Fluorescent probing of the ligand-binding ability of blood plasma in the acute-phase response.

The acute-phase response alters the composition of carrier proteins in plasma, which may affect the blood deposition and transport of biomediators and drugs. The effect of the acute-phase response on the ligand binding ability of plasma was studied in leukemic children with and without systemic inflammation (sepsis and septic shock). To target different transport proteins, differentially charged fluorescent dyes were used: anionic ANS (8-anilinonaphthalene-1-sulfonate), uncharged Nile red, and cationic Quinaldine red. Human serum albumin was a principal carrier for ANS and competed for Nile red binding with lipoproteins. The synchro-scan fluorescence spectra of Nile red in plasma distinguished two species of the dye bound to serum albumin and to low-density and/or very low-density lipoproteins. The binding of Quinaldine red did not correlate with albumin and lipoprotein levels, and was probably determined by alpha(1)-acid glycoprotein. Compared with the control group, leukemia increased Quinaldine red binding by 65% and did not significantly affect the binding of other probes. Sepsis and septic shock did not change the binding of Quinaldine red, but progressively decreased ANS binding, finally by about 33%, and shifted Nile red distribution from serum albumin toward lipoproteins. These changes reflected a modified composition of the three principal transport proteins in plasma in the acute-phase response. Simple and rapid fluorescent tests developed in this study can be used to evaluate the acute-phase response and to optimize drug administration protocols in clinical practice.

[Interaction of bromophenol blue with serum albumin: criteria for using dyes for assessing the state and quantity of protein]. / Vzaimodeistvie bromfenolovogo sinego s syvorotochnym al'buminom: kriterii ispol'zovaniia krasiteliia dliia otsenki sostoianiia i kolichestva belka.

The optical properties of the complexes of the pH-dependent dye bromophenol blue (BPB) with human serum albumin were investigated by the spectrophotometric method. The solvatochromic longwave displacement of bound BPB-2 absorption and BPB-1/BPB-2 redistribution were shown to form the optical signal of complexes. Because of the distortion of the bound BPB-2 signal its quantity was determined as delta A630 = A630 - A660 and the use of lambda max as structural parameter was limited to low pH less than or equal to 3. The conclusion was made that BPB is inapplicable as a structural probe on account of low structural dependence of delta A630 and pH-limitation of lambda max used. The maximal absorption delta Amax = Amax - A660 and its structural independence were obtained in the region of 70-100% occupation of the dye-binding centers of the protein. It is the optimal conditions for the quantitative determination of protein. After maximal dye binding (15-16 molecules of BPB per 1 molecule of albumin) the aggregation and precipitation of the complexes occurred.

[Biophysical mechanisms and kinetics of thrombocyte aggregation]. / Biofizicheskie mekhanizmy i kinetika agregatsii trombotsitov.

The basic stages of platelet interaction and their relationship with kinetics of aggregation are considered. The aggregation is expressed theoretically as adsorbtion of stimulated platelets on the agglutinative surface, including surfaces of single cells and all aggregates that are formed. It is also suggested that the inductor determines the number of activated cells, and that cofactors of aggregation increase the reactivable surface aggregating platelets as a result of interaction with receptors of the platelet membrane. Equations are deduced for describing the dependence of aggregation kinetics from the concentration of inductor and cofactor of aggregation. A method is proposed for determination of binding parameters of inductor and aggregation cofactors with cell membranes by kinetic curves of aggregation.

[Characterization of sensitivity and specificity of fluorescent markers of structural state and binding ability of proteins]. / Kharakteristika chuvstvitel'nosti i spetsifichnosti fluorestsentnykh markerov strukturnogo sostoianiia i sviazyvaiushchei sposobnosti belkov.

Methods for determining the sensitivity of fluorescent probes and their classification with respect to sensitivity and specificity using human serum albumin as a reference protein and 8-anili-nonaphthalenesulfonate, K35 and pyron red as probes are proposed. The indicators of sensitivity and specificity of fluorescent probes as markers of the structural state of proteins are the activation of the probe fluorescence in excess of protein and the constant of its binding to the strong highly affine center of protein sorption. Fluorescent probes as markers of the binding ability are assessed by a protein-dependent increase in fluorescence under the conditions of excess probe and from the constant of its binding to weak secondary binding sites of the protein.

