Method of determination of pyruvate dehydrogenase (pdh) activity and medium of visualisation for implementation in said method

FIELD: biotechnology.

SUBSTANCE: invention relates to determining the activity of pyruvate dehydrogenase complex (PDH complex) by 13C-MP detection (magnetic resonance detection based on the isotope 13C). The essence of the method consists in that the change in activity of PDH complex in the subject to be examined by 13C-MR detection (magnetic resonance detection based on the isotope 13C) is determined using the medium of visualisation comprising hyperpolarised 13C-pyruvate, and when detecting a signal of 13C-bicarbonate or the signal of 13C-bicarbonate and the signal of 13C-pyruvate where the said hyperpolarised 13C-pyruvate is selected from the group consisting of hyperpolarized 13C1-pyruvate, 13C1,2-pyruvate, 13C1,3-pyruvate or 13C1,2,3-pyruvate or any of their combination, the activity of PDH complex is determined.

EFFECT: use of the claimed method enables to determine reliably changes in activity of PDH complex in the subject to be examined.

11 cl, 7 dwg, 5 ex

 

The invention relates to a method for determining the activity of pyruvaldehyde (PDH) by13C-Mr detection (magnetic resonance-based detection of isotope13C) using visualization environments that contain hyperpolarizability13C-pyruvate, and to visualization environments for use in the specified method.

In tissue adenosine triphosphate (ATP) provides energy for the synthesis of complex molecules, and in the muscles for contraction. ATP is formed as a result of the metabolism of energy-rich substrates, such as glucose or long-chain fatty acids. In oxidative tissues such as muscle, the main part of ATP is formed from acetyl-CoA, which enters the citric acid cycle, therefore, the provision of acetyl-CoA is a critical determinant of the production of ATP in oxidative tissues.

Acetyl-CoA is produced either by β-oxidation of fatty acids, either in the metabolism of glucose through the glycolytic biochemical pathway. A key regulatory enzyme in the regulation of the rate of formation of acetyl-CoA from glucose is pyruvaldehyde (PDH), which catalyzes the oxidation of pyruvate to acetyl-CoA and carbon dioxide with concomitant recovery nicotinamide-dinucleotide (NAD) to its reduced forms (NADH). Thus, PDH is a key enzyme in the m in the regulation of the rate of oxidative glycolysis and regulation of the balance between the oxidation of carbohydrate and lipid energy sources.

Recently renewed interest in the structure and functioning of the PDH complex by understanding that the modified activity of the PDH complex is characteristic of many human diseases ranging from relatively uncommon primary PDH deficiency to the main causal factors of morbidity and mortality, such as diabetes, malnutrition, sepsis and Alzheimer's disease.

PDH is an intramitochondrial multienzyme complex consisting of multiple copies of several subunits, including three of the enzymatic activity of E1, E2 and E3 are required to complete the conversion of pyruvate into acetyl-CoA (Patel et al., FASEB J. 4, 1990, 3224-3233). E1 catalyzes the irreversible loss of carbon dioxide from pyruvate; E2 forms acetyl-CoA, and E3 regenerates NAD to NADH. With this set are two additional enzymatic activity: specific kinase, which is capable of phosphorylation of E1 on three serine residues, and loosely coupled-specific phosphatase, which causes a reverse reaction of phosphorylation. Phosphorylation of a single residue of the three serine residues makes E1 inactive. The proportion of PDH in the active (dephosphorylated) state is determined by the balance between the activity of the kinase (PDH-kinase, PDHK) and phosphatase. The activity of the kinase can regulation the change in vivo relative concentrations of metabolic substrates, such as the [NADH]/[NAD+],

[acetyl-CoA]/[COA] and [ATP]/[adenosine diphosphate (ADP)], as well as the availability of pyruvate.

The reaction PDH serve to link metabolism pathways of glycolysis, gluconeogenesis and the synthesis of fatty acids with citric acid cycle. As a consequence, the activity of PDH highly regulated by a number of allosteric effectors and covalent modification.

For painful conditions such as type 1 diabetes and type 2 increased lipid oxidation with concomitant decrease in glucose utilization, which contributes to hyperglycemia. Reduced utilization of glucose in both types of diabetes 1 and 2 are associated with decreased activity of PDH. Furthermore, an additional consequence of the reduced activity of PDH may be that increasing the concentration of pyruvate results in increased availability of lactate as a substrate for hepatic gluconeogenesis. It is reasonable to expect that the increase in PDH activity can increase the rate of glucose oxidation and, consequently, the overall glucose disposal in addition to reduced development of glucose in the liver.

Another factor contributing to the diabetes mellitus is impaired secretion of insulin, which, as shown, is associated with reduced PDH activity in β-cells of the pancreas (Zhou et al., Diabetes 45, 1996, 580-586).

The oxidation of glucose can give bol is the highest yield of ATP per mole of oxygen, than the oxidation of fatty acids. In conditions where energy demand may exceed the energy supply, such as heart failure and some of cardiomyopathy, myocardial ischemia, peripheral vascular disease (including alternating lameness), cerebral ischemia and reperfusion, muscle weakness, hyperlipidemia, Alzheimer's disease and atherosclerosis, it can be expected that the shift in the balance of substrate utilization in favor of glucose metabolism by enhancing the activity of PDH will improve the ability to maintain levels of ATP and, therefore, functions.

As mentioned above, in diabetic state may be useful PDH activation by inhibiting gluconeogenesis and stimulation of consumption of glucose in peripheral tissues. Preliminary data confirming this assumption were obtained using dichloracetate (DHA). The search for new low-molecular PDHK inhibitors, enabling improved efficiency and specificity, is underway now for several years.

On the basis of the above, it is obvious that the determination of PDH activity plays a key role in the diagnosis of certain disorders and diseases. In addition, the determination of the activity of PDH is critical in the evaluation of treatment response, such as response to treatment medicines is mi, which affect the activity of PDH, namely increase it, as well as for screening of drugs that affect the activity of PDH.

Various methods for determining the activity of PDH, which in General can be divided into in vitro tests and in vivo.

In WO-A-2004/021000 disclosed are antibodies that are specific to the PDH, which can be used for thus PDH from a sample from the patient in an active state. The number and/or the active state of the PDH can be determined in vitro immunological analysis.

Tests PDH activity in vitro additionally disclosed in WO-A-99/62506. These tests are either the in vitro with the selected enzymes, which include training, time-consuming, such as selection by polymerase chain reaction (PCR) and cloning PDH-kinase, or cellular assays, which require isolation of primary cells.

The PDH activity in vivo can be determined in the analysis of ex vivo by extracting tissue samples (e.g., muscle or liver tissue), which is extracted as described in WO-A-99/62506. Part of the extract process PDH-phosphatase derived from porcine hearts, and the activity of the untreated sample is compared with the activity dephosphorylating sample thus obtained Stansbie et al., Biochem. J. 154 (1976), 225.

Therefore, there is a need for new and improved methods for determining the activity of PDH, especially PDH activity in vivo.

Presently discovered that hyperpolarizability13C-pyruvate can be used as an agent for determining the activity of PDH in vivo and in vitro through the use of13C-Mr detection.

As mentioned above, the pyruvate is a precursor in the citric acid cycle, and PDH catalyzes the oxidation of pyruvate to acetyl-CoA and carbon dioxide (CO2), which is in rapid equilibrium with bicarbonate (HCO3-).

Found that the metabolic conversion hyperpolarizing13C-pyruvate its metabolite hyperpolarizability13C-lactate, hyperpolarizability13C-bicarbonate (only in case of13C1pyruvate,13C1,2pyruvate,13C1,3pyruvate or13C1,2,3pyruvate) and hyperpolarizability13C-alanine can be used to study metabolic processes in the human body and an animal other than human, using Mr.13C1-pyruvate has T1relaxation (relaxation time) in whole human blood at 37°C for approximately 42 seconds, but found that the conversion hyperpolarizing13C-pyruvate in hyperpolarizability13C-lactate, hyperpolarizability13C-bicarbonate and hyperpolarizability13C-alanine is fast enough, that is the substance of the detection capability of the signal source connection 13C-pyruvate and its metabolites. The number of alanine, bicarbonate and lactate depends on the metabolic status of the tissue under study. The intensity of the Mr signal hyperpolarizing13C-lactate, hyperpolarizing13C-bicarbonate and hyperpolarizing13C-alanine is associated with a number of these compounds and the degree of polarization remaining in the moment of detection, therefore, by monitoring the conversion hyperpolarizing13C-pyruvate in hyperpolarizability13C-lactate, hyperpolarizability13C-bicarbonate and hyperpolarizability13C-alanine possible to study metabolic processes in vivo in humans or animal other than man, by using non-invasive Mr imaging or Mr spectroscopy.

In addition, it was found that the amplitude of the Mr signal arising from the different metabolites pyruvate vary depending on the tissue type. Unique painting metabolic peak formed by alanine, lactate, bicarbonate and pyruvate can be used as a characteristic trait of the metabolic state of the tissue studied and, thus, enables us to distinguish healthy tissue from tumor tissue. Use hyperpolarizing13C-pyruvate to visualize the tumor, where the tumor tissue prowl is no high metabolic activity, described in detail in WO-A-2006/011810.

In addition, the use hyperpolarizing13C-pyruvate for visualization of the heart is described in WO-A-2006/054903.

Thus, in the first aspect of the invention, a method for determining the activity of PDH by13C-Mr detection using the display environment containing hyperpolarizability13C-pyruvate, which detect the signal13C-bicarbonate and possible13C-pyruvate.

The term "determination of the activity of PDH" means the initial measurement of PDH activity, including measurement of initial velocity and the determination of the rate constant.

