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.

Polymorphism of a platelet polypeptide.

Polymorphism of a platelet protein is described. The gene products are represented by two peptides of MW around 30 kD. The allele frequency was estimated to 0.85 and 0.15, the common variant being of slightly higher MW and about 2 charge units more acidic than the other. The peptides were neither released nor phosphorylated, and subcelluar fractionation indicated localization to the cytosol. Attempts to raise antibodies failed, and further characterization could not be done, but the peptides seem to differ from all reasonably well characterized platelet proteins so far.

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.

Marker polypeptide differences between spontaneous strongly and weakly metastatic cancer cells identified by 2-dimensional gel electrophoresis.

A recent 2-dimensional gel electrophoresis study revealed several polypeptide differences between a strongly metastatic and a weakly metastatic clone from a single chemically induced murine fibrosarcoma (Jellum et al., 1984). To exclude the possibility that this was merely coincidental, the study is extended here to 2 other fibrosarcomas recently and similarly induced in mice of the same inbred strain. Metastatic potential was defined by the number of lung metastases spontaneously formed from a transplanted primary footpad tumor. One strongly (or moderately) metastatic cell line and I weakly metastatic line from each of the 3 fibrosarcomas were examined in the same experiments. In confirmation of our previous results, the same polypeptides consistently occurred in considerably greater amounts in the weakly metastatic than in the strongly metastatic cells. One of these marker polypeptides was absent from the strongly metastatic cell lines. In comparison with 2 of the 3 most metastatic lines, the third line was only moderately metastatic, and differed least strongly from the 3 weakly metastatic cell lines with regard to expression of th marker polypeptides. Marker polypeptide expression showed no consistent correlation with tumorigenicity. No other consistent polypeptide differences between strongly metastatic and weakly metastatic cells could be identified among the approximately 2,000 cellular polypeptides separated on the gels.

Marker polypeptides distinguishing between cancer cell clones with high and low potential for spontaneous metastasis in murine fibrosarcoma cells.

Consistent differences in expression of specific proteins were observed between numerous cancer cell clones with high and low potential for spontaneous metastasis. Seventeen clones from two unrelated murine fibrosarcomas were examined concomitantly for spontaneous formation of lung metastases and for occurrence of individual polypeptide differences by high resolution two-dimensional gel electrophoresis. One of the identified marker polypeptides, designated Hi:2, was very strongly expressed by all 10 strongly metastatic clones, but was absent from 6 and only weakly expressed by one of the 7 weakly metastatic clones. Another marker polypeptide, designated Lo:6, was consistently most strongly expressed by the weakly metastatic clones. Among the approximately 2000 individually separated cellular polypeptides, only these two marker polypeptides consistently distinguished between the strongly and weakly metastatic clones from both fibrosarcomas. Five other polypeptides also distinguished between highly and weakly metastatic clones, but not as stringently.

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.

High resolution two-dimensional protein electrophoresis in clinical chemistry.

High resolution two-dimensional (2D)-electrophoresis has been developed to the stage that it is possible to resolve several thousand proteins in cells and tissue, and over six-hundred proteins are separated in human serum. The 2D-technique has been applied to analyses of serum from patients with multiple myeloma, macroglobulinemia and other gammopathies, and to separate apolipoproteins and study abnormalities and polymorphism of these proteins. Cerebrospinal fluid from patients with various neurological diseases has been studied by 2D-electrophoresis and seems to yield information on multiple sclerosis. The 2D-technique has been applied to normal and malignant cells and biopsies, and offers a possibility to detect disease-related proteins. Protein spots from 2D-gels may be used to raise monoclonal antibodies which subsequently can be used to develop simple clinical chemical tests for disease markers. The 2D-electrophoretic method is, however, not yet suitable as a typical routine analysis in the clinical chemistry laboratory, but is primarily a research tool of considerable potential.

Mapping of proteins from cultured fibroblasts of synovial and subcutaneous origin by high resolution two-dimensional polyacrylamide gel electrophoresis.