[Parameters of binding of fluorescent probe pyrrone red with human serum albumin]. / Opredelenie parametrov sviazyvaniia fluorestsentnogo zonda pirronovogo krasnogo s syvorotochnym al'buminom cheloveka.

The constant of binding of a new fluorescent probe pyrrone red to human serum albumin (Kb = 4.7.10(5) M-1) and the number of binding sites (N = 2) were determined by the method of double fluorimetric titration at a Fex/Fem intensity ratio of 560/625 nm. The affinity of pyrrone red for albumin was by 20% lower than that of 8-anilinonaphthalenesulfonate and 2.4 times higher than that of another probe K35. Thus, pyrrone red is almost identical to amlinonaphthalenesulfonate in affinity for albumin and can be considered as a long-wavelength "red" analogue of anilinonaphthalenesulfonate.

[Accumulation of methylene blue by erythrocytes and determination of its maximum sensitivity to cell damage]. / Nakoplenie metilenovogo sinego éritrotsitami i opredelenie ego maksimal'noi chuvstvitel'nosti k povrezhdeniiu kletok.

The accumulation of methylene blue in native and damaged erythrocytes treated by cetyltrimethylammonium bromide at different initial concentrations (c0) of methylene blue and different volume ratios between external solution and cells (Vs/Vc) was studied. It was shown that at low methylene blue concentrations (c0 < 200 microM), the sorption of the dye by cells made the main contribution to its accumulation. As a result, the internal concentration of methylene blue exceeded manifold its external concentration and their ratio Q was 4-6 for native and 6-9 for damaged cells. As Vs/Vc decreased and especially as c0 increased, the diffusion of methylene blue inward the cells increased concentration gradient, and Q sharply fell to 0.9-1.0. The optimal values of c0 and Vs/Vc that provide the maximum sensitivity of Q to cell damage were determined. The advantages of using Q over other parameters of methylene blue accumulation were shown.

[Prevention of binding of Nile red with hydrophobic proteins and surface cuvettes using detergents]. / Predotvrashchenie sviazyvaniia nil'skogo krasnogo s gidrofobnymi belkami i poverkhnost'iu kiuvety s pomoshch'iu detergenta.

The nile red fluorescence in solutions containing Triton X-100, human serum albumin and hexadecane in different combinations was investigated. The dye fluorescence in phosphate buffer after 2-4 min incubation strongly decreased because of the dye aggregation and was stable in a detergent solution. The intensity of nile red fluorescence in the detergent solution was not changed after the addition of equal weight concentration of albumin and increased more than 2 times after the addition of the same concentration of hexadecane. The detergent eliminated the nile red sorption by cuvette surface. It is concluded that the detergent addition increases nile red selectivity as a lipid probe in the lipid-protein systems.

[Psoralen-photosensitized formation of free lipid radicals at 77 degrees K]. / Fotosensibilizirovannoe psoralenami obrazovanie svobodnykh radikalov lipidov pri 77 K.

It is shown by ESR method that 8-methoxypsoralen (8-MOP) under low temperature UV-irradiation has no influence on free radical formation of saturated fatty acids (FA). Under the same conditions the quantum yield of free radical photogeneration for unsaturated FA (oleate and limolenate) after addition of 8-MOP increased 7.5-15 times. The ESR spectrum of free radicals generated after interaction of 8-MOP with polyunsaturated FA, may be presented as superposition of signals-CH-(CH = CH)2-, -CH2CHCH2- and -CH2CH2. The molecular mechanism of photosensitized reactions is discussed.

[A comparative analysis of the immunochemical determination of gentamycin by fluorescence polarization and quenching]. / Sravnitel'nyi analiz immunokhimicheskogo opredeleniia gentamitsina po poliarizatsii i tusheniiu fliuorestsentsii.

