Biomedical subjects

A Bult

Publications and source records attributed to A Bult.

At least 55 records · Page 3Linked to original sources

PubMed1998

Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides.

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.

Angiotensin II↗

PubMed1998

Oxidation of recombinant methionyl human granulocyte colony stimulating factor.

The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9-7.5, the kobs value for the oxidation process is constant. Above pH 7.5, kobs tends to increase with increasing pH. In the pH range 1.9-11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met121 oxidized, while the dioxidation product has oxidized Met121 and Met126 residues. The trioxidation products are the proteins with Met121, Met126 and Met137 or Met0, Met121 and Met126 oxidized.

Amino Acid Sequence↗

PubMed1998

High-performance liquid chromatographic analysis of the investigational anticancer drug 9-aminocamptothecin, as the lactone form and as the total of the lactone and the hydroxycarboxylate forms, in micro-volumes of human plasma.

A high performance liquid chromatographic (HPLC) assay is described for the determination of the investigational anticancer drug 9 aminocamptothecin (9-AC) as the lactone form (9AC(lac)) and as the total of the lactone and hydroxycarboxylate forms (9AC-(tot)), in micro volumes of plasma. The analytical methodology reported here involves a protein precipitation step with cold methanol (-30 degrees C) as sample pretreatment procedure. The methanolic extract is used for the determination of 9AC-(tot). The intact (active) lactone form of 9-AC is separated from the hydroxycarboxylate form in the methanolic plasma extract by solid phase extraction within 48 h after sampling and deproteination. After evaporation to dryness (nitrogen, 40 degrees C) the extract can be stored at -70 degrees C for at least 3 weeks. The drug is analysed by reversed-phase liquid chromatography on a Zorbax SB RP-18 column, using methanol-water eluent (pH 2.2) and fluorescence detection. The presented assay is linear over a concentration range 0.2-100 ng.ml-1 with a detection limit and a limit of quantitation of 0.05 and 0.2 ng.ml-1, respectively, for both 9-AC(tot) and 9-AC(lac) using a 100 ml plasma sample. The proposed method has been implemented in a phase I clinical trial for pharmacokinetic evaluation of this potential new drug.

Aged↗

PubMed1998

Pharmaceutical development of a parenteral lyophilized formulation of the investigational antitumor neuropeptide antagonist [Arg6, D-Trp7,9, MePhe8]-Substance P [6-11].

The aim of this study was to develop a stable parenteral dosage form for the investigational cytotoxic drug [Arg6, D-Trp79,MePhe8]-Substance P [6-11] (Substance P Antagonist G; Antagonist G). Antagonist G bulk drug was structurally and analytically characterized. The drug exhibits excellent aqueous solubility, although relatively poor aqueous stability characteristics. Lyophilization was, therefore, selected as the manufacturing process. Differential scanning calorimetry studies were conducted to determine the freeze-drying cycle parameters which resulted in a stable, lyophilized formulation of Antagonist G. The prototype, containing 50 mg Antagonist G per vial, was found to be the optimal formulation in terms of solubility, length of the freeze-drying cycle, stability, and dosage requirements in the planned phase I clinical trials. Quality control of the freeze-dried formulation showed that the manufacturing process does not change the integrity of Antagonist G. Shelf life studies demonstrated that the formulation is stable for at least 3 years, when stored at 2-8 degrees C in a dark environment. Oxidative degradation products of Antagonist G were isolated and structurally characterized by mass spectrometry, nuclear magnetic resonance spectroscopy, and infrared spectroscopy.

Amino Acid Sequence↗

PubMed1998

Degradation kinetics of three gonadorelin analogues: developing a method for calculating epimerization parameters.

PURPOSE: To develop a method for calculating epimerisation parameters, find out if the kinetics of the independent reactions can be established, and elucidate primary structure-chemical degradation relationships in the degradation kinetics of three gonadorelin analogues. METHODS: The influences of pH, temperature, and buffer concentration on the degradation of the three gonadorelin analogues buserelin, goserelin, and triptorelin were investigated using RP-HPLC. A method was developed to calculate epimerisation and hydrolysis rate constants independently. RESULTS: Explicit structure-degradation mechanism relations were found in the degradation of all three compounds. The L-serine residue was found to be involved in both a solvent-catalysed backbone hydrolysis and a hydroxyl-catalysed epimerisation whereas, the O-tertiary butyl D-serine residue was only involved in proton-catalysed ether hydrolysis. The kinetics of identical reactions in different analogues were generally comparable. CONCLUSIONS: The degradation of the gonadorelin analogues is located at a relatively small number of chemical residues and prediction of the degradation mechanisms and kinetics of other peptides with similar structural elements appears to be possible.

