Biomedical subjects
Publications and source records attributed to A I Gusev.
An analysis of results of the operative treatment of 36 patients who received and did not receive nutritional support in the pre- and postoperative periods has shown that the nutritional support improves the course of the postoperative period.
The effect of liquid chromatographic separation on matrix-related signal suppression in electrospray ionization mass spectrometry (LC-ESI-MS) was investigated. A method incorporating on-line two-dimensional liquid chromatography mass spectrometry (LC/LC-MS) was developed to compensate for matrix effects and signal suppression in qualitative and quantitative analysis. The LC/LC-MS(MS) approach was successfully applied for single-component and multicomponent analysis in a variety of complex matrixes. It was demonstrated that matrix-related signal suppression could be induced solely by (i) column overload, (ii) matrix component-analyte coelution, or a combination of each. Application of on-line orthogonal LC/LC separations can be effective in reducing both causes of matrix-related signal suppression effects i.e., column overload and matrix-analyte coelution for a variety of LCn/MSn applications.
The effect of liquid chromatography separation on liquid chromatography-tandem mass spectrometry (LC-MS-MS) signal response for the characterization of low-molecular-mass compounds in a complex matrix was investigated. Matrix induced signal suppression appears throughout the entire LC-MS-MS analysis of wheat forage extract, with greatest suppression occurring at early retention times. Experimental results show that co-elution of matrix components and analytes from the LC column may be most strongly attributed to column overloading rather than similar analyte and matrix retention behavior. As a result, two-dimensional (LC-LC) separation can be a highly effective approach to address signal suppression effects for the quantitative LC-MS-MS analysis of complex matrix samples.
The application of LC separation and mobile phase additives in addressing LC-MS/MS matrix signal suppression effects for the analysis of pesticides in a complex environmental matrix was investigated. It was shown that signal suppression is most significant for analytes eluting early in the LC-MS analysis. Introduction of different buffers (e.g. ammonium formate, ammonium hydroxide, formic acid) into the LC mobile phase was effective in improving signal correlation between the matrix and standard samples. The signal improvement is dependent on buffer concentration as well as LC separation of the matrix components. The application of LC separation alone was not effective in addressing suppression effects when characterizing complex matrix samples. Overloading of the LC column by matrix components was found to significantly contribute to analyte-matrix co-elution and suppression of signal. This signal suppression effect can be efficiently compensated by 2D LC (LC-LC) separation techniques. The effectiveness of buffers and LC separation in improving signal correlation between standard and matrix samples is discussed.
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Experimental and clinical investigations enabled the authors to develop a method of hernioplasty using a pi-shaped flap from aponeurosis of the external oblique abdominal muscle on the feeding pedicle. In this case the anatomo-functional interrelations of the tissues in the ilio-inguinal area get minimum injuries. It does not make a substantial obstacle for the following regeneration and restitution and so the method is rather efficient and can be used in the everyday practice.
Automation of data collection in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry using a correlative analysis algorithm is demonstrated. This algorithm was employed to compensate for mass spectral jittering in MALDI data collection (e.g., peak shifts along the m/z axis, signal intensity deviations, etc.). Several important parameters for performing correlative analysis, such as the minimum correlation coefficient to be used and number of mass spectra to acquire prior to correlation, have been investigated and optimized. In addition, the correlation algorithm improved mass resolution of low- and high-molecular-weight compounds by as much as a factor of 4. Signal reproducibility in MALDI quantitative analysis also is improved when correlation is employed for data collection. This data collection algorithm can be used in conjunction with other instrumental optimization programs to allow for fully automated MALDI analysis, which is required for the routine applications carried out in many analytical laboratories.