A polarization fluorescence immunoassay (PFIA) for gentamicin with using a set of reagents made in this country was developed. One ml of fluorescein-labeled gentamicin (50 nM) and 100 microliters of antiserum are added to 50 microliters of the sample and the fluorescence polarization is measured. The time of the assay is 10 to 15 minutes, the range of the measurable concentrations is 0 to 800 ng/ml, the sensitivity of the method is 5 ng/ml and the accuracy is 5.8-10.3 per cent. A fluorescence quenching immunoassay (FQIA) for gentamicin was also developed. Determination of gentamicin by the FQIA does not require the use of a specific polarization fluorimeter. Its linear calibrating dependence is more convenient. However, its accuracy and sensitivity are 3 times lower than those of the PFIA.

[Methods of determining lipid peroxidation products in the serum using a thiobarbituric acid test]. / Analiz metodov opredeleniia produktov perekisnogo okisleniia lipidov v syvorotke krovi po testu s tiobarbiturovoi kislotoi.

Main principles for carrying out of the thiobarbituric acid (TBA)-test of blood serum were characterized. These principles involved incubation of the sample at pH 1.3-1.6, extraction of the reaction products with butanol and spectrofluorimetric or spectrophotometric estimation. Content of TBA-active products in blood serum of healthy donors constituted 3.86 +/- 0.66 nmol malonic dialdehyde per ml of blood serum as shown in fluorimetric estimation by means of the procedure developed.

[Diagnosis and prevention of diabetic angiopathies]. / Diagnostika i profilaktika diabeticheskoi angiopatii.

The paper presents a new method to diagnose the I preclinical stage of diabetic angiopathy permitting identification of the risk groups and design of preventive measures. The method implies evaluation of the biochemical index characterizing serum lipids structural defense against lipid peroxidation.

[Lipid peroxidation in the blood and spinal fluid of patients with craniocerebral injuries]. / Perekisnoe okislenie lipidov v krovi i spinnomozgovoi zhidkosti u bol'nykh s cherepno-mozgovoi travmoi.

The content of the products of lipid peroxide oxidation in the plasma of blood flowing from the brain was higher in patients with moderate and severe craniocerebral injury than in patients with no marked functional and metabolic disorders. The concentration of these products in the cerebrospinal fluid was still higher. In patients with severe craniocerebral injury there was also an increase in the content of free fatty acids in blood flowing from the brain and in the content of ferrous iron ions in the cerebrospinal fluid. The intensification of the processes of lipid peroxide oxidation in the brain may play an essential role in determining the severity of the traumatic damage.

[Determination of tyrosine- and tryptophan-containing peptides in plasma by absorption in UV spectral region]. / Opredelenie tirozin- i triptofansoderzhashchikh peptidov v plazme krovi po pogloshcheniiu v UF-oblasti spektra.

A method for determining the cumulative concentration of tyrosine- and tryptophan-containing peptides (TSP and TPP) in blood plasma: 0.2 ml of blood plasma is added 0.1 ml of 1.8 M chloric acid, the sample is centrifuged, the supernatant (0.2 ml) is added 1.8 ml of 0.7 M NaOH and the optic density is measured at 290 nm in the quarts or plastic flask. Equations were derived to calculate separately the concentrations of TSP and TPP, whose normal values are 1.28 +/- 0.31 and 0.25 +/- 0.06 mmol/l, respectively. The increasing total content of peptides in thyrotoxicosis was shown to be mainly conditioned by a higher TSP concentration (a 2.4-fold increase). Therefore, TSP is a more sensitive marker in case of endogenous intoxication versus the generally known index of the cumulative content of oligopeptides.

[Assessment of body intoxication as a balance between accumulation and binding of toxin in plasma]. / Otsenka intoksikatsii organizma po narusheniiu balansa mezhdu nakopleniem i sviazyvaniem toksinov v plazme krovi.

A novel approach to assessing endogenic intoxication (EI) as an imbalance between toxin accumulation and binding by albumin in blood plasma is proposed. The intoxication criterion (IC) is determined by the ratio of the content of medium-weight molecules (MWM) and effective albumin concentration (EAC): IC = MWN/EAC. In children with oncohematological diseases the development of EI is associated with a 4-fold drop of MWM content and a twofold decrease of EAC, and hence, the imbalance between these two values increases 9-10 times. The proposed approach notably improves the sensitivity of diagnosis of the early stages of EI. Application of a new fluorescent marker pirrone red for measuring albumin binding capacity is validated and an algorithm of EAC determination using calibration curve of probe binding to standard albumin solution is described.