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PubMed1978

Application of Henderson equation for the determination of weakly acidic pharmaceutical compounds. Part 1: Determination of sulfathiourea.

A new method based on the Henderson equation is proposed for the determination of sulfathiourea. The determination is achieved in mixed organic solvent/water media. To apply the Henderson equation, the dissociation constant (pKa) of sulfathiourea in such media should be known. Thus, the values of pKa as function of the composition of the media have been determination potentiometrically according to Albert and Serjeant. Equations have been derived to calculated the pKa of sulfathiourea in any medium. The pKa of sulfathiourea in water and the other solvents, methanol, ethanol, isopropanol and acetone, are reported. The proposed method is simple and rapid and gives reliable results. The mean percent recoveries of the powder in 50% solvent: methanol, ethanol, isopropanol and acetone are: 97.87 +/- 1,30, 100.85 +/- 1.35, 102.01 +/- 1.39 and 99.35 +/- 0.29 respectively. For the tablets, the mean percent recovery in 50% acetone is 97.95 +/- 0.60. The mean percent recovery of added standard sulfathiourea to the tablet powder is 101.55 +/- 0.83.

Chemical Phenomena↗

PubMed1981

[Comparison of in vitro methods for the determination of the activity of fosfomycin with the efficacy determined in experimentally infected mice (author's transl)].

Effective doses of fosfomycin against 17 strains of bacteria were determined in therapeutic experiments using infected mice. The peak serum levels obtained after administration of these doses were compared with the minimum inhibitory concentrations determined in vitro. Reasonable minimum inhibitory concentrations were obtained only with Mueller-Hinton agar containing glucose-6-phosphate: 1. Correlation of in vivo and in vitro efficacy is much better in comparison with the use of media without glucose-6-phosphate. 2. Minimum inhibitory concentrations below the levels obtained after therapeutic doses were found only in Mueller-Hinton agar containing glucose-6-phosphate. 3. Minimum inhibitory concentrations are better reproducible when determined on solid media than in broth.

Animals↗

PubMed1977

Determination of cryoglobulins as lipoprotein-autoantibody immune complexes and antigenic determinants against antilipoprotein autoantibody.

Serum IgG-antilipoprotein-autoantibody activity (at 4 degrees C) of a plane xanthoma patient was shown by double-immunodiffusion method. Cryoglobulins in the serum were dissociated to polyclonal IgG and alpha- and beta-lipoproteins by acidification and were reconstructed by neutralization. IgG fraction of the cryoglobulins precipitated with lipoproteins. The cryoglobulins were thus demonstrated to be immune complexes of polyclonal IgG-antilipoprotein-autoantibody and both alpha- and beta-lipoproteins. A part of the lipoprotein-autoantibody immune complexes was not cryoprecipitable. Antigenic determinants for the autoantibody existed in the lipid moieties of lipoproteins, in contrast to the apoproteins which determined the specificity to heteroimmune antilipoprotein antibody. The presence of more than nine different antigenic determinants against the autoantibody indicated that lipoproteins were immunologically heterogeneous depending upon the lipid moieties. Lipoproteins reactive with the autoantibody varied quantitatively in normal individuals and were not detected in a primary hyper-beta-lipoproteinaemia patient and in a primary biliary liver cirrhosis patient with much lipoprotein-X. The absence of antigenicity in the two patients' sera is most likely caused by abnormal lipid moieties of lipoproteins.

Adult↗

PubMed1977

[Use of serologic reactions for determining the concentration of the determinants of an antigen and its valence].

By the serological tests with the erythrocytic diagnostic agents it is possible to estimate the concentration of the determinants in the antigenic preparation under study. For this purpose it is necessary to have at one's disposal the data on the molecular weight and concentration of the reference antigen, valence of erythrocytes loaded with the antigen or antibodies, and antibody heterogeneity index. If these values are known, then one can calculate the concentration of antigenic determinants in terms divided by the valence of the reference antigen. With the aid of pure antibodies preparation obtained with the immunosorption procedure it is possible to measure the determinant concentration of the antigen tested and the valence of the reference antigen. For the capsular antigen of plaque bacilli the valence was estimated by two independent methods and in both cases it was close to 10. The valence of the reference capsular antigen permits to calculate the concentration of the antibody molecules in the hyperimmune sera without isolation of pure antibodies.