The term "13C-Mr detection means13C-MRI visualization or13C-Mr spectroscopy, or a combination of13C-MRI visualization and13C-Mr spectroscopy, that is,13C-Mr spectroscopic imaging. This term additionally means13C-Mr spectroscopic imaging at different points in time.

The term "visualization" means a liquid composition containing hyperpolarizability13C-pyruvate as an Mr-active agent, i.e. the agent visualization.

The imaging medium used in the method according to the invention, can be used as the display environment13C-Mr detection in vivo, i.e. in living humans and animals, other than humans. In addition to the, the imaging medium used in the method according to the invention, can be used as the display environment13C-Mr detection in vitro, for example, in cell cultures, samples of the body, such as blood or cerebrospinal fluid, tissue ex vivo, for example tissue ex vivo, obtained by biopsy, or isolated organs, all of which have the origin from human or animal other than man.

The term "13C-pyruvate" means salt13C-pyruvic acid, which is enriched isotope13C, in which the number of isotope13C higher than its natural abundance.

Isotopic enrichment hyperpolarizing13C-pyruvate used in the method according to the invention, is preferably at least 75%, more preferably at least 80% and especially preferably at least 90%, and most preferably isotopic enrichment of more than 90%. Ideally, the isotopic enrichment is 100%.13C-pyruvate used in the method according to the invention, should be enriched isotope at least C1-position (hereinafter denoted as13C1-pyruvate), because the C1-atom of pyruvate is part of the carbon dioxide (and therefore bicarbonate), formed as the result of PDH-rolled the dummy oxidation of pyruvate. In addition,13C-pyruvate used in the method according to the invention, can be enriched isotope of C1 - and C2-position (hereinafter denoted as13C1,2-pyruvate), at the C1 - and C3-position (hereinafter denoted as13C1,3-pyruvate) or C1-, C2 - and C3-position (hereinafter denoted as13C1,2,3-pyruvate). The isotopic enrichment of only S1-position is preferable, because13C1-pyruvate easily accessible and has a favorably high relaxation time T1in whole human blood at 37°C (about 42).

The terms "hyperpolarizing" and "polarized" hereafter used interchangeably and refer to their level nuclear polarization of more than 0.1%, more preferably more than 1% and most preferably more than 10%.

The level of polarization, for example, be determined by measurements13C-NMR solid state solid hyperpolarization13C-pyruvate, for example in solid hyperpolarization13C-pyruvate obtained by dynamic polarization of nuclei (DPJ)13C-pyruvate. Measurement13C-NMR solid state is preferably in the recording of NMR with a simple pulse sequence using the low angle of the magnetization vector. The intensity of the signal hyperpolarizing13C-pyruvate in the NMR spectrum is compared with intensive the STU signal 13C-pyruvate in the NMR spectrum, made up of the polarization process. Then the level of polarization is calculated from the ratio of intensities of the signal before and after polarization.

Similarly, the level of polarization for dissolved hyperpolarizing13C-pyruvate can be determined by measuring the liquid state NMR. Again the signal intensity of the dissolved hyperpolarizing13C-pyruvate compared to the signal intensity of the comparative sample of known composition, such as liquid pyruvic acid or pyruvate sodium, dissolved in aqueous solution. Then the level of polarization is calculated from the ratio of the integrals of the signal hyperpolarizing13C-pyruvate and known comparative sample, possibly adjusted for relative concentration. Polarization can also be determined by comparison with the signal of thermal equilibrium of the same sample13C-pyruvate after attenuation of hyperpolarization.

The hyperpolarization NMR active13C-cores can be achieved in various ways, which, for example, described in WO-A-98/30918, WO-A-99/24080 and WO-A-99/35508, which are incorporated in this application by reference, and the methods of hyperpolarization represent the polarization transfer from the inert gas, "polarization pumping", the polarization cooling spins, Pravoberezhny SPO is about and the dynamic polarization of nuclei (DPJ).

To obtain hyperpolarizing13C-pyruvate preferably either polarize13C-pyruvate directly, or to polarize13C-pyruvic acid and to convert the polarized13C-pyruvic acid in the polarized13C-pyruvate, for example, by neutralizing base.

One of the suitable ways to get hyperpolarizing13C-pyruvate is the polarization transfer from hyperpolarizing inert gas, which is described in WO-A-98/30918. Inert gases with non-zero nuclear spin, can be hyperpolarization by using light with circular polarization. Hyperpolarizability inert gas, preferably Not or Xe, or a mixture of such gases can be used to implement the hyperpolarization13C-cores. Hyperpolarizability gas may be in the gas phase, or it can be dissolved in a liquid/solvent or himself hyperpolarizability gas can serve as a solvent. Alternatively, the gas can condense on the cooled solid surface and used in this form, or give the possibility of sublimation. Preferably mixing hyperpolarizing gas with13C-pyruvate or13C-pyruvic acid. Therefore, if polarize13C-pyruvic acid,which is liquid at room temperature, hyperpolarizability gas is preferably dissolved in the liquid/solvent, or it serves as a solvent. If polarize13C-pyruvate, hyperpolarizability gas is preferably dissolved in the liquid/solvent which also dissolves pyruvate.

Another suitable way to obtain hyperpolarizing13C-pyruvate is that the polarization give13C-cores by thermodynamic equilibrium at a very low temperature and strong field. Hyperpolarization compared to the working field and temperature NMR spectrometer is carried out by use of very strong fields and very low temperatures (the polarization of the pump). Used the magnetic field strength should be high as possible, respectively, above 1 T, preferably above 5 T, more preferably above 15 Tons or more, and particularly preferably 20 Tons or more. The temperature should be very low, for example 4.2 K or less, preferably To 1.5 or less, more preferably of 1.0 or less, especially preferably 100 microns or less.

Another suitable way of obtaining hyperpolarizing13C-pyruvate is a method of polarization cooling of spins. This method covers the polarization of nuclear spins of a rigid connection or by cooling. The system is administered qualifying the crystalline paramagnetic substances, such as ions Ni2+, lanthanide or actinide with the axis of symmetry of order three or more, or thoroughly mix with them. The equipment is simpler than required for the DPJ, without the need for a uniform magnetic field as the field excitation of the resonance is not attached. This process is carried out by physical rotation of the sample around an axis perpendicular to the direction of the magnetic field. The premise of this method is that the paramagnetic particles are highly anisotropic g-factor. The rotation of the sample electron paramagnetic resonance will come in contact with nuclear spins, leading to a reduction in temperature of the nuclear spins. Rotation of the sample hold as long as the polarization of the nuclear spins will not reach a new equilibrium.

In the preferred embodiment to obtain hyperpolarizing13C-pyruvate is used DPJ (dynamic polarization of nuclei). When the DPJ polarization Mr active nuclei in the connection, which must be polarized, impact agent polarization or the so-called DPJ agent, a compound containing unpaired electrons. During the DPJ provide energy, usually in the form of microwave radiation source which excites the DPJ agent. Upon decay to the ground state is the polarization transfer from unpaired electric is on the DPJ agent on the NMR active nuclei of the connection, which must be polarized, for example on core13C13C-pyruvate. As a rule, during the DPJ to use the average or strong magnetic field and very low temperature, for example, by carrying out the process of the DPJ in liquid helium and in a magnetic field of about 1 T or higher. Alternatively, you can use the average magnetic field and any temperature at which achieved a sufficient strengthening of the polarization. Methodology the DPJ additionally described, for example, in WO-A-98/58272 and in WO-A-01/96895, both documents incorporated in this application by reference.

To polarize the connection method for preparing a mixture of compounds, which should be polarized, and the DPJ-agent ("sample"), which either freeze and bring in the DPJ-polarizer for polarization, or contribute in the DPJ-polarizer in liquid form, and it freezes within a specified polarizer due to the very low ambient temperature. After polarization frozen solid hyperpolarizing the sample is quickly transferred to the liquid state or by thawing, or by dissolving it in a suitable medium dissolution. Dissolution is preferred, and the process of dissolution of the frozen hyperpolarizing sample and a suitable device, therefore, described in detail in WO-A-02/37132. The process of thawing and convenient device for defrosting the ferocious, for example, in WO-A-02/36005.

With the aim of obtaining a high level of polarization in the connection, which must be polarized, the specified connection and the DPJ-agent need to be brought into close contact during the DPJ. It is unacceptable that the sample crystallized by freezing or cooling. In order to avoid crystallization or requires that the sample was attended by stekloobrazovanie or polarization it is necessary to select compounds which do not crystallize during freezing, but more likely to form a glassy substance.

As mentioned above,13C-pyruvic acid or13C-pyruvate are suitable initial materials for the production of hyperpolarizing13C-pyruvate.

Enriched isotopes13C-pyruvate commercially available, for example, as13C-pyruvate sodium. Alternatively, it can be synthesized as described S.Anker, J. Biol. Chem 176, 1948, 133-1335.

In the art there are several methods of synthesis13C1-pyruvic acid. In short, the authors of Seebach et al., Journal of Organic Chemistry 40(2), 1975, 231-237 described the path of synthesis, which is based on the protection and activation of carbonyl-containing source materials, such as S,S-acetal, for example 1,3-dition or 2-methyl-1,3-dition. Dition metallinou and subjected to interaction with methylcobalamin connection and/or13 CO2. By the use of enriched isotopes13C-component, as outlined in this link, it is possible to get the13C1-pyruvate or13C1,2-pyruvate. Then the carbonyl functional group release by using the conventional methods described in the literature. Another way of synthesis begins with acetic acid, which is first converted into acetylmuramic, and then subjected to interaction with Cu13CN. The resulting nitrile is converted into pyruvic acid via amide (see, for example, S.H.Anker et al., J. Biol. Chem. 176 (1948), 1333 or J.E.Thirkettle, Chem Commun. (1997), 1025). In addition,13C-pyruvic acid can be obtained by protonation commercially available13C-pyruvate sodium, for example, by the method described in US 6232497, or the method described in WO-A-2006/038811.

