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In 1990, the worldwide accepted Shackleton method, which provides a possibility of determining the steroid metabolites from urine, was adopted in our laboratory. The procedure is very useful in the diagnosis of different endocrine diseases and in the recognition of dysfunction or absence of enzymes with an important role in steroid metabolism, and it gives possibility to control the treatment in patients with these diseases. Besides the proximate clinical application, the method gives a convenient tool to study the steroid background of these disorders, helping us understand the mechanism of their development. In the last few years, we have examined the steroid profile of patients with hair (androgen alopecia /AA/, effluvium /E/), psychiatric problems (major depression /MD/, eating disorders /EDS/, especially anorexia nervosa and bulimia) and osteoporosis (OP). In all of the examined hair loss diseases, the levels of main androgen metabolites were increased, and elevated 5alpha-reductase activity were found. We could observe the alteration of the activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzyme and marked gender differences in the changes of the steroid metabolism in patients with major depression (MD). In women with OP, the significantly decreased level of certain metabolites points to the role of testosterone, androstenedione and DHEA in postmenopausal bone loss in women. Our experiences contribute to the knowledge of the nature and steroid background of some endocrine and psychiatric diseases.

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Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low-level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.