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Counting blood corpuscles in biological fluids, by using slide trays is a current, rapid, and convenient method. For their adequate calculation, it is necessary to know the volume of biological material under the chamber array of a slide tray, the total volume of the chamber in microl, and the degree of biological material concentration. When the Nechiporenko test reveals single urinary costs, it is recommended to count the latter in the whole chamber of a slide tray, followed by division of their count by the total volume of the chamber in microl. All required data must be given in an instruction for slide trays.

The present review describes the diagnostic value of the metabolites of epinephrine, norepinephrine, dopamine, and serotonin in the laboratory diagnosis of drug abuse. The metabolism and effects of these substances in health and drug addiction are considered. A procedure for their analysis is presented.

The authors have studied the species-specific composition of major periodontopathogenic bacteria (P. gingivalis, P. intermedia, B. forsythus, A. actinomycetemcomitans, and T. denticola), by applying the Russian reagent kit developed and made by HPF "GENETECH", and some fungi, microorganisms, and viruses among the Moscow Region inhabitants with chronic generalized periodontitis in 2006-2008. The studies have shown it possible to use molecular genetic methods for diagnosis, drug therapy monitoring, and epidemiological studies in periodontology and implantology.

The purpose of the study was to elucidate whether hepatitis C virus (HCV) RNA was present in the blood of patients from a Moscow therapeutic-and-prophylactic institution with uncertain results of a study of anti-HCV that were obtained using the algorithm developed for mass screening. The two-stage scheme of testing the serum for anti-HCV, which was obligatory in accordance with the Order of the Ministry of Health of the Russian Federation, was supplemented by a study the samples having a low optical density samples and those with controversial results in the test systems with the expanded spectrum of detectable anti-HCV, the instructions of which comprises criteria for an uncertain result. Sera with uncertain results of anti-HCV tests were assessed by two polymerase chain reaction (PCR) techniques for HCV RNA. The cDNA fragments complementary to HCV RNA were detected in the sera obtained from two elderly persons in PCR when a signal was recorded in agarose gel. The other real-time PCR failed to detect RNA in none sera with an uncertain result as to anti-HCV.

A multiplex analytical system has been developed to carry out microformat latex agglutination tests with video digital registration. The system makes it possible to apply less than 1 microl drops of latex and samples in the matrix format to a carrier and to make the test in 30 points at once, as well as to record and to interpret the results of the test, by keeping analytical information for each sample. Comparison of the results of determination of the serum levels of C-reactive protein, rheumatoid factor, and antistreptolysine in the system developed by the authors with the results obtained in the macroagglutination test by the immunoturbidimetric technique leads to the conclusion that the methods are comparable. The proposed type of a latex agglutination test based on a matrix approach and miniaturization assures efficient production and the quality of laboratory studies.

A microarray analytical system for performing tests of latex agglutination reaction in microformat with digital image registration was developed. The system allows the application of latex microdrops to the surface of the carrier in the form of a regular microarray and mixing of the latex droplets with the individual samples in each droplet of the microarray. The reaction is performed in a total mixture volume of about 1 microl for each of 30 samples simultaneously with video registration and interpretation of the results using a scanning device and specially developed software. The results of the semi-quantitative determination of C-Reactive Protein, Rheumatoid Factor and Anti-Streptolysin O concentrations by traditional macro- and proposed micro-arrayagglutination method were compared with the immunoturbidimetric measurements used as reference method. It was concluded that the suggested method for performing latex agglutination reactions on the basis of a microarray approach with digital image evaluation of results can provide a high throughput and reliable results and also offers significant advantages to the traditional latex agglutination tests with visual interpretation. Comprehensive documentation and objectification of readouts show a siginificant improvement to the present methodology.

Plasma trace elements (TE) were determined by atomic absorption spectrometry with electrothermal atomization and by mass spectrometry with inductively bound plasma. These techniques are highly effective and may be used to determine TE in biological samples. In children, chronic gastrointestinal diseases are accompanied by mineral metabolic disorders. In children with chronic gastrointestinal diseases whose age was 6 to 15 years and whose weight was varying, the scores of some TEs depend on a child's body weight and may be used to evaluate the degree of a pathological process.