Buffers↗

PubMed1998

O-glucuronidation, a newly identified metabolic pathway for topotecan and N-desmethyl topotecan.

During topotecan analysis of clinical urine samples, an additional peak eluting just after the solvent front was observed. This potential metabolite was isolated by chromatographic methods. Mass spectrometry data along with chromatographic retention data and fluorescence characteristics showed that the isolated fractions contained two compounds, i.e. topotecan-O-glucuronide and N-desmethyl topotecan-O-glucuronide. The concentrations of the metabolites in human urine were relatively low. When topotecan was given as a 30 min infusion at a dosage of 1.5 mg/m2 daily for five consecutive days every 3 weeks, the maximal metabolite concentrations in a 24 h urine sample were approximately 10% of topotecan-O-glucuronide and 3.5% of N-desmethyl topotecan-O-glucuronide with respect to the concentration of topotecan in the urine. This is the first report demonstrating that glucuronide metabolites of topotecan are present in the urine of treated patients.

Antineoplastic Agents↗

PubMed1997

Liposomes and immunoassays.

Various aspects of the application of liposomes as a label in immunoassays are reviewed. Methods for the preparation of liposomes, from the basic film method to the more advanced dehydration-rehydration method, are discussed. Furthermore, the markers used in liposome labels, as well as the methods to conjugate liposomes to antigens or antibodies, are summarized. Liposome immunoassays are applied as homogeneous or heterogeneous assays. Homogeneous assays often rely on the lytic activity of complement on antibody-associated liposomes. Another group of homogeneous assays utilizes the inhibitory action of antibodies on the activity of conjugates of mellitin (a bee venom protein) with a hapten. Free mellitin conjugates are able to lyse liposomes effectively. Heterogeneous liposome immunoassays, performed either competitively or non-competitively, resemble more closely standard enzyme linked immunosorbent assays, with the enzyme being replaced by a liposome label. Washing steps are used to separate antigen-specifically bound liposomes from unbound liposomes. All bound liposomes are lysed with a detergent, giving an instantaneous amplification. Flow-injection liposome immunoassays and liposome immunosensors are also described as examples of other possible immunoassay formats.

Biosensing Techniques↗

PubMed1997

Chromatographic analysis of the enantiomers of ifosfamide and some of its metabolites in plasma and urine.

The enantiomers of the cytostatic drug ifosfamide and the two metabolites 2- and 3-dechloroethylifosfamide were isolated from plasma and urine by liquid-liquid extraction with ethyl acetate, resolved on a Chirasil-L-val gas chromatographic column and detected by a nitrogen-phosphorus-selective flame ionisation detector. Resolution of the racemic compounds for identification purposes was also accomplished with high-performance liquid chromatography on a chiral column. The validated gas chromatographic method was suitable to determine the total concentrations and the enantiomeric composition of ifosfamide and its dechloroethylated metabolites in plasma and urine samples from treated patients. Some metabolic preferences in the metabolism of ifosfamide were found.

Adult↗

PubMed1997

Pre-, on- and post-column derivatization in capillary electrophoresis.

This survey gives a short overview of the various reagents and procedures that can be used for pre-, post- and on-column derivatization in capillary electrophoresis. First there is an introduction about capillary electrophoresis as an analytical technique; this is followed by a discussion of the pros and cons of the various modes of derivatization and a comparison with liquid chromatography. In the following paragraphs the reagents for a number of functional groups are discussed. The emphasis is on derivatization of the amino group. Most of the information on the reagents and derivatization procedures is listed in tables together with information on the detection mode, analytes, sensitivity and samples. In addition to the amino group, information is given on labeling of aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups.