The in vitro metabolism of tacrolimus (TAC, FK 506) was investigated in the liver microsomes prepared from normal rats as well as rats treated with dexamethasone (DEX) and rifampin (RIF). The rate of tacrolimus metabolism was similar in control and RIF treated rat liver microsomes, whereas it significantly increased in microsomes obtained from dexamethasone treated rats. Seven different possible metabolites were identified in the microsomal preparations from rats treated with rifampin or dexamethasone whereas the microsomes from the control rats failed to produce the mono-demethylated and monohydroxylated metabolite of TAC (TAC+2, m/z = 805.5). There was an apparent difference in the amount of individual metabolites formed in different groups. This indicates quantitative differences in the induction of cytochrome P450 3A, an enzyme sub family known to be primarily responsible for tacrolimus metabolism. Lack of induction of tacrolimus metabolism by rifampin can be attributed to the lack of effect of rifampin in inducing cytochrome P450 3A in rats.
The identification and simultaneous quantification of Tacrolimus and its hepatic metabolites in baboons has been achieved using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry and static secondary-ion mass spectrometry (TOF-SIMS). Little fragmentation, high sensitivity and tolerance to contamination are the major advantages of these methods, allowing facile identification and quantification of metabolites produced in vitro with minor analyte isolation. Based on the MALDI and TOF-SIMS results, seven metabolites have been identified: de-methylated, di de-methylated, hydroxylated, di hydroxylated, de-methylated hydroxylated, dihydrodiol, and di de-methylated hydroxylated. The concentrations of the parent drug and its major metabolites (e.g. de-methylated, di de-methylated) were measured using Rapamycin as an internal standard. The time course of Tacrolimus and its major metabolites as a function of incubation time was calculated. Good correlation between SIMS and MALDI results was obtained.
The protocol and various matrices were examined for quantification of biomolecules in both the low ca. 1200 amu and mid mass 6000-12000 amu ranges using an internal standard. Comparative studies of different matrices of MALDI quantitative analysis showed that the best accuracy and standard curve linearity were obtained for two matrices: (a) 2,5-dihydroxybenzoic acid (DHB) combined with a comatrix of fucose and 5-methoxysalicylic acid (MSA) and (b) ferulic acid/fucose. In the low mass range, the quantitative limit was in the 30 fmol range and in the mid mass range the quantitative limit was in the 250 fmol range. Linear response was observed over 2-3 decades of analyte concentration. The relative error of the standard curve slope was 1.3-1.8% with correlation coefficients of 0.996-0.998.The main problem for quantitative measurement was suppression of the signal of the less concentrated component (analyte or internal standard) by the more concentrated component. The effect was identified with saturation of the matrix by the analyte. The threshold of matrix saturation was found to be in the range of 1/(3000-5000) analyte/matrix molar ratio. To avoid matrix saturation the (analyte+internal standard) to matrix molar ratio should be below this threshold. Thus the internal standard concentration should be as low as possible.DHB/MSA/fucose and ferulic acid/fucose matrices demonstrated good accuracy and linearity for standard curves even when the internal standard had chemical properties different from the analyte. However, use of an internal standard with different chemical properties requires highly stable instrumental parameters as well as constant (analyte+internal standard)/matrix molar ratio for all samples.
The fast-evaporation method of sample preparation has been applied for quantitative analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. An instrumental protocol focusing on improvement of shot-to-shot repeatability and compensation for signal degradation has been developed for quantification of angiotensin II using the fast-evaporation technique and an internal standard. The fast-evaporation method was compared to the standard method of sample preparation (using a multicomponent matrix) in the quantitative analysis of angiotensin II, and found to be superior in several respects. Improvement in sample homogeneity using the fast-evaporation method enhanced both point-to-point repeatibility and sample-to-sample reproducibility. The relative standard deviations of the analyte/internal standard ratios (point RSD) were decreased by a factor of three compared to those obtained using the multicomponent matrix method. The average point RSD was found to be ca. 5% for the fast-evaporation technique. Two internal standards were evaluated for quantification of angiotensin II. The better one, 1-SAR-8-Ile angiotensin II, yielded a relative standard deviation of the standard curve slope of ca. 2.2% over two orders of magnitude of concentration (45 nM to 3000 nM), an improvement by a factor of two over the standard preparation method. Renal microdialysate samples, spiked with angiotensin II and the internal standard 1-SAR-8-Ile angiotensin II, were also analyzed using the fast-evaporation technique. The detection limit was calculated to be in the high attomole range (675 amol). Furthermore, the accuracy for a single determination of angiotensin II concentration in these samples was found to be 13.9% with a relative error of 8.19%.