Animals↗

PubMed1977

Quantitative determination of drugs by means of densitometry of thin-layer chromatograms. Part 6 Determination of 1-benzhydril-4-allypiperazine dihydrochloride in biological media.

A quantitative method for determination of 1-benzhydril-4-allypiperazine dihydrochloride (As-2) in blood is proposed, based on direct densitometry of t.l.c. plates. The main advantage of the method consists in the fact that it can be applied for determination of As-2 in biological samples in the presence of its metabolites and other components of the investigated samples. The statistical treatment of results obtained from ten analyses shows that the method has no systematic error and the relative percentage error is +/- 8.5%. The lowest concentration which can be determined is 5 microng/1 ml plasma. It has been established that As-2 administered intravenously in the maximally tolerable dose of 22 mg/kg can be detected in the blood after the first min (RF = 0.45). Ten min after application one major metabolite (RF = 0.35) is observed under UV-light. When administered per os in the maximally tolerable dose of 100 mg/kg, As-2 is established in the blood after 15 min while the metabolite mentioned above is detected by the thirtieth min.

Animals↗

PubMed1997

A new fast and standardless method for direct determination of metals associated with particulate matter in air: avoiding errors in the determination of Pb in an urban environment.

We describe a new method and the relevant instrumentation necessary for its implementation in the analysis of metals associated with particulate matter in air. The procedure can be divided into two steps: in the first step the sample is accumulated in a device through electrostatic precipitation whose center is a graphite tube; in the second step the graphite tube itself is used as an atomization device for the determination of the metals present in the sample through the electrothermal atomic absorption technique. The method is simple, fast, accurate, and inexpensive. Moreover, if the experimental conditions are well chosen, there is no need for calibration, which is very convenient in the case of samples such as particulate matter in the air. The elements that can be determined with the present apparatus are Hg, Cd, Tl, Ag, Mg, and Mn. These are highly or medium volatile because the materials used cannot reach very high temperatures for long periods. The experiments are confined to air, but other gases, in which a corona discharge is possible, would give the same results. With the method proposed, it was possible to show that the official method for Pb determination in the urban environment of Bologna presents a negative systematic error of about 25%.

Lead↗

PubMedDate not supplied

Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor.

The antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen-binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by humoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen-specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour-associated antigen mucin-1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen-specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity.

Antibody Specificity↗

PubMed2003

The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9].

Despite 12 yr since the discovery of SRY, little is known at the molecular level about how SRY and the SRY-related protein, SOX9 [SRY-related high-mobility group (HMG) box 9], initiate the program of gene expression required to commit the bipotential embryonic gonad to develop into a testis rather than an ovary. Analysis of SRY and SOX9 clinical mutant proteins and XX mice transgenic for testis-determining genes have provided some insight into their normal functions. SRY and SOX9 contain an HMG domain, a DNA-binding motif. The HMG domain plays a central role, being highly conserved between species and the site of nearly all missense mutations causing XY gonadal dysgenesis. SRY and SOX9 are architectural transcription factors; their HMG domain is capable of directing nuclear import and DNA bending. Whether SRY and SOX9 activate testis-forming genes, repress ovary-forming genes, or both remains speculative until downstream DNA target genes are identified. However, factors that control SRY and SOX9 gene expression have been identified, as have a dozen sex-determining genes, allowing some of the pieces in this molecular genetic puzzle to be connected. Many genes, however, remain unidentified, because in the majority of cases of XY females and in all cases of XX males lacking SRY, the mutated gene is unknown.

Amino Acid Sequence↗

PubMed2002

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody.

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.