Nine different cell lines of human (A) rheumatoid and (B) normal synovial fibroblasts and of (C) normal skin fibroblasts were obtained from tissue explants and grown as monolayers. The cellular protein synthesis was studied by high resolution two-dimensional polyacrylamide gel electrophoresis. Between the fourth and 10th passage of the cells in culture apparently no consistent differences were found which could be ascribed to the origin of the cells, to the rheumatoid state of the synovial tissue, or to the increasing age of the cultured fibroblasts.

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.

Two-dimensional electrophoretic analysis of human leukocyte proteins from patients with rheumatoid arthritis.

Human leukocyte proteins from more than 150 patients with rheumatoid arthritis, together with age- and sex-matched controls, were analyzed by use of the ISO-DALT technique in two-dimensional polyacrylamide gel electrophoresis. Patients with ankylosing spondylitis, polymyalgia rheumatica, psoriatic arthritis, calcium tendinitis, post-infectious arthritis, and asymmetrical seronegative arthritis were also included as positive controls. Synthesis of several proteins, referred to by number as members of the "Rheuma" set, is shown to increase in the leukocyte preparations from patients with classical rheumatoid arthritis. Several of these proteins are specific to monocytes or granulocytes; others are of unknown cellular origin, but appear to be unique to rheumatoid arthritis. The Rheuma proteins appear to be indicators of disease activity, because their increased synthesis can be correlated with sedimentation rate and other clinical indices of rheumatoid disease activity.

Clinical applications of two-dimensional electrophoresis.

High-resolution two-dimensional electrophoresis has been used in our institute to study sera from patients with multiple myelomas; cerebrospinal fluid from patients with different neurological disorders; blister fluid, dermis, and epidermis from patients with skin diseases; and biopsies from brain tumors; and to search for tumor-associated proteins in cancer biopsies and pre-cancer sera. The method differentiates between all main types of immunoglobulins and gives rise to protein patterns for cerebrospinal fluid that may carry diagnostic information. The pattern of proteins in serum and blister fluid is almost identical, confirming that the latter is a filtrate of serum. We also analyzed sera collected from patients several years before it was clinically recognized that they had cancer (JANUS serum bank), in an attempt to find tumor-associated proteins in serum at an early stage of the disease. We encountered difficulties in detecting trace proteins, due to masking effects of such major serum-protein constituents as albumin. A combination of computerized gas chromatography-mass spectrometry and high-resolution two-dimensional electrophoresis is capable of mapping compounds of both low and high molecular mass. Used in combination with pattern-recognition analysis, the complete multicomponent analytical system opens the possibility of diagnosing diseased cells solely on the basis of differences in their biochemical composition.

Two-dimensional electrophoretic patterns of proteins of normal mucosa, polyps, and carcinomas of the large intestine.

Two-dimensional protein maps of biopsies from normal mucosa, tubular adenomas (polyps), and colonic carcinomas in humans have been studied in order to obtain information on the possible precancerous role of intestinal polyps. The maps are complex and several hundred protein spots are seen after staining with Coomassie Blue. Most of the spots are common to these three tissue types, but there are also differences. Polyps and carcinomas had a strikingly similar protein pattern, different from that of normal mucosa. There were negligible differences in the two-dimensional protein maps of tubular adenomas from patients who also had colonic cancer, as compared with adenomas from persons without carcinomas. The results lend further support to the hypothesis that polyps may be precursors of carcinomas of the large intestine.

Biochemical composition of suction blister fluid determined by high resolution multicomponent analysis (capillary gas chromatography--mass spectrometry and two-dimensional electrophoresis).

The biochemical composition of blister fluid was compared with serum and with blister fluid from erythematous lesions induced by ultraviolet irradiation. By using glass capillary gas chromatography--mass spectrometry over 100 metabolites were determined and with the aid of two-dimensional high resolution electrophoresis (the ISO-DALT system) several hundred protein spots were seen. The results show that the suction blister fluid qualitatively have a serum-like pattern but that the concentration of each compound was smaller than in serum. Also in suction blisters raised on erythematous reactions induced by ultraviolet light the same pattern was seen. The content of sodium, potassium and chloride was the same in suction blisters raised on erythematous and normal skin as that of serum.