The present paper describes the use of the scanner "Expert-Lab Agglutination" system to record the results of latex-agglutination tests in making a reaction on the lids of 96-well immunoassay plates. The described system provides a possibility of having a primary image of all tests carried out on a carrier. Software offers the prospect of contrasting the images of individual and control samples, increasing the size of images, and comparing the latter. The use of special image treating techniques may also yield objective figures characterizing the rate of a reaction. The possibility of archiving primary information and retrospectively appraising the correctness of the result of an analysis at any required moment is of fundamental importance to the latex-agglutination test.

Whether a commercially available scanner might be used as a vertical photometer of 96-well microplates for enzyme immunoassay (EIA) was studied. The analytical characteristics of the scanner in this regard were assessed, by comparing the findings with the respective photometric data obtained by a "VICTOR" EIA analyzer. It is concluded that the scanner registration system in combination with "Expert-Lab EIA" may be used in all types of EIA. The scanner system provides additional possibilities as compared with the conventional photometric analyzers.

Disorders in restructuring of T-cell receptor gamma-chain in DNA from skin biopsy specimens and peripheral blood lymphocyte of patients with malignant skin lymphomas at different stages of the disease were detected by PCR. Nucleotide sequences of monoclonal T-cell receptors were determined. The study showed that DNA sequences from the skin and lymphocytes did not coincide in some cases.

Histone proteins and nucleic acid precursors were studied in 10 normal subjects and 20 patients with disseminated psoriasis before and after combined therapy including Enkad. A drop in the concentrations of H1 histones was detected in the blood lymphocytes and plasma in parallel with an increase in the concentrations of arginine-rich histones and a many-fold decrease in the acid-soluble fraction representing a pool of nucleic acid precursors. After combined therapy the ratio of lysin- and arginine-rich nuclear proteins in blood lymphocytes changed significantly and approximated the normal value; the distribution of histone-like proteins virtually did not change in the plasma, but the level of plasma and erythrocyte acid-soluble fraction normalized. This indicates positive changes in nucleic acid transport after therapy. The authors demonstrate the possibility of these findings for evaluation of the treatment efficiency.

The pool of free purine derivatives and activities of the key enzymes of purine metabolism (adenosine deaminase, purine nucleoside phosphorylase, and 5'-nucleotidase) in lymphocytes, erythrocytes, and epidermis homogenates were measured in 20 normal subjects and 15 patients with psoriasis by high-performance liquid chromatography. The levels of AMP, GMP, and IMP purine monophosphates are decreased in the epidermis and red cells of psoriasis patients, whereas the final products of hypoxanthine, xanthine, and uric acid metabolism are accumulating, and the activities of ADA and PNP are increased double in the skin, all this indicating purine derivatives catabolism.

The pool of purine compounds was analysed in liver, skeletal muscle and blood of mice during the growth of Ehrlich ascites tumour cells. Three fast isocratic high-performance liquid chromatographic methods were used. (1) Determination of nucleotides by an isocratic ion-pair reversed-phase chromatography with a 10 mM ammonium phosphate buffer containing acetonitrile and tetrabutylammonium phosphate. (2) Separation of nucleosides and nucleobases in cell extracts by a reversed-phase system with methanol and 50 mM potassium phosphate buffer as eluent. (3) Nucleosides and nucleobases in body fluids were analysed by a reversed-phase system with 10 mM potassium phosphate containing methanol. These methods allow the rapid determination of purine compounds in small biological samples from various cell types and body fluids, with high accuracy and sensitivity. The pool of cellular nucleotides increased during the exponential phase of tumour growth. Adenosine accumulated significantly in all tissues in the stationary phase of tumour growth.