Amino Acids↗

PubMed1997

Determination of 4-hydroxyifosfamide in biological matrices by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the determination of 4-hydroxyifosfamide, a metabolite of ifosfamide, in plasma of cancer patients. The analyte is derivatized to 7-hydroxyquinoline, which can be detached fluorimetrically. The calibration graph is linear in the concentration range 0.05-25 microM, the limit of detection being 40 nM. Any inference from acrolein, another metabolite of ifosfamide, was ruled out. 4-Hydroxyifosfamide is very unstable in plasma and a stabilization procedure by adding citric acid has been developed. Thus treated, the samples were stable for 4 days. Analysis of a patient's plasma samples revealed that the 4-hydroxifosfamide concentration did not exceed 10 microM.

Cerebellar Neoplasms↗

PubMed1997

Nesting and fitness: lifetime reproductive success in house mice bidirectionally selected for thermoregulatory nest-building behavior.

To test the hypothesis that large, well-built, nests are an important component of fitness, we kept 12 mating pairs of two high-selected, two control, and two low-selected lines, selected for thermoregulatory nest-building behavior, at 22 and 4 degrees C with access to 10 g of cotton to build a nest, for a period of 180 days. Measurements included number of litters born per family, number of young per litter born and surviving up to 40 days of age, nest type built by the parents, and weight gain of the young from weaning (20 days of age) to 40 days of age. In all lines the production and survival of offspring was substantially decreased at 4 degrees C compared to 22 degrees C, but the high-selected lines produced more and better-quality offspring, surviving up to 40 days of age at both temperatures compared to the control and low-selected lines. This indicates that thermoregulatory nest-building behavior and evolutionary fitness are closely associated.

Animals↗

PubMed1997

No evidence for a Y chromosomal effect on alternative behavioral strategies in mice.

This study takes the first step toward testing a Y chromosomal effect on both aggression and thermoregulatory nest-building behavior in mouse lines either bidirectionally selected for short (SAL) and long (LAL) attack latency or high (HIGH) and low (LOW) nest-building behavior. Using reciprocal crosses between SAL and LAL, and between HIGH and LOW, we found no indications for Y chromosomal effects on thermoregulatory nest-building behavior. As for aggression, we confirmed earlier studies on SAL and LAL, i.e., the origin of the Y chromosome influences attack latency, i.e., aggression. However, we did not find indications for a Y chromosomal effect on aggression in the HIGH and LOW lines. Since aggression and nest-building behavior have been shown to be characteristic parameters of two fundamentally different behavioral strategies, the present data underline the improbability of Y chromosomal genes underlying the genetic architecture of alternative behavioral strategies.

Aggression↗

PubMed1997

Self-assembled monolayers for biosensors.

The use of self-assembled monolayers (SAMs) in various fields of research is rapidly growing. In particular, many biomedical fields apply SAMs as an interface-layer between a metal surface and a solution or vapour. This review summarises methods for the formation of SAMs upon the most commonly used materials and techniques used for monolayer characterisation. Emphasis will lie on uniform, mixed and functionalised monolayers applied for immobilisation of biological components including (oligo-)nucleotides, proteins, antibodies and receptors as well as polymers. The application of SAMs in today's research, together with some applications will be discussed.

Biosensing Techniques↗

PubMed1997

STEP: a family of brain-enriched PTPs. Alternative splicing produces transmembrane, cytosolic and truncated isoforms.

The family of striatal enriched phosphatases (STEPs) consists of protein tyrosine phosphatases (PTPs) that are enriched within the central nervous system. Previous biochemical studies have shown that the STEP family includes transmembrane, as well as soluble, cytosolic proteins. We now extend these findings with the isolation and characterization of a new, truncated member of this family, termed STEP38. The cDNA of STEP38 encodes a protein of 346 amino acids with a predicted mobility of 38 kDa. In contrast to the cytosolic variants, it contains two hydrophobic amino acid sequences at its N-terminus, two sequences enriched in Pro, Glu, Asp, Ser and Thr residues (PEST sequences), and two polyproline domains. We have used differential centrifugation, continuous sucrose gradients, and transfection experiments to clarify the subcellular localization of STEP38 within membrane compartments. These experiments suggest that a pool of STEP38 is targeted to membrane compartments of the endoplasmic reticulum. The STEP38 cDNA contains a stop codon upstream of the catalytic phosphatase domain that is normally present in other STEP variants, and enzymatic assays conform that STEP38 is inactive. Thus, the STEP family consists of cytosolic, transmembrane, and truncated isoforms. These findings are similar to what has been found for some members of the protein tyrosine kinase (PTK) family that uses alternative splicing mechanisms to produce active and inactive variants. By analogy with suggested mechanisms of action for the truncated PTKs, inactive STEP isoforms may participate in signaling events by protecting potential substrates from dephosphorylation by other members of this family.