The hyperpolarization13C-pyruvic acid by the DPJ is described in detail in WO-A1-2006/011809, which is incorporated in this application by reference. In short,13C-pyruvic acid can be used directly for the DPJ, because it forms a glassy substance by freezing. After the DPJ frozen hyperpolarizing13C-pyruvic acid necessary to dissolve and neutralize, i.e. convert to13C-pyruvate. For this conversion is necessary is mo a strong Foundation. In addition, because13C-pyruvic acid is a strong acid, it is necessary to choose the DPJ-agent, which is resistant to this strong acid. The preferred base is sodium hydroxide, and the transformation hyperpolarizing13C-pyruvic acid with sodium hydroxide results in giperplazirovannom13C-pyruvate sodium, which is the preferred13C-pyruvate for environment visualization, which is used for Mr imaging and/or spectroscopy in vivo, that is, Mr imaging and/or spectroscopy, carried out from living persons or animals other than humans.

Alternative,13C-pyruvate, i.e. salt13C-pyruvic acid, can be used for the DPJ. Preferred salts are13C-pyruvate that contain inorganic cation from the group consisting of NH4+, K+, Rb+Cs+Ca2+, Sr2+and VA2+preferably NH4+, K+, Rb+or Cs+more preferably K+, Rb+Cs+and most preferably Cs+as described in detail in WO-A2-2007/111515 included in this application by reference. The synthesis of these preferred13C-pyruvate also disclosed in WO-A2-2007/111515. If hyperpolarizability13C-pyruvate is used in cf is de imaging for MRI imaging and/or spectroscopy in vivo, preferred currency inorganic cation from the group consisting of NH4+To+, Rb+Cs+, CA2+, Sr2+and VA2+on physiologically very good tolerable cation, such as Na4+or meglumine. This can be accomplished by methods known in the art, for example by using a cation exchange column.

More preferred salts are13C-pyruvate organic amine or amino compound, preferably TRIS-13C1-pyruvate or meglumin-13C1-pyruvate, as described in detail in WO-A2-2007/069909 included in this application by reference. The synthesis of these preferred13C-pyruvate also disclosed in WO-A2-2007/069909.

If hyperpolarizability13C-pyruvate used in the method according to the invention, obtained by the DPJ, the model for the polarization of the containing13C-pyruvic acid or13C-pyruvate and the DPJ agent may further comprise an ion of a paramagnetic metal. Found that the result of the presence of paramagnetic ions of the metal in the composition for a polarization by the DPJ are elevated levels of polarization in13C-pyruvic acid/13C-pyruvate, as described in detail in WO-A2-2007/064226, which is incorporated in this application by reference.

In another embodiment of the CPE is and visualization, used in the method according to the invention, contains hyperpolarizability13C-pyruvate and malate. Thus, in the second aspect of the invention, a method for determining the activity of PDH by13C-Mr detection using the display environment containing malate and hyperpolarizability13C-pyruvate, which detect the signal13C-bicarbonate and possible13C-pyruvate.

In the context of this invention, the term "malate" means a salt of malic acid. Malate is not hyperpolarizing.

Malate appropriately add to giperplazirovannom13C-pyruvate after the polarization process. There are several ways to add malate. When the result of the process of polarization is a liquid composition containing hyperpolarizability13C-pyruvate, malate can be dissolved in a specified liquid composition or a liquid composition can be added a solution of malate in a suitable solvent, preferably in an aqueous medium. If the result of the process of polarization is a solid composition containing hyperpolarizability13C-pyruvate or13C-pyruvic acid, for example, when used DPJ, malate can be added to the environment of dissolution, which is used for dissolving the solid composition, and dissolving in it. For example,13C-pyruvate, polarisusa is the way the DPJ, can be dissolved in an aqueous medium such as water or a buffer solution containing water, including malate, or13C-pyruvic acid, polarized way the DPJ, can be dissolved in an environment of dissolution that contains the basis for the conversion of pyruvic acid into pyruvate and malate. Alternatively, malate can be added to the final liquid composition, i.e. in the liquid composition after dilution/thaw, or in a liquid composition after removal of the DPJ-agent and/or a possible ion of a paramagnetic metal. Again malate can be added in the form of a solid substance in a liquid composition or preferably dissolved in an aqueous solvent, e.g. in an aqueous medium such as water or buffer solution. To stimulate the dissolution of malate in the art there are several known methods, such as shaking, stirring, mixing on a vortex or sonication. However, the preferred methods, which are fast and do not require mixing device or contribute to bringing into contact with the liquid composition.

Accordingly, malate added in the form of malic acid or salt of malic acid, preferably the sodium malate. Concentration hyperpolarizing13C-pyruvate and malate in the imaging medium used in the method according to the invention, the example is about the same, or malate is present at lower or higher concentrations than13C-pyruvate. If, for example, the imaging medium contains x M13C-pyruvate, it contains x M or approximately x M or fewer of malate, but preferably not less than one-tenth x M malate or more of malate, but preferably not more than three times x M malate. In the preferred embodiment, the concentration of malate in the imaging medium used in the method according to the invention, approximately equal or equal to the concentration hyperpolarizing13C-pyruvate. The term "approximately equal concentration" means the concentration of malate, which is +/-30% concentration13C-pyruvate, preferably +/-20%, more preferably +/-10%.

Using the imaging medium containing malate and hyperpolarizability13C-pyruvate, it is possible to determine the nature of the regulation of PDH. Stream PDH can be Engibarov or by inactivation of the enzyme complex PDK, as described previously, or instantly through inhibition of the final product. Demonstrated that the increased ratio of NADH/NAD+or acetyl-CoA/COA reduce PDH-mediated oxidation of pyruvate, and the availability of oxaloacetate to include acetyl-CoA in the Krebs cycle is the main determinant of intramitochondrial concentrations of acetyl-CoA. Mala is an intermediate connection oxidative metabolism of glucose and can enter the Krebs cycle in the form of oxaloacetate through anaplerotic sequences of reactions to improve the overall carbon flow. Not wishing to be bound by this hypothesis, the inventors suggest that the introduction of the rendering environment containing malate and hyperpolarizability13C-pyruvate, the degree of inhibition of the final product on PDH may be limited, and in the case of high-PDH activity increased flux of pyruvate via the enzyme complex, which may be determined by the method according to the invention. In situations of low activity of the PDH authors of the invention can assume that the inhibition of the final product is equally important, and that malate is present in the imaging medium, should not affect the flow of pyruvate via the enzyme complex, which may be determined by the method according to the invention.

In yet another embodiment himself malate is not present in the visualization, but it is administered to a subject to be study, that is, a living person or animal, other than man, in cell culture, in the sample of the body, such as blood, tissue ex vivo, such as tissue obtained by biopsy, or a stand-alone body, before the introduction of the rendering environment used in the method according to the invention.

As mentioned above, the imaging medium according to the method according to the invention can be used as a display environment for determining the activity of PDH in vivo by13C-Mr detection, i.e. living people or the animals, other than people. For this purpose, the imaging medium is proposed in the form of a composition that is suitable for introduction into the body of a living person or animal other than man. Such an imaging medium preferably contains, in addition to the Mr active agent13C-pyruvate aqueous carrier, preferably a physiologically tolerable and pharmaceutically acceptable aqueous carrier, such as water, buffer solution or saline. Such an imaging medium may further comprise conventional pharmaceutical or veterinary carriers or excipients, for example additives for preparations that are commonly used for diagnostic compositions in human or veterinary medicine.

In addition, the imaging medium in accordance with the method according to the invention can be used as a display environment for determining the activity of PDH in vitro by13C-Mr detection, for example, in cell cultures, samples of the body, such as blood samples, tissue ex vivo, such as tissue obtained by biopsy, or isolated organs. For this purpose, the imaging medium is proposed in the form of a composition that is suitable for adding, for example, cell cultures, blood samples, tissues ex vivo, such as tissue obtained by biopsy, or isolated organs. Taka is the imaging medium preferably contains, in addition to the Mr active agent13C-pyruvate, the solvent, which is compatible with the cell or tissue in vitro, and used in these analyses, such as DMSO, or methanol, or a mixture of solvents containing an aqueous medium and a nonaqueous solvent, for example a mixture of DMSO and water or buffer solution or methanol and water or buffer solution. As it is obvious to experts in the art, pharmaceutically acceptable carriers, excipients and additives for preparations can be present in such an environment, visualization, but are not required for such purpose.

If the imaging medium used in the method according to the invention, used for determining the activity of PDH in vivo, i.e. in the body of a living person or animal, other than man, the specified imaging medium is preferably introduced into a specified body parenterally, preferably intravenously. Typically, the monitoring body is placed in the MRI magnet. Specialized13C-MRI RF coils have to cover the region of interest. The exact dosage and concentration of the rendering environment will depend on a number of factors, such as toxicity and route of administration. Appropriately, the imaging medium is injected at concentrations up to 1 mmol of pyruvate per kg of body weight, preferably from 0.01 to 0.5 mmol/the g more preferably from 0.1 to 0.3 mmol/kg Less than 400 after administration, preferably less than 120 seconds, more preferably less than 60 s after the introduction of the applied sequence Mr imaging, preferably a sequence that encodes interest amount with a composite frequency and spatial path selection. The exact time sequence of Mr in a high degree depends on the interest amount and the type.

If the imaging medium used in the method according to the invention, used for determining the activity of PDH in vitro, the imaging medium is from 1 mm to 100 mm13C-pyruvate, more preferably from 20 mm to 90 mm, and most preferably from 40 to 80 mm13C-pyruvate.