Isocratic methods are described for the separation of purine compounds in specimens of jejunal mucosa available by peroral biopsy from patients with coeliac disease. The first mode provided separation of both the ionic nucleoside monophosphates and the non-ionic nitrogen bases and nucleosides. The second mode was applicable to analyses of the nucleoside mono-, di- and triphosphates only. These chromatographic procedures were carried out with tetrabutylammonium phosphate as an ion-pair modifier and different concentrations of acetonitrile in the mobile phase. The modes are flexible in that they permit the optimization of the separation of these compounds according to the purpose of the investigation. The results of studies on purine metabolite patterns in coeliac mucosa are discussed.

The pattern of purine derivatives was studied in the erythrocytes of C3HA and ICR mice during Hepatoma 22 and Ehrlich ascites tumor cells growth. Host erythrocytes purine nucleotides, nucleosides and bases were affected by the implanted tumors. The results indicated that the host erythrocytes markedly concentrated adenine and guanine nucleotides on the 5th and 11th-12th day of tumor growth. By contrast, content of nucleosides and bases were sharply decreased during the log growth phase (5th day) with the restoring of these precursors within the 11th-12th day (plateau phase). These observations indicate that aspects of the purine compounds metabolism in host erythrocytes are linked with tumor development.

The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37 degrees C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold. Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [14C]-adenosine yielded no [14C]-inosine ruling out extramitochondrial adenosine deamination. It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested.

The content of purine compounds in red blood cells was measured and compared in 21 neonates with a history of chronic intrauterine hypoxia, depending on the condition at birth and the early period of adaptation. The content of purine compounds in whole blood was measured at the moment of birth and on days 1, 3 and 5 of life. It has been disclosed that the pattern of purine metabolism abnormality in funic red blood cells makes it possible to predict the course of the early period of adaptation. Changes in the content of hypoxanthine and inosine monophosphate appeared to be most remarkable.

The pattern of purine and pyrimidine derivatives was studied in the erythrocytes of C3HA and ICR mice during Hepatoma 22 and Ehrlich ascites tumor cell growth. Concentrations of purine nucleotides, nucleosides and nucleobases of host erythrocytes were changed after the implantation of the tumors. The host erythrocytes markedly concentrated adenine and guanine nucleotides both during logarithmic and plateau phase of tumor growth (5th and 11-12th days after inoculation of the tumor). The contents of nucleosides and nucleobases in the red blood cells were decreased during the logarithmic growth phase but restored within the plateau phase.

Venous blood acid-soluble fraction was investigated by means of high-efficiency liquid chromatography in patients with myocardial infarction and angina pectoris. Myocardial ischemia is shown to result in marked changes of purine and pyrimidine metabolism. A rise in intermediate and end products of purine metabolism (inosine, hypoxanthine, xanthine, uric acid), and the emergence of thymin and thymidine were demonstrated in venous blood of patients with myocardial infarction and angina pectoris.

Alterations in the levels of hypoxanthine and some other purine derivatives occurring in the erythrocytes from umbilical cord blood of newborn infants with perinatal hypoxia were examined. Newborn babies were divided into two groups which included infants with favourable (Group 1) and complicated (Group 2) periods of early adaptation. Purine metabolites of erythrocytes were analyzed by isocratic reversed-phase HPLC. It was shown a reciprocal relation between concentrations of hypoxanthine and IMP in erythrocytes of infants from two groups that was expressed in elevation (Group 1) and a fall (Group 2) of hypoxanthine level while in Group 1 the IMP content had a tendency to decrease and Group 2 was characterized by an elevated level of this compound. The alterations in the concentrations of these interrelated metabolites may be due to the effects of hypoxia upon purine metabolism in erythrocytes.

Acute disturbances of the cerebral circulation occupy the leading place in the structure of neurovascular diseases. Studies on metabolic processes in the patients, body in the acute period of the stroke and, particularly, the metabolism of free amino acids enable one to expand our knowledge of the pathogenetic essence of the stroke and its differential diagnosis, and thus to decide on the therapeutic measures to be taken for correcting the disturbed metabolism. Of great interest in this connection are dynamic determinations of the levels and the spectrum of free amino acids in biological fluids.

