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Biomedical subjects

G J De Jong

Publications and source records attributed to G J De Jong.

7 recordsLinked to original sources
PubMed2002

Mechanisms of copper incorporation into human ceruloplasmin.

Ceruloplasmin is a multicopper oxidase essential for normal iron homeostasis. To elucidate the mechanisms of copper incorporation into this protein, holoceruloplasmin biosynthesis was examined by immunoblot analysis and (64)Cu metabolic labeling of Chinese hamster ovary cells transfected with cDNAs encoding wild-type or mutant ceruloplasmin. This analysis reveals that the incorporation of copper into newly synthesized apoceruloplasmin in vivo results in a detectable conformational change in the protein. Strikingly, despite the unique functional role of each copper site within ceruloplasmin, metabolic studies indicate that achieving this final conformation-driven state requires the occupation of all six copper-binding sites with no apparent hierarchy for copper incorporation at any given site. Consistent with these findings a missense mutation (G631R), resulting in aceruloplasminemia and predicted to alter the interactions at a single type I copper-binding site, results in the synthesis and secretion only of apoceruloplasmin. Analysis of copper incorporation into apoceruloplasmin in vitro reveals that this process is cooperative and that the failure of copper incorporation into copper-binding site mutants observed in vivo is intrinsic to the mutant proteins. These findings reveal a precise and sensitive mechanism for the formation of holoceruloplasmin under the limiting conditions of copper availability within the cell that may be generally applicable to the biosynthesis of cuproproteins within the secretory pathway.

Animals↗
PubMed1991

Evaluation of eluents in thermospray liquid chromatography-mass spectrometry for identification and determination of pesticides in environmental samples.

The influence of different eluents in positive and negative ion mode thermospray liquid chromatography-mass spectrometry was studied with several groups of pesticides, including carbamates, chlorotriazines, phenylureas, phenoxy acids and organophosphorus and quaternary ammonium compounds, and the corresponding degradation products. Using the positive ion mode in combination with reversed-phase eluents the base peaks generally corresponded either to [M + H]+ for the chlorotriazines and their hydroxy metabolites or to [M + NH4]+ for the carbamates, the phenylureas, the organophosphorus pesticides and their oxygen analogues. In the negative ion mode different processes such as (dissociative) electron-capture and anion attachment mechanisms occurred. Fragment ions such as [M - CONHCH3]- for the carbamates, [M - H]- for the chlorotriazines, phenylureas and chlorinated phenoxy acids and [M].-, [M - R]- (R being a methyl or ethyl group) for organophosphorus pesticides were usually formed. Depending on the eluent additive used (ammonium acetate, ammonium formate and/or chloroacetonitrile), three different adduct ions were formed: [M + CH3COO]-, [M + HCOO]- and [M + Cl]-. Normal-phase eluents with cyclohexane, n-hexane and/or dichloromethane provided more structural information and enhanced the response of several compounds. The positive ion mode was useful for the detection of chlorinated phenoxy acids and chlorophenols which could not be detected in the positive ion mode using reversed-phase systems. The base peaks generally corresponded to [M].+, [M + H]+ or [M - Cl]+. For the characterization of difenzoquat, a quaternary ammonium pesticide of which trace level analysis is troublesome, a post-column ion-pair extraction system was used. An aqueous mobile phase with a sulphonate-type counter ion was applied and an extraction solvent containing cyclohexane-dichloromethane-n-butanol (45:45:10) was used in thermospray liquid chromatography-mass spectrometry. Illustrative examples of the determination of residue levels of pesticides in soil matrices are shown.

Carbamates↗
PubMed1990

Naphthalene- and anthracene-2,3-dialdehyde as precolumn labelling reagents for primary amines using reversed- and normal-phase liquid chromatography with peroxyoxalate chemiluminescence detection.

Naphthalene-2,3-dialdehyde (NDA) and anthracene-2,3-dialdehyde (ADA) were applied as pre-column labelling reagents for the peroxyoxalate chemiluminescence detection of primary amines. The advantages of these labels are the selective derivatization reaction with primary amines and the good chemiluminescence properties. A serious disadvantage is the formation of cyanide-induced side-products which are major interferences in reversed-phase chromatography. For normal-phase chromatography, the excess of reagent was removed by adding a polar amine after derivatization, with subsequent extraction of the labelled analyte with an apolar solvent. The detection limit for NDA-labelled fluvoxamine, an anti-depressant, was in the low femtomole range in standard solutions and in urine samples. For ADA-labelled analytes difficulties were obtained with linearity in peroxyoxalate chemiluminescence detection, probably owing to oxidation of the derivative by hydrogen peroxide.

Amines↗
PubMed1989

Chemiluminescence detection for high-performance liquid chromatography of biomedical samples.

During recent years, much progress has been made in the development of high-performance liquid chromatographic (HPLC) detection systems based on chemiluminescence (CL). CL is now one of the most sensitive detection methods in HPLC. For many compounds detection limits in the femtogram to picogram range have been obtained. Several on-line post-column reactions have been used for chemical excitation of the analytes. Some theoretical aspects of CL detection are presented and special attention is devoted to the coupling of CL to flow systems. The influence of the kinetics of the reaction on the sensitivity of the detection system is stressed. The mechanisms and detection systems of the peroxyoxalate, luminol and lucigenin CL reaction are described. A few examples of the use of bioluminescence for HPLC detection are given, and some less common CL reactions used in flow systems are also mentioned. Many biomedical and related applications are shown. Possibilities and limitations of the various reactions and detection systems are evaluated.

Chemistry, Clinical↗