Amino Acid Sequence↗

PubMedDate not supplied

Application of covalent affinity chromatography with thiol-disulphide interchange for determination of environmental exposition to heavy metals based on the quantitative determination of Zn-thionein from physiological human fluids by indirect method based on analysis of metal contents.

Intoxication with heavy metals results in numerous poisonings and diseases. They disturb metabolism of the system, are the source of cancer, degeneration changes and others. As a result of kidney damage the urine of people exposed to heavy metals contains different low molecular weight proteins, oligopeptides and amino acids, indicating pathological changes. One of the proteins is a very specific metallopolythiopolypeptide--metallothionein (MT). Based on earlier investigations, a very good correlations has been found between the contents of metallothionein in urine and plasma and the concentration of heavy metals in the blood, urine, kidneys, liver and brain and general in level of exposition to heavy metals. The aim of our investigations was to carry out quantitative isolation of Zn-thionein (Zn-Th), in order to determine the level of exposition to heavy metals. For Zn-Th protein isolation by covalent affinity chromatography with thiol-disulphide interchange (CAC-TDI) was applied, which is a modern technique of separation of a high affinity, good repeatability and reproducibility, allowing specific isolation of the thiol-proteins CAC-TDI gel was used as a solid-phase extraction (SPE) support for preconcentration of Zn-Th protein and Zn bonded with Zn-Th from water, rine, plasma and breast milk samples. The investigations showed unfavourable effect of the support on separation of thiol proteins and good correlation between the concentration of MTs protein added to water, plasma and urine and the concentration of protein indirectly determined via atomic absorption spectrometric (AAS) method, by preconcentration on SPE support metals formerly bound with MT protein and absorbed on CAC-TDI gel and calculated from metals concentration. The present paper is a continuation of earlier experiments on quantitation of Hg-thionein and Cd-thionein in physiological fluids and homogenates.

Chromatography, Affinity↗

PubMed2000

Direct NMR-spectroscopic determination of active-enzyme concentration by titration with a labeled inhibitor: determination of the k(cat) value of almond beta-glucosidase.

A new method for the determination of active-enzyme concentration of a glucosidase by using (13)C NMR spectroscopy is reported. The method consists of quantifying the binding between a (13)C-labelled, strong competitive inhibitor, [5-(13)C]-1-azafagomine (1), and the enzyme. The concentration of free inhibitor 1 is measured in a series of binding experiments from the intensity of its NMR signal relative to that of a reference. From a plot of the concentrations of bound vs. free inhibitor 1, the amount of specifically bound 1, that is, the amount of active sites, is determined. From this value, active-enzyme concentration and k(cat) value can be calculated.

Binding, Competitive↗

PubMedDate not supplied

A flow injection chemiluminescence method using Cr(III) as a catalyst for determining hydrogen peroxide. Application to H(2)O(2) determination in cultures of microalgae.

Flow injection analysis has been applied to the determination of hydrogen peroxide produced by some different species of microalgae. The method is based on the luminol-H(2)O(2) chemiluminescence reaction using Cr(III) as a catalyst. Optimum experimental conditions for the method have been studied and trace amounts of hydrogen peroxide determined with detection limits of 4 10(-8) mol/L. The method using Cr(III) was compared with that using horseradish peroxidase as the catalyst.

Catalysis↗

PubMedDate not supplied

Determination of sterilization effectiveness by measuring bacterial growth in a biological indicator through firefly luciferase determination of ATP.

A bioluminescence procedure for measurement of microbial ATP allows a rapid determination of the effectiveness of autoclave sterilization. This determination is achieved faster than detection of acid production in a biological indicator via a pH indicator. Bacterial outgrowth from spores on test strips of the biological indicator was detected by measurement of ATP using the firefly luciferase reaction. A measureable increase in ATP was found after 5 hours of incubation of a biological indicator that had been treated under sterilizing conditions that produced 75% sterility of the biological indicator as measured by acid production. This is a marked improvement over the 24-48 hours of incubation currently required.

Adenosine Triphosphate↗

PubMed1996

Direct determination of glutathione in human blood by micellar electrokinetic chromatography: simultaneous determination of lipoamide and lipoic acid.

