**2. Methods**

A pathomorphological study was carried out using autopsy material of 68 infants aged under 1 who died during the follow-up period, as well as 10 infants of the same age whose death was not caused by cardiac problems. All TGA patients were broken down in 2 groups according to patients' anatomical type: the first group included patients with a simple form of TGA – TGA with atrial septum defect (ASD) and intact ventricular septum (IVS) – 37 patients (19 aged under 1 month and 18 aged 1 to 6 months), while 31 patients having TGA with ASD and VSD (7 aged under 1 month, 13 aged 1 to 6 months and 11 patients aged 6 to12 months) were assigned to the second group.

Biopsy samples were preserved in 4 % phosphate-buffered formalin and then washed off in a distilled water solution followed by processing in cryoprotectans (solutions of saccharose: 5 % for 2 hours, 10 % for 2 hours, 15 % for 12 hours). Fluorometry of histologic specimens was carried out by using an Axioskop 40FL microscope and an AxioCamHRc camera. To get a good computerized image of each histologic specimen, a Zeiss Plan-Neofluar x 40 lens was used for 20 s at a +24 °С room temperature. The images obtained were processed by AxioVision 3.1 software (Carl Zeiss).

Fluorescent probes containing ethidium bromide and chlortetracycline were used to perform fluorometry of the myocardium. Staining myocardium slices with ethidium bromide was done in a phosphate buffer pH=7.4 that contained 5.0 \* 10 3 g/l of ethidium bromide for 5 minutes at a temperature of 25 °С, while chlortetracycline, also done in a phosphate buffer pH=7.4 containing 2.6 \* 10 2 g/l of chlortetracycline, was applied for 1 minute at a temperature of 25 °С. Fluorometry of histological preparations was then carried out. In the case of ethidium bromide, uptake was equal to 510-523 nm, emission – to 595-605 nm, while in the case of chlortetracycline, those parameters came to 400 nm and 520 nm respectively. In addition, myocardium samples were stained with antibodies for Monoclonal Anti Skeletal Myosin (FAST) Clone MY-32 skeletal myosin. FITS-conjugated secondary antibodies were used as a secondary marker.

The following properties were measured: muscle fibre diameter, relative area of muscle tissue, its apparent density, number of nuclei, mean area of a nucleus, nucleus-cytological relations and number of intramyocardial vessels.

Microsoft Excel 2000 was used to perform statistical processing of the results. T-tests were employed to provide the reliability of differences of mean quantities and correlation relationships. Differences p<0.05 were considered as reliable.

from newborns to 1-year-old babies? 3. What pathomorphological distinctions are typical for 2 anatomical types of TGA: with intact ventricular septum (IVS) and with ventricular

The purpose of this research is to study the content of chemical elements and the morphological structure of the myocardium in infants with different TGA types. Three tasks were set to achieve this goal: 1. To investigate some features of the content of CE and the structure of cardiomyocytes in 3 age groups: newborns aged 1 to 6 months and babies aged 6 to 12 months. 2. To study the concentration of CE in different parts of the heart in infants with TGA and in patients whose death was not caused by cardiac problems (control group). 3. To compare the features of CE and pathomorphological structure of 2 anatomic types of TGA: with atrial septal defect (ASD) and intact ventricular septum (IVS) and with atrial

A pathomorphological study was carried out using autopsy material of 68 infants aged under 1 who died during the follow-up period, as well as 10 infants of the same age whose death was not caused by cardiac problems. All TGA patients were broken down in 2 groups according to patients' anatomical type: the first group included patients with a simple form of TGA – TGA with atrial septum defect (ASD) and intact ventricular septum (IVS) – 37 patients (19 aged under 1 month and 18 aged 1 to 6 months), while 31 patients having TGA with ASD and VSD (7 aged under 1 month, 13 aged 1 to 6 months and 11 patients aged 6

Biopsy samples were preserved in 4 % phosphate-buffered formalin and then washed off in a distilled water solution followed by processing in cryoprotectans (solutions of saccharose: 5 % for 2 hours, 10 % for 2 hours, 15 % for 12 hours). Fluorometry of histologic specimens was carried out by using an Axioskop 40FL microscope and an AxioCamHRc camera. To get a good computerized image of each histologic specimen, a Zeiss Plan-Neofluar x 40 lens was used for 20 s at a +24 °С room temperature. The images obtained were processed by

