Anne Karine Thorsrud

Capillary electrophoresis for diagnosis and studies of human disease, particularly metabolic disorders

High-performance capillary electrophoresis (HPCE) has been used in a multicomponent analytical system designed to diagnose and study human diseases, particularly metabolic disorders. Comparative analyses, using HPCE, high-performance liquid chromatography (HPLC) and an automated amino acid analyser, were carried out on urine and blood samples from patients with homocystinuria, cystinuria, glutathione synthetase deficiency and adenylosuccinase deficiency. HPCE of the sulphur-containing amino compounds, derivatized with monobromobimane and detected by fluorescence spectroscopy, was a quick and simple alternative to classical amino acid analysis. The detection of the characteristic succinylpurines associated with adenylosuccinase defect was equally well achieved with HPLC and HPCE (absorbance detector). Owing to the possible connection between deficiency of taurine (2-amino-1-ethanesulphonic acid) in the heart and the development of cardiomyopathy and heart failure, a simple HPCE method was developed for the determination of taurine in sub-milligram samples of biopsies of the myocardium. The homologue 3-amino-1-propanesulphonic acid was the internal standard, and derivatives of 9-fluorenylmethyl chloroformate and fluorescence detection were used. It is suggested that the potential of HPCE to analyse small volumes should be exploited in biomedicine and clinical diagnosis to analyse sub-milligram samples of tissue biopsies and cells.

Quantitative analysis of polycyclic aromatic compounds in diesel exhaust particulate extracts by combined chromatographic techniques

Abstract High resolution gas chromatography—flame ionization detection (GCFID) with on-column injection was used for quantitative analysis of oxy-polycyclic aromatic hydrocarbons (oxy-PAH), nitro-PAH, and phthalate esters in the extracts of three different diesel exhaust particulate samples. Various compounds in the extracts were sorted into different compound classes by high-performance liquid chromatography fractionation and then, components were tentatively identified by GC—mass spectrometry and retention indices. Combined with the quantitative results obtained in previous studies, the comparative quantitative bar diagrams with more than one hundred polycyclic aromatic compounds were constructed for these three diesel particulate extracts. Some results of the quantitative analysis can be related to response in a toxicity test of the original extracts.

Protein mapping of two metallothionein-rich cell strains and their parent lines, using high-resolution two-dimensional electrophoresis☆

A high-resolution two-dimensional gel electrophoresis (2-D) technique was used to characterize one human and one murine cadmium-resistant substrain and their parental wild-type lines. The substrains are cultured on 100 microM cadmium and contain high levels of the cysteine-rich protein metallothionein (MT). All four cell lines were labeled with [35S]methionine during growth. A remarkable consistency was found in the protein maps of the resistant strains compared to those obtained from their corresponding wild-type lines. Thus, in the maps from the human substrain only two spots were detected which were not found in the parent cells. In the murine substrain, two spots were more abundant and two diminished compared to the parent cells. No distinct spots corresponding to authentic MT were detected in any of the autoradiographs from the cadmium-resistant cells. The reason for this was found to be failure of the protein to focus in the first dimension. Purified [35S]cystine-labeled MT appeared as a diffuse labeling over the entire gel, and subsequently as wide horizontal bands in the second dimension. These bands were also clearly visible in the protein maps when MT-rich cells had been labeled with [35S]cysteine. This study shows that the standardized 2-D gel system used in many laboratories cannot be used to screen cell populations for MT.

Profiling cells and body fluids - chromatography and two-dimensional electrophoresis as complementary techniques

Recent results on the use of gas chromatography-mass spectrometry, high-performance liquid chromatography with diode array detector, amino acid analysis and high-resolution two-dimensional (2-D) electrophoresis to study body fluids and cells in health and disease are surveyed. The chromatography profiling techniques are particularly suitable for the diagnosis of inborn errors of metabolism, with DNA technology as a complementary tool for prenatal diagnosis. Both chromatography and electrophoresis were utilized to study pre-diagnostic sera from the JANUS serum bank and to classify certain bacteria. Protein profiling by 2-D electrophoresis was employed to identify marker proteins associated with the metastatic properties of cloned cancer cells. The electrophoretic technique is also appropriate as a preparative tool for isolating sufficient amounts of marker proteins for microsequencing of amino acids. Chromatography and protein profiling were complementary tools for evaluating the toxicity and mutagenicity of environmental samples in a new test utilizing living human leukocytes and fibroblasts.

Mapping of synovial fibroblast proteins by high resolution two-dimensional polyacrylamide electrophoresis.

The highly sensitive protein separation technique two-dimensional polyacrylamide gel electrophoresis was used to study the cellular protein synthesis in 1) cultured human rheumatoid and 2) normal synovial fibroblasts and 3) normal skin fibroblasts. No reproducible differences were found that could be ascribed to cellular origin, the disease or in vitro cellular aging. The reasons for that probably were related to the cell cultures and not to failure of protein detection.