Chalone isolated by the Verly method was injected into animals of different age (2 groups) under partial hepatectomy. The percentage of hepatocytes in S-, G2-, M- and post-M-periods was determined by morphoautoradiography. These parameters were used for the reconstruction of the extent and dynamics of cell proliferation during 36-48 hours of regeneration. The suppressing effect of chalone on hepatocyte proliferation was more pronounced in young mice. The lack of complete inhibition of DNA synthesis and of the effect of proliferative synchronization was observed in both groups of mice.

The purine metabolism was studied in the thymus and spleen lymphocytes of C3HA mice during hepatoma 22 growth in comparison with the transport characteristics of hypoxanthine and inosine in lymphocytes. An increase in the transport of these metabolites from erythrocytes was observed in the early hepatoma growth period. It may be associated with a certain pattern in the dynamics of purine metabolic pool in the lymphocytes. The inverse relationship is found between the rates of hypoxanthine transport into thymocytes and its intracellular concentration.

The authors studied the metabolism of purine compounds in the skeletal muscle of 129 Re mice with hereditary muscular dystrophy (MD). The study showed impairment of purine metabolism which was expressed in a sharp decrease in ATP levels and an increase in the content of AMP, IMP and uric acid. No changes were revealed in the pool of purine nucleotides in murine red blood cells. A study of some physical properties of the red blood cells in mice with myopathy showed no alterations in the osmotic resistance of erythrocytes, yet there was a reduction in their pliability as compared to control. Examination of the temperature resistance revealed anomalies of red blood cells in myodystrophic mice at 50 degrees C. The detected changes of some physical properties of erythrocytes seem to be related to abnormalities of the sumbembranous contractile apparatus of these cells.

A method is described for the simultaneous analysis of twelve major nucleotides, certain biochemically essential nucleosides and nitrogen bases. The separation was achieved using an ion-pair reversed-phase high-performance liquid chromatographic (HPLC) system with a radially compressed NovaPak C18 cartridge and tetrabutylammonium phosphate (TBA) as a mobile phase modifier under acetonitrile gradient conditions. The effects of TBA and acetonitrile on the retention of major nucleotides were studied. The retention of nucleotides increased significantly with an increase in the TBA concentration from 0 to 2 mM and remained constant up to 5 mM TBA. The logarithm of the capacity factor decreased in proportion to the concentration of acetonitrile in the mobile phase. The separation of nitrogen bases, nucleosides and some of their deoxy forms was performed on an isocratic ion-pair reversed-phase HPLC system with pentanesulphonic and heptanesulphonic acid using a mu Bondapak C18 column. The described chromatographic procedures were applied to the separation of various biological extracts.

Procedures are presented for the analysis of profiles of purine and pyridine compounds in human and rabbit red blood cells by reversed-phase high-performance liquid chromatography and in Ehrlich ascites tumour cells of mouse by ion-pair high-performance liquid chromatography. These compounds are present in rabbit erythrocytes in higher concentrations than in human blood cells, and in rabbit reticulocytes the concentration of purine compounds is still higher. During glucose-free incubation, human red cells accumulate adenosine and adenine in the presence of coformycin owing to the inhibition of adenosine and AMP deamination. Ehrlich ascites tumour cells lose major portions of purine mono-, di- and triphosphates between the seventh and eleventh day after inoculation into mouse peritoneal cavities.

The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37 degrees C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine-guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.

The levels of DNA and RNA in gastric mucosa were assayed in 50 patients suffering various morphological variants of chronic gastritis and 73 cases of stomach cancer. Nearly identical rise in nucleic acid concentrations was identified in both atrophic gastritis and stomach cancer as compared to atrophy-free gastritis and normal condition. A link between atrophic gastritis and gastric cancer was suggested.