Fluorescent probes containing ethidium bromide and chlortetracycline were used to perform fluorometry of the myocardium. Staining myocardium slices with ethidium bromide was done in a phosphate buffer pH=7.4 that contained 5.0 \* 10 3 g/l of ethidium bromide for 5 minutes at a temperature of 25 °С, while chlortetracycline, also done in a phosphate buffer pH=7.4 containing 2.6 \* 10 2 g/l of chlortetracycline, was applied for 1 minute at a temperature of 25 °С. Fluorometry of histological preparations was then carried out. In the case of ethidium bromide, uptake was equal to 510-523 nm, emission – to 595-605 nm, while in the case of chlortetracycline, those parameters came to 400 nm and 520 nm respectively. In addition, myocardium samples were stained with antibodies for Monoclonal Anti Skeletal Myosin (FAST) Clone MY-32 skeletal myosin. FITS-conjugated secondary

The following properties were measured: muscle fibre diameter, relative area of muscle tissue, its apparent density, number of nuclei, mean area of a nucleus, nucleus-cytological

Microsoft Excel 2000 was used to perform statistical processing of the results. T-tests were employed to provide the reliability of differences of mean quantities and correlation

septum defect (VSD)?

septal defects (ASD) and (VSD).

to12 months) were assigned to the second group.

AxioVision 3.1 software (Carl Zeiss).

antibodies were used as a secondary marker.

relations and number of intramyocardial vessels.

relationships. Differences p<0.05 were considered as reliable.

**2. Methods** 

The concentrations of CE were determined by X-ray fluorescence analysis with synchronous radiation (SRXRF). All measurements were carried out at the station of X-ray fluorescent elemental analysis in the Siberian Centre of Synchrotron and Terahertz Radiation (Budker Institute of Nuclear Physics SB RAS). The parameters of the storage ring VEPP-3 and experimental station are as follows: Eex = 2 GeV, B = 2 T, Ie = 100 mA; chamber for the analysis is made from elconait; maximum diameter of the sample is 30 mm; the spot size is 1 ÷ 30 mm2; exposure time is 10 ÷ 1000 c; the excitation energy is from 12 to 45 keV; elements determined: from S to U; X-ray fluorescence from the sample is registered by 10mm2 Si(Li) detector (OXFORF, Oxford Instruments Inc., USA) with energy resolution 150 eV at 5.9 keV, respectively (Trounova et al., 1998).

The advantages of the application of SR as a primary source of excitation are as follows: the high intensity → the better peak/background ratio → analysis of samples with low masses (down to 0.5 mg, dry weigh); linear polarization → lower background → lower detection limits (down to 0.02 ppm for organic matrices); the wide spectrum of radiation → optimization of excitation energy, the possibility to measure samples, as well as varying the excitation energy.

The concentrations of CE in the samples of heart muscle and vessels were calculated by the external standard method (different certified reference materials [CRM]) were used). The corresponding approaches were elaborated upon, using different certified reference materials with similar matrices: the applicability of different standards and the absorption characteristics of their matrices were investigated (Trunova et al., 2008). All spectra obtained were processed by the AXIL programme (Canberra Packard, Benelux). The samples investigated are the fragments of myocardium tissue with masses from 2 to 10 mg (dry weigh). At one of the steps of the sample procedure they are dried for 48 hours and longer to obtain a dry sample with a flat surface.

The content of CE was measured in 40 samples of myocardium of TGA infants aging from 1 to 4.5 months (mean age 3.0+0.7 months, heart's mass 54.0+5.0 g, body mass 4.2+0.3 kg). The concentrations of the following 14 CE were studied: S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br, Rb, Sr by SRXRF (Okuneva et al., 2010). By using X-ray fluorescence analysis with synchronous radiation (XFA SR), concentrations of the following 14 CE were studied: S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br, Rb, Sr (Okuneva et al., 2010). Myocardium samples were taken from ventricles and atria not later than 24 hours after death. Overall, more than 270 X-ray fluorescence spectra of CE were obtained. The content of CE was determined on the basis of 1 µg per 1 g of tissue.
