the initial (parental) cell lines and did not depend on the origin of the parental lines. At the same time, the expression of the membrane-bound form of Fas decreased considerably; 2) variations of the balance between membrane-bound and soluble forms of Fas in selected in vivo variant cells and the expression of the [H(2)O(2)(CA) + PGE(S)]-phenotype by these cells (this phenotype determines one of the essential mechanisms of the protection of a tumor cell in vivo) possibly represent independent secondary changes acquired during tumor progression in vivo.

Lonene-coated sulfonated silica as a packing material in the packed-capillary mode of electrochromatography.

An aliphatic ionene (2-10-ionene) has been selected as a modifier to prepare a novel polymer-coated packing material for capillary electrochromatography. The packing material was produced by dynamical modification of a commercially available sulfonated silica Exsil-100 SCX. Strong ion-exchange interactions in the capillary packed with ionene-modified sulfonated silica have been demonstrated by the example of the retention of p-aminobenzoic acid. The total calculated anion-exchange capacity of the sorbent in the capillary was about 4 x 10(-9) mol. A fast separation (about 15 min) of several aromatic acids was achieved with the packing material. The highest number of theoretical plates obtained was about 120,000. Limits of detection of the aromatic acids were 2-5 microg/ml. The advantages and lacks of the approach are discussed briefly.

Ion-chromatographic selectivity of polyelectrolyte sorbents based on some aliphatic and aromatic ionenes.

An aliphatic ionene with hydroxyl group (2HP-8 ionene), mixed aliphatic-aromatic ionenes (3-X and 6-X ionenes), aromatic ionene (Ph-X ionene), and viologen (Dp-X ionene) - polymers with quaternary nitrogen atoms in the main chain served as modifiers in synthesising polyelectrolyte sorbents for ion chromatography. The selectivity of produced and several previously prepared anion exchangers was compared with those of aliphatic ionenes. It was found that aromatic ionenes having a rigid structure of polymer chains are similar to their aliphatic analogues with shorter chains with a high charge density. Polyelectrolyte sorbents based on aromatic ionenes show higher selectivity to aromatic acids (e.g., 1-naphthalenesulfonic acid) as compared with aliphatic ionenes due to specific pi-pi interactions.

Polyelectrolyte sorbents based on aliphatic ionenes for ion chromatography.

2-4, 3-4, 2-8, 3-8, 3-6, 4-6, 6-8, 6-10-ionenes (polymers with quaternary nitrogen atoms in the main chain) served as modifiers in synthesizing polyelectrolyte sorbents for ion chromatography. The approaches to the synthesis and their stability are discussed. Cluster analysis was applied to separate aliphatic ionenes to three groups as chromatographic modifiers, namely hydrophilic, hydrophobic, and intermediate. Each group is characterized by a certain selectivity to sulfate, perchlorate, iodide and thiocyanate. The sorbents show high selectivity and efficiency up to 15,000 theoretical plates per meter.