Analytical methods have been developed for the detection and quantification of cyclosporin A (CsA) in blood using time-of-flight secondary-ion mass spectrometry (TOF-SIMS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). Linear calibration curves were obtained for both methods in the range 25-2000 ng/mL in whole blood. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 7 and 23 ng/mL, respectively, for the TOF-SIMS method; the LOD and LOQ were 10 and 33 ng/mL, respectively, for the MALDI/TOF-MS method. Coefficients of variation ranged from 3 to 5% for the TOF-SIMS method and from 4 to 8% for the MALDI/TOF-MS method. Blood samples were also analyzed by a nonspecific method, fluorescence polarization immunoassay (FPIA), and by a specific method, high-performance liquid chromatography (HPLC). The TOF-MS results are in good agreement with the HPLC results, but not with the FPIA results. The TOF-MS methods can also provide information about CsA metabolites.
A variant of the counterflow isotachophoresis on nitrocellulose membranes (NCM) was applied for the analysis of the epitope specificity of monoclonal antibodies (Mabs). Different Mabs against mouse and human alpha-fetoprotein were fixed on the membrane and the immunoreagents were transferred consequently to these dots by the electroendo-osmotic flow. The epitope specificity of the Mabs was estimated by the competition for the homologous antigen between the Mab added to the antigen and the one fixed on the membrane.
Immunolocalization of albumin was investigated in normal and regenerating adult mouse liver after perfusion fixation with saponin. Light and electron microscopy have demonstrated that in normal liver the intralobular activity of albumin synthesis was distributed along the gradient decreasing from the portal tract to central vein. The uniform intensive staining of all hepatocytes was observed in regenerating liver. In addition, albumin synthesis was expressed in the epithelial cells of portal bile ducts.
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Splenocytes of rats immunized with mouse alpha-fetoprotein (MAFP) and cells of mouse myeloma NS-I were used to obtain hybridomas that synthesize antibodies to MAFP (anti-MAFP). Antibody activity was determined by the two tests: solid-phase ELISA and indirect immunoperoxidase staining of the regenerating liver sections of mice poisoned with CCl4. After recloning of one of the hybridomas a clone that effectively synthesized anti-MAFP was selected and reproduced. Anti-MAFP were isolated from cultural fluid of the clone by sorption-elution on CnBr-activated sepharose 4B conjugated with purified MAFP. Monoclonal origin of anti-MAFP has been demonstrated by electrophoretic homogeneity of the preparation, and by its appurtenance to one of the subclasses of rat IgG. Anti-MAFP does not precipitate MAFP but distinctly inhibits the formation of the precipitation line by MAFP and rabbit antibodies to MAFP in the zone of diffusion of monoclonal antibodies. Monoclonal anti-MAFP were high-effective in the reaction with MAFP in both tests. Anti-MAFP specificity is directed to one of the MAFP determinants that is common to rat and human alpha-fetoprotein.
Ultrastructural localization of alpha-fetoprotein (AFP) was studied with the use of the indirect immunoperoxidase technique in the regenerating liver of three strains of mice after CCl4 poisoning. Upon the use of monoclonal and monospecific antibodies AFP synthesis was revealed in part of mature hepatocytes and in smaller cells. No analogues of small cells were found in the normal liver. It is noted that there is structural similarity of atypical AFP-synthesizing cells to precursor cells of hepatocytes during chemical hepatocancerogenesis in rats. Approaches to identification of atypical cells are discussed.
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