The PDH activity may be determined in accordance with the method according to the invention by detecting signal13C-bicarbonate and possible signal13C-pyruvate. This definition is based on the following reaction, which is illustrated for13C1pyruvate; * denotes13C-tag:

According to the scheme 1 reduced activity of PDH is manifested in reduced formation of carbon dioxide and, consequently, in reduced signal13C-bicarbonate. At physiological pH balance of CO2/bicarbonate of sdin is in the direction of bicarbonate.

The term "signal" in the context of the invention refers to the amplitude of the Mr signal to either the integral or the area of the peak relative to the background in the spectrum of13C-Mr, which is13C-bicarbonate and possible13C-pyruvate. In the preferred embodiment of the signal represents the peak area.

In the preferred embodiment of the detect signals13C-bicarbonate and13C-pyruvate.

In the preferred embodiment of the method according to the invention the above-mentioned signal13C-bicarbonate and possible13C-pyruvate is used to generate a metabolic profile, which is a measure of the activity of PDH. If the method according to the invention is carried out in vivo, i.e. in a living person or animal, other than man, the specified metabolic profile may be removed from the entire body, for example, obtained by13C-Mr detection in vivo in the body. Alternatively, the specified metabolic profile, create an interest area or volume, i.e. with a specific tissue, organ or part of the specified human or animal other than man.

In another preferred embodiment of the method according to the invention the above-mentioned signal13C-bicarbonate and possible13C-pyruvate is used to generate a metabolic profile of cells in cell culture, samples of org the mechanism, such as samples of blood, tissue ex vivo, such as tissue obtained by biopsy, or a stand-alone body, originating from human or animal other than man. Specified metabolic profile then create by13C-Mr detection in vitro.

Thus, in the preferred embodiment, a method for determining the activity of PDH by13C-Mr detection using the display environment containing hyperpolarizability13C-pyruvate, which detect the signal13C-bicarbonate and possible13C-pyruvate, and where the specified signal or the specified signal is used to generate a metabolic profile.

In the preferred embodiment to create the specified metabolic profile using signals13C-bicarbonate and13C-pyruvate.

In one embodiment to generate the metabolic profile using the intensity of the spectral signal13C-bicarbonate and possible13C-pyruvate. In another embodiment to generate the metabolic profile using the integral of the spectral signal13C-bicarbonate and possible13C-pyruvate. In another embodiment to generate the metabolic profile using the intensity signals from the individual images13C-bicarbonate and possible13C-pyruvate. In another embodiment of interest is cunosti signal 13C-bicarbonate and possible13C-pyruvate receive two or more than two time points to calculate the rate of change13C-bicarbonate and possible13C-pyruvate.

In another embodiment, the metabolic profile includes the use of data processed signals13C-bicarbonate and possible13C-pyruvate or derived from them, such as relationships of signals, the corrected signals or information on dynamic or the metabolic rate constant, derived on the basis of the picture signals of multiple Mr detections, i.e. spectra or images. Thus, in the preferred embodiment of the adjusted signal13C-bicarbonate, i.e. the ratio of signal13C-bicarbonate signal13C-pyruvate, include in the metabolic profile or use to create it. In the following preferred embodiment of the signal13C-bicarbonate, adjusted according the total signal13C-carbon, include in the metabolic profile or use to create it, where the total signal13C-carbon is the sum of the signals13C-bicarbonate and13C-pyruvate. In a more preferred embodiment the ratio of signal13C-bicarbonate signal13C-pyruvate include in the metabolic profile or use for his POPs the project.

Metabolic profile created in the preferred embodiment of the method according to the invention, is an indicator for the PDH activity of the examined body part, body, cells, tissue sample from the body and so on, and found the information can be used at a later stage for a variety of purposes.

One of these goals may be the evaluation of compounds that alter the activity of PDH, preferably compounds that increase the activity of PDH. Compound that increases the activity of PDH may potentially be valuable in the treatment of painful conditions associated with impaired glucose utilization, such as diabetes, obesity (Curto et al., Int. J. Obes. 21, 1997, 1137-1142) and lactacidemia. In addition, it can be expected that this compound is useful in diseases where tissue supply of energy rich substrates is limited, such as peripheral vascular disease (including alternating lameness), heart failure and some of cardiomyopathy, muscle weakness, hyperlipidemia, and atherosclerosis (Stacpoole et al., N. Engl. J. Med. 298, 1978, 526-530). Compound that activates PDH, may also be useful in the treatment of Alzheimer's disease (Gibson et al., J. Neural. Transm. 105, 1998, 855-870).

In one embodiment of the method according to the invention is carried out in vitro, and the resulting information used in OC is the NCA the efficacy of potential drugs, which alter the activity of PDH, for example, in drug development and/or screening process. In this embodiment of the method according to the invention can be implemented in suitable cell cultures or tissue. Cells or tissue is brought into contact with a potential drug, and the activity of PDH is determined by13C-Mr detection according to the method according to the invention. Information about the efficacy of potential drugs can be obtained by comparing the activity of PDH treated cells or tissue with the PDH activity of untreated cells or tissue. Alternatively, the variation of PDH activity can be determined by determining the activity of PDH cells or tissue before and after treatment. This assessment of efficacy of a drug can be accomplished, for example, on the microplate, which allow parallel testing of various potential drugs and/or different doses of potential medicines and, therefore, provide an opportunity for screening with high output.

In another embodiment of the method according to the invention is carried out in vivo, and the resulting information used in assessing the effectiveness of potential drugs that modify the activity of PDH in vivo. In this embodiment of the method can be carried out, for example, on pogopin the x animals or volunteers in a clinical trial. Experimental animal or volunteer impose potential drug and determine the activity of PDH by13C-Mr detection according to the method according to the invention. Information about the efficacy of potential drugs can be obtained by determining the variation of PDH activity before and after the introduction of, for example, for a certain period of time when the re-introduction. This assessment of efficacy of a drug can be in pre-clinical research (experimental animals) or in clinical trials.

In another embodiment of the method according to the invention is carried out in vivo or in vitro, and the resulting information used to assess response to treatment and/or to determine the efficacy of treatment in patients undergoing treatment for this disease. If, for example, a patient with diabetes treated with antidiabetic medicine that, as expected, increases the activity of PDH, the PDH activity can be determined according to the method according to the invention. Appropriately, the activity of PDH is determined by the method according to the invention before treatment specified antidiabetic drug, and then after that, for example, for a certain period of time. By comparing the initial activity with PDH PDH activity during and after treatment to assess possible does antidiabetic drug any positive effect on the activity of PDH, and, if show, then to what extent. For implementing the method according to the invention for the above purpose, in vitro, of course, you want to be able to obtain suitable samples from the patient undergoing treatment, such as tissue samples or samples from the body, such as blood samples.

As described above, the information obtained by the method according to the invention, can be used at a later stage for a variety of purposes.

Another goal may be to obtain reliable information about medical conditions, such as identifying patients at risk, early detection, assessment of disease progression, severity and complications associated with the disease.

Thus, in one embodiment of the method according to the invention is carried out in vivo or in vitro, and the resulting information used to identify patients at risk of developing the disease and/or preventive measures to avoid the development of disease. Diagnosis of type 2 diabetes is often delayed, until not appear complications (Harris et al., Diabetes Metab. Res. Rev. 16, 2001, 230-236). Early treatment prevents some of the most damaging complications, but because modern methods of treatment of type 2 diabetes remain inadequate warning is highly preferred. There remains a need for defined and optimal approaches to the identification of patients at risk and/or candidates for preventive measures such as lifestyle changes, including a low-calorie diet with a low fat diet and physical activity. Conventional approaches include tests for glucose tolerance and measurement of plasma glucose on an empty stomach, but high-risk patients has not yet suffer from hyperglycemia and, therefore, they do not identify these tests. It would therefore be useful to have a method, useful for the identification of patients at risk for development of type 2 diabetes and identify candidates for preventive measures. The method according to the invention can provide the necessary information to carry out this identification. In this embodiment of the method according to the invention can be applied to determine the initial activity of PDH in the first time and for further definitions of PDH activity over a period of time with a certain frequency, for example every six months or annually. It can be expected that the reduction in the activity of PDH will indicate an increased risk of progression of type 2 diabetes, and rate of descent can be used by the doctor to make the decision on early prevention and/or treatment. In addition, the results of determining the activity of PDH in time can be combined with the results of tests on glucose tolerance and measurement of plasma glucose on an empty stomach, and the combined results can be used DL the decision on prevention and/or treatment. For implementing the method according to the invention for the above purpose, in vitro, of course, you want to be able to obtain suitable samples from the patient undergoing treatment, such as tissue samples or samples from the body, such as blood samples.

In another embodiment of the method according to the invention is carried out in vivo or in vitro, and the resulting information used for early detection of diseases. For some neurodegenerative diseases, including Alzheimer's disease, reported reduced activity of PDH. For Alzheimer's disease, this effect is specific to certain areas of the brain, and it is most pronounced in the parietal and temporal lobes. Early diagnosis of neurodegenerative diseases such would enable early intervention. The method according to the invention can provide the necessary information for the implementation of early diagnosis. In this embodiment of the method according to the invention can be applied to determine the initial activity of PDH and comparing it with the normal activity of PDH, for example with the activity of PDH in healthy subjects, or to determine the initial activity of PDH in certain areas of the brain, which we know that it affected certain neurodegenerative disease, and comparing it with PDH activity in the brain regions known that they n is affected by the specified disease. The activity of PDH can preferably be used for early diagnosis in conjunction with other clinical markers and/or symptoms, for example, for Alzheimer's disease. For implementing the method according to the invention for the above purpose, in vitro, of course, you want to be able to obtain suitable samples from the patient undergoing treatment, such as spinal fluid.