Alternative Splicing↗

PubMed1996

STEP61: a member of a family of brain-enriched PTPs is localized to the endoplasmic reticulum.

The STEP family of protein tyrosine phosphatases is highly enriched within the CNS. Members of this family are alternatively spliced to produce both transmembrane and cytosolic variants. This manuscript describes the distinctive intracellular distribution and enzymatic activity of the membrane-associated isoform STEP61. Transfection experiments in fibroblasts, as well as subcellular fractionations, sucrose density gradients, immunocytochemical labeling, and electron microscopy in brain tissue, show that STEP61 is an intrinsic membrane protein of striatal neurons and is associated with the endoplasmic reticulum. In addition, structural analysis of the novel N-terminal region of STEP61 reveals several motifs not present in the cytosolic variant STEP46. These include two putative transmembrane domains, two sequences rich in Pro, Glu, Asp, Ser, and Thr (PEST sequences), and two polyproline-rich domains. Like STEP46, STEP61 is enriched in the brain, but the recombinant protein has less enzymatic activity than STEP46. Because STEP46 is contained in its entirety within STEP61 and differs only in the extended N terminus of STEP61, this amino acid sequence is responsible for the association of STEP61 with membrane compartments and may also regulate its enzymatic activity.

Amino Acid Sequence↗

PubMed1996

Analysis of ifosforamide mustard, the active metabolite of ifosfamide, in plasma.

Ifosforamide mustard is the active metabolite of ifosfamide, a cytostatic drug. In this study a sensitive and selective method for the analysis of ifosforamide mustard in plasma is described. The method consists of direct derivatisation of ifosforamide mustard in plasma with diethyldithiocarbamate and subsequent solid-phase extraction of the resulting derivative. The analysis of the derivatisation product was performed by high-performance liquid chromatography with UV detection. The calibration graph was linear in the concentration range 0.45-45 microM and the minimum detectable concentration was 0.45 mumol. The samples were stabilised by addition of semicarbazide and sodium chloride. A patient's plasma sample was analysed by means of the described method. The ifosforamide mustard concentration was 2.3 microM.

Adult↗

PubMed1996

Application of xanthine oxidase-catalyzed luminol chemiluminescence in a mouse interleukin-5 immunoassay.

A chemiluminescent substrate reagent for use in a sandwich immunoassay for the model antigen mouse interleukin-5 (IL-5) was developed using xanthine oxidase and luminol. Various parameters involved in this chemiluminescent reaction have been studied, including the substrate hypoxanthine, luminol and the Fe(II)-EDTA complex. Addition of the Fe(II)-EDTA complex enhances the chemiluminescence signal considerably. The xanthine oxidase-catalyzed chemiluminescent immunoassay was compared to horseradish peroxidase-linked immunoassays with luminol as chemiluminescent, and tetramethyl benzidine as colorimetric substrate. The detection limit of the xanthine oxidase-luminol assay was found to be about 0.6 pg/ml IL-5, whereas the peroxidase-catalyzed immunoassays have detection limits of about 1.3 (HRP-TMB) and 2.9 pg/ml (HRP-luminol) IL-5.

Animals↗

PubMed1996

Precolumn fluorescence derivatization of the antagonist [Arg6,D-Trp7,9,MePhe8]-substance P[6-11] with benzoin in high-performance liquid chromatography and selective detection of arginine-containing fragments in its degradation products.

Precolumn fluorescence derivatization for the determination of the antagonist [Arg6,D-Trp7,9,MePhe8]-Substance P¿6-11¿ (antagonist G) using benzoin in HPLC was studied. Under the conditions chosen (0.067 M NaOH, heating at 100 degrees C for 10 s), a good yield of fluorescent derivatives was obtained and no methodology-related degradation occurred. The detection limit of antagonist G was 0.21 nmol/ml. The method has been applied to the selective and sensitive detection of arginine-containing fragments in degradation products of antagonist G.

Amino Acid Sequence↗