On the molecular level biological (physiological) research is directed toward identification and quantification of compounds exerting biological (physiological) effects. The compounds involved may be both of low and high molecular mass; in the latter category it may be attempted to reveal structural changes caused under physiological or pathophysiological conditions (ageing, disease), which manifest themselves as functional changes. At this level of physiological study we may see an interactive link between analytical chemistry and physiology (it is possible to use the term "analytical physiology"). The work of this Department is aimed at connection of these areas and can be categorised as follows.

1. Instrumentation progress in the area of separation methods.
2. Methodological progress and development of new separation methods applicable to the estimation of physiologically important compounds (capillary electrophoresis and HPLC/MS).
3. Changes underlying the modified physiology of structural proteins (mainly connective tissue)

As is obvious from the above introduction all three areas are interconnected and it is not simple to categorise a particular problem to a particular area. The good analytical background (modern instrumentation such as capillary electrophoresis and HPLC/MS) of this department makes it possible to carry out analyses for other department of the institute. An example of good cooperation may be a the one with the Department of Epithelial Physiology.

Regarding the instrumentation area it is possible to mention development and construction of ultramicroanalytical systems  chip analysis or development of an offline combination of synchronous fluorescence spectroscopy and capillary electrophoresis.

Considerable effort was dedicated to the development of new methods for the identification and quantification of physiologically important compounds. The most frequently studied compounds were steroids. A broad spectrum of analytical methods such as capillary electrophoresis in microemulsion and micellar modes (so called electrokinetic chromatography) with diode array detection and highperformance liquid chromatography joined with mass spectrometry (HPLC/MS) were developed. These methods allowed us to detect corticosterone metabolites and offered further insight into steroid metabolism in animals (mammalian and avian intestine). This work was done in close collaboration with the Department of Epithelial Physiology.

Methods for the identification and quantification of a wide spectrum of other compounds were also developed: vitamins, pigments, dicarbonyl sugars, fatty acids, coenzyme A, amino acids, peptides and proteins. Preferably capillary electrophoretic techniques and HPLC/MS were used for this purpose. This was partly done in cooperation with other departments. For example, we identified and quantified a new pigment of the avian eggshell (zinccontaining protoporphyrin IX). Identification of the binding site of Na+/K+ATPase for pyrene isothiocyanate represents another example. New separation modes of electromigration techniques were also intensively studied  microemulsion electrokinetic chromatography and nonaqueous capillary electrophoresis.

A separate area is represented by the analysis of proteins, mainly proteins of connective tissue. The more significant results were the following.

Methods which were developed for the separation of collagen fragments (capillary electrophoresis in acidic buffers) were successfully applied to quantitation of collagen type I, III and V in tissues.

All these analyses aimed at better understanding the physiological role of spontaneous nonenzymic (chemical) reactions between the free amino group (lysine, arginine, Nterminal amino acid) of slowly metabolised proteins and the oxo group of endogenous compounds (e.g. aldehydic sugars, lipid peroxidation products). These reactions have a significant effect in normal physiological (ageing) or pathophysiological (diabetes) situations, i.e. in changes of the biological functions of proteins. The next aim was the elucidation of the role of toxic substances capable of protein modification in the environment, in foods as well as revealing in the metabolically inert deposits of such compounds in tissues. A related aspect was a study of the kinetics of low molecular mass metabolites (including inorganic ions) and their binding to the extracellular matrix components. For this purpose a method capable of determining the binding of lead and zinc to collagen (or its fragments) was developed. It was demonstrated that two categories of binding sites exist in the collagen molecule, the number of which correlates rather well with the available aspartic and glutamic acid residues. It was also described that lead (after its administration in water) is accumulated in collagencontaining tissues (placenta and chorionic membranes).

High fat diet feeding of laboratory rats yielded a considerable increase in the concentration of reactive carbonylcontaining compounds in rat heart reperfusates (i.e. it resulted in an increase of reactive carbonyl compounds capable to react with proteins present in the vascular walls). The concentration of these metabolites can be brought back to control level when the high fat diet is switched back to standard pelleted diet.

Metabolites arising from posttranslational modifications (glycation) were found in specific (two) collagen fragments (revealed by capillary electrophoresis with offline coupled synchronous fluorescence spectroscopy). Collagen glycation and modification with lipidderived metabolites was studied in ageing and hypertriglyceridemic (HTG) animals. It was found that pentosidine (glycationspecific amino compound measured by HPLC) concentration significantly increases with age in collagen from rat skin and tail tendon but fluorescence (measured at four wavelengths typical of sugar or lipidderived adducts) significantly increase with age only in skin collagen. Significant differences between HTG and control animals were observed only in the pentosidine content for young animals; in old animals the values were identical. We noccluded that longlived proteins in different nocnective tissues are differently available for posttranslational modifications and that results obtained with tail tendons (as a typical connective tissue model) cannot be automatically applied to other soft connective tissues in the body.

Posttranslational modifications of proteins by alcohol intake were also investigated. This research was done in cooperation with the Institute of Forensic Medicine, University of Verona, Italy. There were monitored and discovered changes in structure (composition) of hair proteins (keratins) and serum transferrin which were induced by alcohol. These peptide modifications in transferrin have forensic (diagnostic) consequences that may be practically useful.

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