In yet another embodiment of the method according to the invention is carried out in vivo or in vitro, and the resulting information used to monitor the progression of the disease. This can be useful for diseases or disorders where the disease has not progressed to the level where shown or recommended treatment, for example, due to severe side effects associated with the specified treatment. In this situation, the choice of action is a "watchful waiting", i.e. the patient carefully monitored for disease progression and early detection of deterioration. In this embodiment of the method according to the invention can be applied to determine the initial activity of PDH and for further definitions of PDH activity over a period of time with a certain frequency. It can be expected that the reduction in the activity of PDH will indicate the progression and worsening of the disease, and this decrease can be used in atom for a decision about the beginning of treatment. For implementing the method according to the invention for the above purpose, in vitro, of course, you want to be able to obtain suitable samples from the patient undergoing treatment, such as tissue samples or samples from the body, such as blood samples.

In yet another embodiment of the method according to the invention is carried out in vivo or in vitro, and the resulting information used to determine the severity of the disease. Often the disease progress from their appearance over time. Depending on the type of symptoms and/or detect specific clinical markers of the disease are characterized by certain stages, such as early (mild) stage, medium (moderate) stage and severe (late) stage. More sophisticated stages are common to several diseases. It is known that a number of clinical markers are used to determine the stage of the disease, including more specific, such as certain enzymes or protein expression, as well as more General, such as blood counts, electrolytes, and so on. In this context, the PDH activity may represent a clinical marker, used alone or in combination with other markers and/or symptoms, to determine the stage of the disease and, consequently, the severity of the disease. Therefore, it may be possible the application of SP the soba according to the invention for determining the activity of PDH in a patient by quantitative and based on the value of the activity of PDH determine the disease stage of the patient. Ranges PDH, which is characteristic of a particular stage of the disease, can be established by determining the activity of PDH according to the method according to the invention in patients suffering from, for example, by disease in the early, middle and late stages, and determine the range of PDH activity, which is characteristic of a particular stage.

In yet another embodiment of the method according to the invention is carried out in vivo or in vitro, and the resulting information used for the identification and assessment of complications associated with the disease. Some diseases, such as diabetes can cause many complications, not only sharp, such as hypoglycemia, ketoacidosis or nemetona hyperosmolar coma, but also long-term complications related to the organs, including cardiovascular disease, kidney damage and/or failure and damage to the retina. Depending on how that affects whether diabetes and to what extent affects organs such as the heart or kidneys, you need to modify the treatment of the disease so that it was directed to this damage and cause them to reverse development. In the method according to the invention it is possible to determine the activity of the PDH-specific organs, for example, by13C-Mr detection in vivo carried out with a surface coil placed over the heart or kidneys. You can expect h what about the low activity of PDH in the heart or the kidney is an indicator of the destruction of this body, for example, diabetes (Huang et al., Diabetes 52, 2003, 1371-1376).

Because the activity of PDH is influenced by several factors, such as nutritional status, availability/condition of oxygen, insulin, and a number of cofactors, it is important to control these factors, for example, by providing patients a diet plan or standardized food before implementation of the method according to the invention. Also discovered that the patient should not starve, as this may result in a reduced signal13C-bicarbonate.

In one aspect of the invention, the activity of PDH purposefully and adjustable modulate by oral or parenteral administration, for example, glucose, fatty acids or ketone Oxygen phone status can be modulated by exposure to respirable gas to the implementation of the method according to the invention or pharmaceutically through induction of stress or changes in perfusion.

In another embodiment of the PDH activity determined in the described manner, but sequentially or simultaneously with the quantitative determination of fatty acid metabolism, or in front of him. As described above, the acetyl-CoA is formed as a result of glycolysis or fatty acid metabolism, and shift from one to another is part of many painful conditions. In addition to the direct determination of the activity of the PDH method according to the invention, an indirect measure and the activity of PDH by measuring the metabolism of fatty acids would be complementary and important. Fatty acid metabolism can be quantified by introducing the display environment containing hyperpolarizability13C-acetate, and13C-Mr detection of the signals from the metabolite13C-azetilcarnitin and possible13C-acetyl-CoA or13C-acetyl-CoA and original connection13C-acetate.

Thus, another aspect of the invention is a method for determining the activity of PDH by13C-Mr detection using the display environment containing hyperpolarizability13C-pyruvate and hyperpolarizability13C-acetate, which detect signals13C-bicarbonate and possible13C-pyruvate and signals13C-azetilcarnitin and possible13C-acetyl-CoA or13C-acetyl-CoA and13C-acetate.

Another aspect of the invention is a method for determining the activity of PDH by13C-Mr detection using the display environment containing hyperpolarizability13C-pyruvate, which detect signals13C-bicarbonate and possible13C-pyruvate, and where before or after that13C-Mr detection exercise13C-Mr detection using the display environment, which contains hyperpolarizability13C-acetate, and find where the signals13C-azetilcarnitin and possible13C-acetyl-CoA or13C-acetyl-CA and 13C-acetate.

13C-pyruvate and13C-acetate can be hyperpolarization and enter at the same time as expecting the imaging medium containing hyperpolarizability13C-pyruvate and hyperpolarizability13C-acetate, gives a more accurate and complete determination of PDH activity.

Anatomical and/or, where appropriate, perfusion information may be included in the method according to the invention when performing in vivo. Anatomical information may be, for example, obtained by removing a proton or13C-Mr image with or without the use of a suitable contrast agent before or after a method according to the invention.

Wednesday Mr-imaging, containing malate and hyperpolarizability13C-pyruvate, as discussed above, is new, therefore, in another aspect of the invention proposed environment Mr-imaging, containing malate and hyperpolarizability13C-pyruvate.

In addition, the imaging medium containing hyperpolarizability13C-pyruvate and hyperpolarizability13C-acetate, as discussed above, is new, therefore, in another aspect of the invention proposed environment Mr-imaging, containing13C-pyruvate and hyperpolarizability13C-acetate.

As mentioned and discussed in detail above, the environment Mr imaging according to the invention, i.e. with the food Mr-imaging, containing malate and hyperpolarizability13C-pyruvate, and environment Mr-imaging, containing13C-pyruvate and hyperpolarizability13C-acetate, can be used in the method of determining the activity of PDH by13C-Mr detection.

The imaging medium according to the invention can be used as the display environment in vivo, i.e. in living humans and animals, other than humans. For this purpose, the imaging medium is proposed in the form of a composition suitable for introduction into the body of a living person or animal other than man. Such an imaging medium preferably contains, in addition to the Mr active agent13C-pyruvate or13C-pyruvate and13C-acetate or malate and Mr active agent13C-pyruvate, water carrier, preferably a physiologically tolerable and pharmaceutically acceptable aqueous carrier, such as water, buffer solution or saline. Such an imaging medium may further comprise conventional pharmaceutical or veterinary carriers or excipients, for example additives for preparations that are commonly used for diagnostic compositions in human or veterinary medicine.

In addition, the imaging medium according to the invention can be used as the display environment in vitro, i.e. in cellular cool the rounds, samples from the body, such as blood samples, tissues ex vivo, such as tissue after biopsy, or isolated organs. For this purpose, the imaging medium is proposed in the form of compositions suitable for adding, for example, cell cultures, blood samples, tissues ex vivo, such as tissue after biopsy, or isolated organs. Such an imaging medium preferably contains, in addition to the Mr active agent13C-pyruvate or13C-pyruvate and13C-acetate or malate and Mr active agent13C-pyruvate, the solvent, which is compatible with the cell or tissue in vitro, or which is used, for example DMSO or methanol, or a mixture of solvents containing an aqueous medium and a nonaqueous solvent, for example a mixture of DMSO and water or buffer solution or methanol and water or buffer solution. As is obvious to a specialist in the art, pharmaceutically acceptable carriers, excipients and additives for preparations can be present in such environments, visualization, but not required for this purpose.

A brief description of graphic materials

Figure 1 shows a comparison of the relationship of the amplitude peak13C-bicarbonate to13C-pyruvate to ("STZ-pre") and after ("STZ-post") injections streptozotocin (STZ) rats for induction model of type 1 diabetes. "*" indicates p=0.01.

On Fig shows the effect of starvation ("erh") on the ratio of the peak amplitude 13C-bicarbonate to13C-pyruvate in rats. "**" indicates p<0,0001.

Figure 3 shows the change in the peak amplitude13C-bicarbonate to13C-pyruvate-time (2 weeks and 4 weeks) in rats on a diet high in fat ("HFF") compared to the basal level. "#" indicates p<0,002, "##" indicates p<0,005.

Figure 4 shows the ratio of active/total PDH (%) fed rats ("power"), the hungry rats (control fasting"), rats on a diet with a high fat diet (High Fat Fed") and rats with diabetes ("STZ").

Figure 5 shows the correlation between the activity of PDH, measured on the heart tissue ex vivo Protocol described previously by the authors Seymour et al (Seymour, A.M. & Chatham, J.C. (1997) J Mol Cell Cardiol 29, 2771-2778), and determination of PDH activity according to the method according to the invention by measuring the relationship of the amplitude peak13C-bicarbonate to13C-pyruvate.

Figure 6 shows the upper part of the active average Mr spectra taken in rats before ("basal") and after the induction of hyperthyroidism (7 days - T3) and in comparison with Mr-spectra taken in the control group (7 days control). In the lower part 6 shows a comparison of the relationship of the amplitude peak13C-bicarbonate to13C-pyruvate on day 7 group C disease (T3) and control group (Control) (grey triangles) compared with the basal level (black diamonds).

p> 7 shows a comparison of the relations13C-bicarbonate to13C-pyruvate from eating rats and starving rats after injection hyperpolarizing13C-pyruvate (light gray columns) or a mixture hyperpolarizing13C-pyruvate and malate (dark gray columns).