Simultaneous separation of the main ribonucleotides, nucleosides and bases was carried out using tetrabutyl ammonium phosphate (TBA) hetaerons with a reversed-phase (C18) packing material. Chromatographic properties of purine and pyrimidine metabolites in radially compressed NovaPak C18 system was studied at various concentrations of acetonitrile and TBA in the mobile phase. The optimal conditions for gradient separation of nucleotides, nucleosides and bases were evaluated. The procedure was used for analysis of blood from healthy persons and patients with acute myocardial infarction.

Under examination there were 29 patients (14 females and 15 males aged 20 to 40 years) with various forms of progressing myodystrophies (PMD). The control group consisted of 9 patients (2 females and 7 males) of the same age group. The content of free amino acids in the blood plasma and erythrocytes was determined by the method of column ion-exchange chromatography. The changes of the plasma and erythrocyte levels of most amino acids were found to be diverse in all forms of the PMD. Differences between primary PMD forms and denervation amyotrophies were revealed. The results of the studies show that content of free amino acids in the plasma and erythrocytes reflects both the general adaptation changes in the body in the conditions of the myodystrophic process, and the specificity of amino acid metabolism disturbances in various PMD forms.

The distribution of purine compounds in the skeletal muscles of the anterior and posterior limbs of 129/Re mice with hereditary muscular dystrophy (HMD) was investigated in a comparative study. The results revealed unidirectional metabolic disorders in both groups of muscles which was manifested in a quantitative redistribution of phosphorus-containing purine components and a decrease in the pool of adenylates in muscle tissue. Elevated concentrations of purine metabolites (inosine, hypoxanthine, xanthine, and uric acid) indicated an augmented metabolism of purines in this abnormality. Impairment of metabolic transformations of purines in the muscle tissue in HMD may be one of the factors responsible for the development of the pathological process in the muscle in the given disease.

The content of the precursors and metabolites of nucleic acids in the brain and liver of normal rabbits, rabbits with hypothyrosis (thyroidectomy) and hyperthyrosis (100-150 Mg of L-thyroxine per 1 kg of body mass daily for 10-12 days) was determined by liquid chromatography with a high resolution. A decrease in the content of deoxyguanosine, thymine, hypoxanthine, cytosine, thymidine and an increase in the content of inosine, adenine and adenosine in the liver of animals with hypothyrosis were shown. In the brain of these rabbits the level of cytidine was lowered, and the levels of deoxyguanosine, thymine, inosine, hypoxanthine, adenine and uracil were on an increase. An increase in the levels of deoxyguanosine, thymine, adenine, thymidine and a decrease in the levels of cytidine, hypoxanthine, cytosine, adenine and uracil in the liver were noted in hyperthyrosis. In the brain of rabbits with hyperthyrosis an increase in the levels of deoxyguanosine, cytidine, inosine and adenine was combined with a decrease in the levels of thymine and hypoxanthine. These thyroid-dependent changes in the content of registered metabolites and precursors of nucleic acids in tissues suggest that the observed shifts illustrate one of many mechanisms of the regulation of cell metabolism by thyroid hormones.

The content of 4 fractions of nuclear proteins (histones, acid chromatin protein, globulins and chromatin-free acid protein) in diploid and polyploid hepatocytes from intact and regenerating liver of mice is studied. These types of nuclei are found to differ in the protein content and in the protein/DNA ratio. Synthesis of all classes of nuclear proteins was intensified at the G1-stage, and synthesis of DNP non-histone proteins at the end of S- and G2-stage. Possible role of different nuclear proteins in the regulation of cell multiplication is discussed.

Various methods are described for the analysis of purine derivatives in biological samples by ion-pair high-performance liquid chromatography (HPLC) with both gradient and isocratic systems. A new approach is proposed that is suitable for the separation of nuclei acid constituents in different cells with a specific enzymatic activity pattern. The ion-pair HPLC methods were developed for the analysis of erythrocytes, lymphocytes and mitochondria acid-soluble fractions in clinical and experimental studies of normal and altered nucleotide metabolism. The results of studies of purine metabolite redistribution in mouse liver mitochondria during a 30-min incubation at 37 degrees C and data on purine metabolic alterations in mouse thymocytes during hepatoma growth are discussed.

