(directory “SOFTWARE“; folder “Chemie“; subfolder “Dentistry“; subfolder “Medical Chemistry“).

10.06.2011

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List of PhD theses

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Department of medical chemistry and biochemistry (DMCB) provides screening of a biologically active substance/extract/phytopreparation (on model reactions, mammalian cells and animal models) and consulting services for pharmaceutical and food industry, research institutes and individual research groups.

Based on the needs of the customer, services supplied by DMCB may range from single measurements to complete studies. Assays may be set up according to the requirements of the client. If necessary, we also assist in preparation of the material for publication of results.

The competence of DMCB is achieved and maintained by their active research. At present, research and development activities at DMCB are focused on oxidative stress and antioxidant functions.

We can assist our clients e.g. to investigate effects of plant extracts, phytochemicals, functional food and dietary supplements, on oxidation of LDL (® atherosclerosis) or DNA (® cancer), on cellular defence functions, and to explore antioxidant effects of intact plant/extract/ component/dietary supplement.

Examples of available cell systems:
Rat Hepatocytes
Human Hepatocytes
Rat Neonatal Cardiomyocytes
Human Umbilical Vein Endothelial Cells – HUVEC
Human Keratinocytes
Human Fibroblasts
Human Osteoblasts

Research activities of DMCB:

1.   Antioxidant testing

2.   Lipid metabolism

3.   Cellular defence functions

4.   DNA damage

5.  Effects on cytochrome P450 system

6.  Cell cytotoxicity/cytoprotectivity

7. Safety evaluation on experimental animals

8. Safety evaluation on human volunteers

Contact:

Prof. RNDr. Jitka Ulrichová, CSc.
Department of Medical Chemistry and Biochemistry (DMCB)
Hněvotínská 3, 775 15 Olomouc
Czech Republic

Tel/Fax    +420 585 632 312
E-mail   jitkaulrichova@seznam.cz

(i) Assessment of antioxidant potency

Focus

(i)  Scavenging of free radicals
(ii) Prevention of lipid peroxidation

Models

(i)  Screening of potency of the test compound in vitro
(ii) In vivo screening  ® demonstration of the effect on an animal model (rat, mouse) ® placebo controlled studies with human volunteers
(iii) The use of oxidized LDL as bioindicators

Why antioxidant testing?

Purposeful and directed production of reactive oxygen species (ROS) is a key element of important physiological functions (signal transduction, apoptosis). Excessive production of ROS (e.g. due to chronic inflammation, ischemia, drugs, pollutants, irradiation, cigarette smoke etc.) may cause oxidative damage to cell constituents. Such changes may lead to mutagenesis, membrane disruption, receptor malfunction, altered enzyme activities and mitochondrial damage. ROS are known to be implicated in ageing processes, and in diseases the incidence of which typically increases with age (cancer, cardiovascular disease, neurodegenerative disorders). The potential beneficial effects of antioxidant compounds have led to intensive search for novel antioxidant molecules to be used as components of dietary supplement/functional food.

Assessment of antioxidant effect in vitro

Tests:

(i) Scavenging of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH)
(ii) Scavenging of superoxide anion
(iii) Scavenging of peroxyl radical
(iv) Metal-chelating properties
(v) Electrochemical behaviour
(vi) Prevention of lipid peroxidation

Aim: To find out if the test compound/extract/phytopreparation can inactivate free radicals and/or prevent lipid peroxidation.

Procedure: DPPH radical, superoxide anion and peroxyl radical are produced by standard methods. Detection of free radicals is based on sensitive chemiluminescense techniques. Studies will be done with several concentrations of the test compound and repeated on separate days. The results will be presented as comparison of the potency of the test compound with that of a simultaneously tested known antioxidant. As the model of lipid peroxidation, peroxidation is induced chemically in biological lipids (e.g. lipids from rat liver).Assesment of antioxidant effect in vivo

Tests:

(i) Total antioxidant capacity of plasma and liver tissues
(ii) Level of reduced glutathione in erythrocyte and liver cells
(iii) Level of lipid peroxidation products
(iv) Activities of antioxidant enzyme in erythrocytes and liver (SOD, glutathione peroxidase)

Aim: To find out if the test substance/extract/phytopreparation has antioxidant effects in vivo.

Procedure: Effect of the test compound/extract/phytopreparation will be investigated after induction of experimental oxidative stress (by vitamin E deficiency and/or chemical prooxidant) to mice/rats. The test compound will be administered with feed or drinking water. Markers of oxidative stress in various tissues will be determined at the end of the test period. Determination of test compounds in tissues, cells and extracellular fluid. Determination of total antioxidant capacity of plasma and tissues.

(i) Atherosclerosis, cardiovascular disease, injury process

Focus

(i) Oxidized LDL
(ii) Plasma lipids and lipoproteins

Models

(i) Screening

®  animal model

Why LDL oxidation?

For several years it has been known that development of atherosclerosis is related to the level of the ”noxious” low-density lipoprotein (LDL). More recent studies have revealed that ultimate atherogenic agents are in fact modified, mainly oxidized, forms of LDL. Clinical studies have shown that LDL-peroxidation products (LDL-PP) are closely related to coronary, carotid and brachial atherosclerosis. Indeed, as an indicator of the risk of atherosclerosis and cardiovascular disease (CVD) LDL-PP clearly exceeds sensitivity and specificity of the common lipid markers MDA/TBARS. Therefore, focusing on LDL oxidation will not only help in diagnosis and follow up of treatment of CVD, but also enables targeted development dietary supplements for prevention or decreasing the risk of atherosclerosis and CVD.

Prevention of LDL oxidation in vitro

Tests:

(i) Direct prevention of LDL oxidation (conjugated dienes, LDL lag phase)
(ii) LDL-incorporation.

Aim: To find out (i) if the test compound can prevent oxidation of human LDL in vitro, and (ii) if the test compound can incorporate into LDL particles.

Procedure: Studies are performed with LDL fraction isolated from human plasma. Oxidation is induced by copper and quantified by the common diene conjugation method. Studies will be done with several concentrations of the test compound and repeated on separate days. The results will be presented as comparison of the potency of the test compound with that of a simultaneously tested known antioxidant. Incorporation of test compounds into LDL to study the effect on LDL stabilisation.

Animal model: in vivo LDL oxidation and development of atherosclerotic lesions

Tests:

(i) Level of oxidized LDL
(ii) Stability of LDL against chemically induced oxidation
(iii) Effect of test compounds on atherosclerotic lesions

Aim: To find out how the test substance/extract/phytopreparation affects LDL oxidation and the progression of atherosclerosis in experimental animals

Procedure: Knockout LDLR mice (lacking the LDL receptor), fed an atherogenic diet, are used as an experimental model for atherosclerosis. The test compound will be administered in feed or drinking water. The level of LDL oxidation (circulating oxidized LDL) will be analyzed in blood samples. Atherosclerotic lesions will be recorded in histopathological investigations.

Effects on LDL oxidation in human volunteers

Tests:

(i) Level of total cholesterol
(ii) Level of LDL cholesterol
(iii) Level of oxidized LDL

Aim: To find out how the test compound affects LDL oxidation (and other lipid risk factors) in human body.

Procedure: Human volunteers with modestly elevated serum cholesterol level will be given the test compound or placebo after twelve-weeks run-in period. Blood samples will be taken at one to two week intervals during the study, and analyzed for LDL oxidation and the conventional plasma lipids.