Examples

Further, the terms pyruvate,13C-pyruvate and13C1-pyruvate are used interchangeably, and all they mean13C1-pyruvate. Similarly, the terms pyruvic acid,13C-pyruvic acid and13C-pyruvic acid are used interchangeably, and all they mean13C1-pyruvic acid.

Example 1. Getting the display environment containing hyperpolarizability13With1-pyruvate obtained by way of the DPJ

Tris(8-carboxy-2,2,6,6-(Tetra(hydroxyethyl)-benzo-[1,2-4,5']-bis(1,3)dithiol-4-yl)-methyl-sodium salt (trailovic radical), which is synthesized in accordance with example 7 of WO-A1-98/39277, was added to the13C-pyruvic acid (40 mm) in vitro with obtaining a composition comprising 15 mm trifilova the radical. Next was preparing an aqueous solution of Gd-chelate of 1,3,5-Tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-were)-[1,3,5]triazine-2,4,6-trione (ion of a paramagnetic metal), which is synthesized in accordance with example 4 of WO-A-2007/064226, and 0.8 µl (14,6 mm) was added in PR the tag with 13C1-pyruvic acid and trifilova radical.

The composition is transferred from the tube into the cell, and this cell for the sample was made in the DPJ-polarizer. The composition was subjected to polarization in terms of the DPJ at 1.2 K in a magnetic field at 3.35 T under microwave irradiation (93,89 GHz) for 45 minutes

Then the composition was dissolved in aqueous sodium hydroxide solution, Tris buffer, and EDTA at a pressure of 10 bar (1 MPa) and a temperature of 170°C. the resulting imaging medium contained 80 mm hyperpolarizing13C1-sodium pyruvate at pH of 7.2 to 7.9 with a polarization of about 30% at the time of introduction.

Example 2. Determination of PDH activity according to the method according to the invention in animal models of the disease diabetes

This study included three groups of male Wistar rats to study as diabetes type I, or insulin resistance, pre-diabetes type II.

Source PDH activity (basal level) was determined in the first group of 6 rats according to Example 3. Then all the rats induced type I diabetes using a single intraperitoneal injection of freshly prepared streptozotocin (STZ; 50 mg/kg body weight) in 50 mm cold citrate buffer (pH 4.5). Five days after the induction of STZ-diabetes again determined the activity of PDH, where each rat served as its own experimental to what stralem. Comparison of the relationship of the amplitude peak13C-bicarbonate to13C-pyruvate before and after the injection of STZ clearly shows the reduction of the specified relation and, consequently, a decrease in PDH activity (Figure 1).

Then rats were restored and were killed after 1 h using intraperitoneal injection of pentobarbital sodium for analysis of tissue preparations and blood plasma. Heart, lung, liver and soleus and gastrocnemius muscles were quickly excised, immediately frozen using chilled N2aluminum tongs, and kept at -80°C for subsequent analysis. Approximately 3 ml of blood was collected from the thoracic cavity after cutting the heart. The blood was immediately centrifuged (3200 rpm for 10 min at 4°C) and separated plasma. Was separated by a 200 μl aliquot of plasma was added inhibitor of lipoprotein lipase activity tetrahydrolipstatin (THL) for analysis neeterificirovannah fatty acids (nezhk). All plasma samples were immediately frozen and kept at -80°C. AWH Pentra 400 (Horiba ABX Diagnostics, Montpelier, France) was used for analysis on the plasma glucose, nezhk (Wako Diagnostics, Richmond, USA) and 3-p-hydroxybutyrate (Randox, Co. Antrim, UK). The plasma insulin was measured using enzyme-linked immunosorbent assay (ELISA) for rat insulin (Mercodia, Uppsala, Sweden).

The second group of rats (n=12) were divided into 2 subgroups and each subgroup was determined the original PDH activity (basal level) according to P is the iMER 3.

The first subgroup ("erh") fasted overnight before each determination of the activity of PDH, and food was removed at 18.00 hours on the day prior to determination. This corresponded to starvation for 14-18 h after removal of food. The effect of fasting on the ratio of the peak amplitude13C-bicarbonate to13C-pyruvate is shown in figure 2.

In the second subgroup (after feeding) PDH activity was determined in the state after feeding food produced with free access. After defining the basal level of activity of PDH all of the rats were restored and were killed after 1 h for analysis of tissue preparations and plasma, as described above.

In the third group of rats (n=7) PDH activity was determined according to Example 3 at 3 points in time: original PDH activity (basal level), 2 and 4 weeks. After the initial determination of PDH activity (basal level) of all 7 rats were placed on a diet high in fat, where 55% of the calories is a saturated fat, for the induction model of metabolic syndrome, pre-diabetes type 2. Food has always been freely available. After determining the activity of PDH in time 4 weeks the rats were restored and were killed after 1 h for analysis of tissue preparations and levels of metabolites in plasma, as described above. Figure 3 shows the change in the peak amplitude13C-bicarbonate to3 C-pyruvate in time.

Fabric hearts from all animals were analyzed to determine the active and total fractions of the PDH enzyme (PDHaand PDHtaccording to the Protocol described previously Seymour et al. (Seymour, A.M. & Chatham, J.C. (1997) J Mol Cell Cardiol 29, 2771-2778). Figure 4 shows the proportion of enzyme PDH in the active form. There is strict compliance with the results of the activity of PDH, which are measured on the basis of the ratio between the amplitudes of peaks13C-bicarbonate to13C-pyruvate in all three groups. This is further confirmed by figure 5, which shows a strong correlation between PDH activity measured ex vivo in tissue of the heart and measured on the basis of the ratio between the amplitudes of peaks13C-bicarbonate to13C-pyruvate.

Example 3.13C-Mr detection

Example 3A. Preparation of animals

All rats were subjected to anesthesia using isoflurane (2% in oxygen), and kept in heated bedding. Watched so that the body temperature was maintained at 37°C. In tail vein injected with a catheter, and then the rats were placed in a hand-crafted system holding the animal. Monitored ECG, breathing rate and body temperature and provide air heating. The state of anesthesia was maintained by izoflurana (1.7%)are delivered through a nose cone.

Example 3b. Introduction and dosing hyperpolarizing13C-Perova is and

1 cm3the display environment, prepared in Example 1 were injected with under anesthesia, the rat within 10 through the catheter tail vein.

Example 3b.13With Mr imaging/spectroscopy

Handcrafted1H/13C the triangular coil was installed over the chest of the rat, localizing the signal from the heart. Rats were placed in a 7 T MRI scanner in the form of a horizontal cylinder associated with use interface with remote control Varian Inova. The correct position was confirmed by removing the axial proton flash image (TE/TR=1,17/2,33 MS, matrix size = 64×64, FOV (field-of-view) = 60×60 mm, slice thickness = 2.5 mm, the deflection angle of the magnetization vector of the excitation = 15°). The adjustment of the synchronization of the heart was performed to reduce the width of the proton band up to about 120 Hz.

Immediately before injection initiated synchronized ECG13C-Mr pulse sequence spectroscopy. 60 individual cardiac spectra was taken 1 minute after injection (TR=1 s, the deviation angle of the vector magnetization excitation = 5°, swing = 6000 Hz, shot points = 2048, frequency centered at the signal pyruvate).

A series of cardiac spectra13C-Mr) were analyzed using the AMARES algorithm performed jMRUI software (Naressi et al., Computers in Biology and Medicine, 31(4), 2001, 69-286 and Naressi et al., Magnetic Resonance Materials in Physics, Biology and Medicine, 12(2-3), 2001, 141-152). The spectra were combined, and then the baseline and DC corrected on the basis of the last half of the shot points. Peaks corresponding to pyruvate and bicarbonate were approximatively with previous data to fit the shape of the Lorenz curve, the frequencies of the peaks, the relative phases and widths of the bands.

The maximum peak area of pyruvate was calculated for each series of spectra and used to calculate the maximum ratio of bicarbonate/pyruvate. This helped to normalize variations in polarization between each data series. Also calculate the parameters describing the kinetics of progression bicarbonate, namely the time before appearing, time to peak and time decay to half of the maximum.

Example 4. Determination of PDH activity according to the method according to the invention in animal models of the disease hyperthyroidism

This study included twelve male Wistar rats (2 groups of 6) to study the effects of hyperthyroidism on cardiac metabolism.

Source PDH activity (basal level) was determined in all rats according to Example 3. Then hyperthyroidism was induced in 6 rats 7 daily intraperitoneal injection of freshly prepared triiodthyronine (T3; 0.2 mg/kg body weight/day). The other six rats, which served as controls, p who were given 7 daily intraperitoneal injections of saline water (0.9 per cent). After 7 days the introduction of the T3 again determined the activity of PDH in each of the 12 rats according to the method according to the invention. The ratio of the peak amplitude13C-bicarbonate to13C-pyruvate was compared in rats, which were injected T3 versus control rats both at the basal level and on day 7. The results clearly show that the introduction of T3 causes a decrease of the ratio of the peak amplitude13C-bicarbonate to13C-pyruvate, and this gives a reduction in the activity of PDH (6).

Rats were killed 24 hours later by intraperitoneal injection of sodium pentobarbital to obtain tissue preparations. Hearts were quickly excised and cut into two approximately equal halves. One half was immediately frozen using chilled N2aluminum tongs, and kept at -80°C for subsequent biochemical analysis. Intact mitochondria were isolated from the other half of the heart and used to assess mitochondrial function.

Example 5. Determination of PDH activity according to the method according to the invention using the display environment containing malate and hyperpolarizability13C-pyruvate

Six male Wistar rats were studied in each of the 4 experimental conditions to determine whether the infusion hyperpolarizing13C-pyruvate non-invasive way to assess the nature of the regulation of PDH.