Utilization of ion-air reagents in a reversed-phase chromatographic system allows solving a number of problems related to the separation of purine and pyrimidine derivatives. Simultaneous analysis of nucleotides, nucleosides and their bases was carried out by acetonitrile gradient elution using tetrabutyl ammonium phosphate as a counterion in the mobile phase. Besides, optimal conditions were selected for isocratic separation of adenine nucleotides and their metabolites. Furthermore, isocratic separation of certain purines and pyrimidines was achieved by modifying the stationary C18-phase with pentane- and heptane sulphonic acids.

Using reverse phase ion pair high performance liquid chromatography, the levels of free adenosine, inosine, adenine, xanthine, hypoxanthine, guanine and deoxycytidine in thymocytes and splenic T- and B-lymphocytes of C3HA mice, were studied under normal conditions and at different times (5 hrs, 1, 2, 3, 4, 5, 8 and 20 days) after transplantation of solid hepatoma 22a. The adenosine and inosine levels in thymus and spleen lymphocytes were 5 to 10 times as low as that of purine bases. Inosine was totally absent in T-and B-lymphocytes. The absolute content of adenine and guanine in thymus and spleen lymphocytes was higher compared to purine bases. It was shown that in all cases studied the decrease in hypoxanthine, xanthine and guanine levels in T- and B-lymphocytes during maximal tumour growth, i.e., on the 5th and 8th post-inoculation days as well as at the terminal period (20th day), was correlated with the decrease in the adenosine deaminase and functional activities of these cells. The level of free adenine in thymocytes and spleen T-lymphocytes during tumour growth showed a 2-4-fold increase in comparison with normal values. A dramatic decrease of intracellular concentration of deoxycytidine was observed in thymocytes and spleen T- and B-lymphocytes beginning with the 5th hour and over the whole subsequent period. The key role of the deoxycytidine decline during tumour growth as a possible cause of simultaneous impairment of DNA synthesis and purine deoxyribonucleoside phosphorylation in lymphocytes is discussed.

Applying a recently developed reverse-phase ion-pair HPLC technique we determined purine compounds in acid extracts of mitochondria isolated from rat and mouse liver, rat renal cortex, and hepatoma. Intramitochondrial purine nucleosides and bases were evaluated quantitatively for the first time. All mitochondria examined contain significant amounts of adenosine, guanosine, inosine, guanine, and hypoxanthine. Moreover, IMP was identified in all extracts. Adenine was detected in traces only. These results are in accordance with the concept of mitochondrial metabolism of purine compounds.

Tonsils from 10 patients (18-40 years old), with chronic tonsillitis were studied by means of histological methods as well as by high pressure liquid chromatography. In 5 patients with decompensated forms of the disease, accompanied by heart impairments during 1-3 years, only slight alterations in epithelial and follicular parts of tonsils were observed, while pronounced destruction of tonsil cells was detected in other 5 patients suffering from repeated anginas. The following purine and pyrimidine derivatives were identified in faucial tonsil tissue of all the patients with chronic tonsillitis studied: adenine, guanine, adenosine, guanosine, inosine and uridine. Distinct increase in content of these derivatives as well as occurrence of xanthine and uric acid (non-identified in the first group of patients with chronic tonsillitis) was noted in the patients with maximal destruction of faucial tonsil cells.

During development of transmitted solid hepatoma 22 pools of purine metabolites were studied as well as the rate of 3H-hypoxanthine and inosine incorporation was measured in acid soluble fraction of liver tissue and erythrocytes of mice C3HA as well as of the tumor tissue. Specific alterations in metabolism of adenine and guanine nucleotides and of their derivatives were detected at various steps of the hepatoma development in the tissues studied. Specific interaction of the hepatoma cells with host tissues appears to be realized via the pool of purine nucleotides.