A reproducible, rapid procedure for the determination of glutathione in human blood by micellar electrokinetic chromatography has been developed. Whole blood samples were deproteinized with 5% w/v sulfosalicylic acid (final concentration). After centrifugation, the supernatant was directly injected for analysis, without further derivatization. Separations were performed by using an uncoated capillary of 30 cm effective length and 50 microns internal diameter (ID), 50 mM Tris-HCl, 30 mM sodium dodecyl sulfate (SDS), pH 7.00, as running buffer, and 10-20 kV. On-line detection was carried out at 214 nm and a detection limit in the range of femtomoles was achieved. Under the same experimental conditions, we resolved a mixed standard solution containing glutathione in its oxidize and reduced forms, lipoamide and alpha-lipoic acid. The corresponding migration times were reproducible. The present method allows rapid determination of these compounds, which play a critical role in oxidative stress, in cellular defense against injurious agents and whose levels are related to the toxicology and metabolism of several toxins and drugs, such as antineoplastic agents.

Benzenesulfonates↗

PubMed1979

[Use of the combined Kjeldahl-wet digestion/Berthelot-reaction for determining nitrogen in biological materials. 2. Design and testing of a partially automated apparatus for routine determination of nitrogen].

The authors describe the design and the mode of operation of a partially automated analyzer system, based on the combination of the Kjeldahl wet digestion with the Berthelot reaction, for the determination of nitrogen in biological materials. The results from three years of operation are discussed from the view-points of variability of the materials to be tested, of accuracy and of precision. This analytical technique (with an accuracy of 98% and a precision of 4%) is suited for determining amino and amido nitrogen as well as non-aromatically bound ring nitrogen. The statistical evaluation of the analytical results revealed that the deviation of the measured values is mainly caused by the high-temperature digestion.

Amides↗

PubMed1979

[Analytical methods for nitrate and nitrite determination in foods. 3. Spectrophotometric determination of nitrate and nitrite using sulphanilic acid/1-naphylamine, and of nitrite using resorcinol/zirconium (IV) oxychloride].

The Nitrate content of foods may be determined by means of azo-dye coupling after reduction of the nitrate with the aid of active cadmium produced in the sample solution. An easy method for determining nitrite consists in the colorimetric estimation of a coloured chelate formed from nitrite, resorcinol and zirconium(IV) oxychloride.

1-Naphthylamine↗

PubMed2003

Adsorptive stripping voltammetric determination of cefepime at the mercury electrode in human urine and cerebrospinal fluid, and differential pulse polarographic determination in serum.

The cephalosporin cefepime has been studied by adsorptive stripping voltammetric on the hanging mercury drop electrode, followed by linear sweep voltammetry (staircase). The adsorptive stripping response was evaluated with respect to preconcentration dependence and other variables. The drug is strongly adsorbed in acid media, with maximum adsorption at pH 5.8. The detection limit found was 4.8 x 10(-10) M, with 120-s preconcentration. The relative standard deviation at the 10(-7) M level was 0.93%. This method was applied to the determination of cefepime in human urine and cerebrospinal fluid. Differential pulse polarography has been applied to determination in human serum.

Anti-Bacterial Agents↗

PubMed2002

Determination of melanotan-II in rat plasma by liquid chromatography/tandem mass spectrometry: determination of pharmacokinetic parameters in rat following intravenous administration.

A method is described for the determination of melanotan-II (MT-II), a synthetic cyclic heptapeptide analog of alpha-melanocyte-stimulating hormone (alpha-MSH), in rat plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analyte is recovered from plasma by solid-phase extraction (SPE) and subsequently analyzed by LC/MS/MS. A SPE procedure using OASIS 96-well plates is used for extraction of MT-II from rat plasma. Using the described approach a limit of quantitation of 5 ng/mL was achieved in rat plasma. This level of sensitivity allowed the determination of pharmacokinetic parameters, following intravenous administration of MT-II in rat.

Animals↗