In this example, the imaging medium, with whom containing a series of malate and hyperpolarizability 13C-pyruvate, used to determine the nature of the regulation of PDH. Stream PDH can be inhibition or inactivation of the enzyme complex PDK, or instantly through inhibition of the final product. Demonstrated that the enhanced relationship NADH/NAD+or acetyl-CoA/COA reduce PDH-mediated oxidation of pyruvate and, of course, the availability of oxaloacetate to include acetyl-CoA in the Krebs cycle is the main determinant of intramitochondrial concentrations of acetyl-CoA. Malate is an intermediate connection oxidative metabolism of glucose can enter the Krebs cycle through anaplerotic sequence of reactions with an increase in the overall carbon flow. Suggested that the use of the display environment containing malate and hyperpolarizability13C-pyruvate, it is possible to reduce the degree of inhibition of PDH final product. In cases of high activity of PDH he should increase the flow of pyruvate via the enzyme complex, as determined by detection13C-bicarbonate using13C-MP. Suggested that in rats on an empty stomach due to the already very low activity of PDH inhibition of the final product was insignificant, and that the joint infusion of malate should not affect detected education13C-bicarbonate.

Each of 6 rats was investigated in compliance and Protocol, described in Example 3, in the fed state and on an empty stomach (to modulate the activity of PDH), with 40 µmol one hyperpolarizing13C-pyruvate and 40 µmol hyperpolarizing13C-pyruvate, induzirovannomu together with 40 μmol of malate (for manipulation of the flow of the Krebs cycle/capture acetyl-CoA). The imaging medium containing hyperpolarizability13C-pyruvate or malate and hyperpolarizability13C-pyruvate, infusional through the tail vein of the rats in the Mr-scanner and removed the heart spectra every second for 1 min were Detected signals13C-pyruvate and13C-bicarbonate, monitored conversion13C-pyruvate in13C-bicarbonate, and the ratio of pyruvate to bicarbonate used as a marker of PDH flux.

Infusion of the rendering environment containing malate and hyperpolarizability13C-pyruvate, increased the flow of PDH by 32% compared to the imaging medium containing one hyperpolarizability13C-pyruvate, indicating that removal of the acetyl-CoA inclusion in the Krebs cycle increases the flow of PDH. The PDH flux was 55% lower in fasting rats, which were injected with one hyperpolarizability13C-pyruvate, compared with rats in the fed state and is not changed when using the display environment containing malate and hyperpolarizability13C-piruw is so Here the low activity of PDH prevent further flow of the enzyme. These results, shown in Fig.7, confirm that the inhibition of the final product restricts the flow of PDH in the fed state, whereas the activity of PDH regulates the oxidation of pyruvate in the fasting state. In conclusion, in this study we obtained evidence that hyperpolarizability Mr can be useful for obtaining detailed information about metabolic regulation than only obtaining information about the state of metabolism.

1. The method of determining changes in activity piruvatdegidrogenazy complex (PDH complex) we want to study the subject by13C-Mr detection (magnetic resonance-based detection of isotope13C) using the display environment containing hyperpolarizability13C-pyruvate, which detect the signal13C-bicarbonate or a signal13C-bicarbonate and a signal13C-pyruvate, where specified hyperpolarizability13C-pyruvate selected from the group consisting of hyperpolarizing13C1pyruvate,13C1,2pyruvate,13C1,3pyruvate or13C1,2,3pyruvate, or any combination thereof.

2. The method according to claim 1, wherein detect signals13C-bicarbonate and13With pyruvate.

3. The method according to claim 1 or 2, predstavlyayushie is a method of determining changes in the activity of the PDH complex in vivo human or animal, other than human.

4. The method according to claim 1 or 2, representing a method of determining changes in the activity of the PDH complex in vitro in cell culture, in samples of body tissues ex vivo, or in a stand-alone body, originating from human or animal other than man.

5. The method according to claim 1, where the specified signal or signals used to generate a metabolic profile.

6. The method according to claim 5, which is carried out in vivo or in vitro, and where the resulting information is used in assessing the effectiveness of potential drugs that modify the activity of the PDH complex.

7. The method according to claim 5, which is carried out in vivo or in vitro, and where the resulting information is used to assess response to treatment and/or to determine the efficacy of treatment in patients undergoing treatment for their disease.

8. The method according to claim 5, which is carried out in vivo or in vitro, and where the resulting information is used to identify patients at risk of developing the disease and/or candidates for preventive measures to avoid the development of disease.

9. The method according to claim 5, which is carried out in vivo or in vitro, and where the resulting information is used for the early detection of diseases.

10. The method according to claim 5, which is carried out in vivo or in vitro, and where the resulting information is used to monitor progressio the project for a disease, determine the severity of the disease or for the identification and assessment of complications associated with the disease.

11. The method according to claim 1, where the specified imaging medium further comprises malate.



 

Same patents:

FIELD: medicine.

SUBSTANCE: method for describing indications for choosing a conservative therapeutic approach to the patients suffered recurrent myocardial infarction involves pre-therapeutic patient's blood examination to analyse blood serum for oxidation-resistant lipoproteids (ORLs), to analyse erythrocytes for the pyruvic acid concentration (PAC), and if the ORL value is equal to 1.89 nmole MDA/mg of protein of β-lipoprotein and lower, while the PAC value is equal to 1.81 mmole/l and higher, the conservative therapy is added with the preparations of lipoic acid.

EFFECT: method is simple, has a broad information value and allows a more objective assessment of the patient's state and identifying a group of the patients in need of the conservative treatment and suffered recurrent myocardial infarction by the preparations of lipoic acid.

3 ex

FIELD: medicine.

SUBSTANCE: taken venous blood is separated into two samples. The first sample is stabilised with a solution of sodium citrate, the second one - with ethylene diamine tetraacetate. The first sample of whole blood is added with adenosine diphosphate as an aggregation inducer and tested for a peak amplitude of thrombocyte aggregation and a peak amplitude of adenosine triphosphate release profile by impedance method. The second sample is used to measure a fraction of thrombocytes and a fraction of blood corpuscles. It is followed by calculating a thrombocyte aggregation potential index by formula I=LmaxPTCΩmaxHTC100%; wherein Lmax is the peak amplitude of adenosine triphosphate release profile, Ωmax is the peak amplitude of thrombocyte aggregation, PCT is the fraction of thrombocytes, HTC is the fraction of blood corpuscles. If the value I is less than 0.5%, the low clinical effectiveness of the antiaggregant therapy is stated, and the value I being 1.5-2.5% shows the high effectiveness thereof.

EFFECT: improving the objective estimation of the clinical effectiveness of the antiaggregant therapy in the patients with acute coronary syndrome, and providing an opportunity for predicting the clinical course of the disease.

1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine. A composition for stimulating the skin stem cell production containing interleukin-1 alpha and a dermatologically acceptable diluent or carrier.

EFFECT: invention provides improving the stem cell stimulation.

2 ex

FIELD: chemistry.

SUBSTANCE: method involves dissolving 855 mg of a crystalline hydrate of copper chloride (CuCl2·2H2O) in 100 ml of distilled water (concentration of Cu2+ ions in the prepared solution is 50 mmol/l) and adding 1 ml of the prepared solution to 100 ml of a standard reagent used in glucose oxidase test. The ascorbic acid oxidant used is copper chloride solution in end concentration in the glucose oxidase reagent of 500 mcmol/l.

EFFECT: method enables correct determination of glucose content.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: workers' blood serum is analysed for interleukin 4, protein S-100β, protein S-100 autoantibodies, voltage-dependent Ca-channel autoantibodies, glutamate receptor autoantibodies, γ-aminobutyrate receptor autoantibodies, dopamine receptor autoantibodies; diagnostic coefficients F1 and F2 are calculated; if the value F1 is less than F2, the early changes of the nervous system are diagnosed for the chronic exposure to vinyl chloride; F1 more or equal to F2 enables stating the absence of any signs of the chronic exposure to vinyl chloride. The developed method may be used in the periodic medical screenings, medical examinations of workers to diagnose some occupational diseases.

EFFECT: use of the invention improves higher accuracy of identifying the various signs of the chronic exposure to vinyl chloride through the use of a complex of the immunological structures of nerve tissue in the chronic exposure to vinyl chloride.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention may be used to predict a developing myocardial dysfunction in the children with acute lymphoblastic leukemia (ALL) at different stages of polychemotherapy (PCT). The method involves the blood examination for the iron metabolism parameters, namely before the beginning of polychemotherapy (1) and after the induction of remission (2), blood serum ferritin, hepcidin and iron are evaluated in the patients; the derived values are inserted into the equations to calculate varying ECG, IMS, B(E-Ea) NT-pro-BNP after the completion of the intensive PCT course (3) and the total coefficient K is calculated by formula K=ECG3* IMS3* B(E-Ea)3* NT-pro-BNP3, wherein a probability of the myocardial dysfunction is stated by the total coefficient, namely: the coefficient K> 0.24 ensures predicting the developing cardiac complications, while K <0.24 show a lower risk of the cardiac complications.

EFFECT: possibility to detect a risk of the developing myocardial dysfunction accompanying the early PCT by the biochemical parameters, namely in terms of iron metabolism.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method consists in determining a characteristic profile of a test sample of a human biological fluid. It is concentrated off-line. Biologically active substances are separated using complexing additives, and a 'reference' is determined. Steroid hormones are taken as the analysed biologically active substances. The hormones are separated by performed by reversed-phase HELC in gradient elution using a diode array detector. The steroid profiles are used to form a matrix of the analytical signal intensities and the retention factors of each steroid. Each sample is graphically imaged by method of principal components, and the graphical images are used to form 'reference' and deviation clusters. The 'reference' and deviation clusters are corrected by soft independent modelling of class analogy taken as a reference. The pathologies are diagnosed by an ability of the patient's image to come with a 'reference' or a deviation.

EFFECT: reliable diagnosis of the pathologies associated with adrenal cortical diseases.

6 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for prediction the efficacy of the anti-TNF therapy in the patients with rheumatoid arthritis on the basis of genetic typing the polymorphisms of TNF-alpha proinflammatory cytokine. The allelic polymorphism of the TNF-alpha gene promoter is studied in position 857. If the heterozygous state (genotype - 857ST) or the homozygous T allele carriers (genotype - 857TT) is identified, a high probability of the successful infliximab therapy is predicted. If identifying the homozygous allele C carrier in position - 857 of the TNF-alpha gene promoter (genotype - 857SS), a high probability of the failed infliximab therapy is predicted.

EFFECT: invention enables the rapid and effective prediction of the clinical outcome of the anti-TNF therapy in the patients with rheumatoid arthritis by one polymorph position.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: menopausal women with an endometrial hyperplastic type having complaints about spotting undergo biopsy of the lining of the uterus to determine endometrial progesterone and testosterone, if observing progesterone falling within the range of 2.0 to 7.0 ng/g of tissue and testosterone falling within the range of 4.0 to 8.8 ng/g of tissue, developing endometrial cancer is predicted, while progesterone within 24.0 to 29.6 ng/g of tissue and testosterone within 16.8 to 22.4 ng/g of tissue enable predicting developing uterine fibroid. The technical and economic effectiveness of the method consists in the fact that the detected levels of progesterone and testosterone in the intact endometrial tissue in the menopausal patients with a hyperplastic type are high-information laboratory indicators of the presence of either malignant, or benign uterine pathology, which can be used to form the groups of patients with the high risk of malignancy in the body of the uterus.

EFFECT: method is available, quick to implement.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: laboratory diagnostic technique for the small-dose poisoning with organophosphorous agents consists in assessing catalytic activity of blood plasma aryl esterase heated to 55°C for 5 min with indophenyl acetate used as a substrate. Catalytic activity of the enzyme and its increase after heating are assessed once after a contact with a organophosphorus agent; the effect of increasing catalytic activity of blood plasma aryl esterase after heating is expressed in %, taking catalyst activity of blood plasma aryl esterase before heating as 100%. In case activity of plasma aryl esterase after heating is increased by more than 20%, the small-dose poisoning with organophosphorus agents is diagnosed.

EFFECT: use of the declared technique enables stating effectively the fact of the small-dose poisoning with OFAs in the absence of any clinical signs of poisoning.

1 tbl, 2 ex

FIELD: medicine, hepatology.

SUBSTANCE: one should detect the level of hepato-specific enzymes (HSE) in blood plasma, such as: urokinase (UK), histidase (HIS), fructose-1-phosphataldolase (F-1-P), serine dehydratase (L-SD), threonine dehydratase (L-TD) and products of lipid peroxidation (LP), such as: dienic conjugates (DC), malonic dialdehyde (MDA). Moreover, one should detect the state of inspecific immunity parameters, such as: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, one should evaluate the state of regional circulation by applying rheohepatography (RHG), the system of microhemocirculation with the help of conjunctival biomicroscopy (CB) to detect intravascular index (II). In case of increased UK, HIS levels up to 0.5 mcM/ml/h, F-1-P, L-SD, L-Td, LP products, CIC by 1.5 times, higher IRI up to 2 at the norm being 1.0-1.5, altered values of regional circulation, increased II up to 2 points at the norm being 1 point, not more one should diagnose light degree of process flow. At increased level of UK, HIS up to 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 1.5-2 times, increased IRI up to 2.5, altered values of regional circulation, increased II up to 3-4 points one should diagnose average degree of process flow. At increased level of UK, HIS being above 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 2 and more times, increased IRI being above 2.5, altered values of regional circulation, increased II up to 5 points and more one should diagnose severe degree of process flow.

EFFECT: higher accuracy of diagnostics.

3 ex

FIELD: medicine, infectology, hepatology.

SUBSTANCE: in hepatic bioptate one should detect products of lipid peroxidation (LP), such as: dienic conjugates (DC), activity of antioxidant enzymes, such as: catalase (CAT)and superoxide dismutase (SOD). One should calculate by the following formula: C = DC/(SOD x CAT)x100, where DC - the content of dienic conjugates, SOD - activity of superoxide dismutase, CAT - activity of catalase. At coefficient (C) values being above 65 one should predict high possibility for appearance of cirrhosis, at 46-645 - moderate possibility and at 14-45 -low possibility for appearance of cirrhosis.

EFFECT: higher accuracy of prediction.

3 ex

FIELD: medicine, clinical toxicology.

SUBSTANCE: at patient's hospitalization one should gather the data of clinical and laboratory values: on the type of chemical substance, patient's age, data of clinical survey and laboratory values: body temperature, the presence or absence of dysphonia, oliguria being below 30 ml/h, hemoglobinuria, erythrocytic hemolysis, exotoxic shock, glucose level in blood, fibrinogen and creatinine concentration in blood serum, general bilirubin, prothrombin index (PTI), Ph-plasma, the state of blood clotting system. The state of every sign should be evaluated in points to be then summed up and at exceeding the sum of points being above "+20" one should predict unfavorable result. At the sum of "-13" prediction should be stated upon as favorable and at "-13" up to "+20" - prediction is considered to be doubtful.

EFFECT: higher accuracy of prediction.

2 ex, 3 tbl

FIELD: medicine, juvenile clinical nephrology.

SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.

EFFECT: higher efficiency of detection.

7 dwg, 1 ex, 6 tbl

FIELD: clinical medicine, pulmonology.

SUBSTANCE: one should carry out complex estimation of interleukin-1β) concentration in blood, saliva, bronchoalveolar liquid. Moreover, one should detect distribution coefficient (DC) for IL-1β as the ratio of IL-1β blood content to IL-1β salivary content. At increased IL-1β blood content by 10 times and more, by 2 times in saliva, unchanged level of bronchoalveolar IL-1β, at DC for IL-1β being above 1.0 one should predict bronchial obstruction. The method enables to conduct diagnostics of the above-mentioned disease at its earlier stages.

EFFECT: higher efficiency of prediction.

2 tbl

FIELD: medicine, diagnostics.

SUBSTANCE: the present innovation deals with genetic trials, with diagnostic field of oncological diseases due to analyzing DNA by altered status of gene methylation that take part in intracellular regulation of division, differentiating, apoptosis and detoxication processes. One should measure the status of methylation in three genes: p16, E-cadherine and GSTP1 in any human biological samples taken out of blood plasma, urine, lymph nodes, tumor tissue, inter-tissue liquid, ascitic liquid, blood cells and buccal epithelium and other; one should analyze DNA in which modified genes of tumor origin or their components are present that contain defective genes, moreover, analysis should be performed due to extracting and purifying DNA out of biological samples followed by bisulfite treatment of this DNA for modifying unprotected cytosine foundations at keeping 5-methyl cytosine being a protected cytosine foundation followed by PCR assay of bisulfite-treated and bisulfite-untreated genes under investigation and at detecting alterations obtained according to electrophoretic result of PCR amplificates, due to detecting the difference in the number and electrophoretic mobility of corresponding fractions at comparing with control methylated and unmethylated samples containing normal and hypermethylated forms of genes one should diagnose oncological diseases. The method provides higher reliability in detecting tumors, detection of remained tumor cells after operation.

EFFECT: higher efficiency of therapy.

1 cl, 3 dwg, 4 ex

FIELD: medicine, gastroenterology.

SUBSTANCE: one should carry out diagnostic studying, moreover, on the 5th -6th d against the onset of exacerbation in case of gastric and duodenal ulcerous disease one should detect the content serotonin, histamine and acetylcholine in blood, then during 2-3 wk one should conduct medicinal therapy to detect serotonin, histamine and acetylcholine level in blood again and at serotonin content being by 2-3 times above the norm, histamine - by 1.15-1.4 times above the norm and acetylcholine - by 20-45% being below the norm one should predict the flow of gastric and duodenal ulcerous disease as a non-scarring ulcer.

EFFECT: higher accuracy of prediction.

3 ex

FIELD: medicine.

SUBSTANCE: method involves taking blood from ulnar vein (systemic blood circulation) and from large vein of the injured extremity proximal with respect to lesion focus (regional blood circulation). Spontaneous NST-test value is determined and difference is calculated in systemic and regional blood circulation as regional-to-systemic difference. The difference value is used for predicting clinical course of pyo-inflammatory disease in extremities.

EFFECT: high accuracy of diagnosis.

4 cl, 2 tbl

FIELD: medicine, gastroenterology.

SUBSTANCE: one should introduce biologically active substance, moreover, in patient's blood serum one should detect the content of acetyl choline and choline esterase activity followed by 2-h-long intragastric pH-metry at loading with biologically active substance as warm 40-45%-honey water solution at 35-40 C, and at increased content of acetyl choline being above 1.0 mM/l, choline esterase being above 0.5 mM/l/30 min and pH level being 6.0-6.9 it is possible to consider apitherapy to be useful for treating ulcerous duodenal disease.

EFFECT: higher efficiency and accuracy of detection.

3 ex

FIELD: medicine, gastroenterology.

SUBSTANCE: it has been suggested a new method to detect pharmacological sensitivity to preparations as acidosuppressors. After the intake of the preparation a patient should undergo fibrogastroduodenoscopy 3 h later, then, through endoscopic catheter one should introduce 0.3%-Congo red solution intragastrically and the test is considered to be positive at keeping red color that indicates good sensitivity to the given preparation, and in case of dark-blue or black color the test is considered to be negative that indicates resistance to this preparation. The suggested innovation widens the number of diagnostic techniques of mentioned indication.

EFFECT: higher efficiency of diagnostics.

2 ex

Up!