WO2007088446A2 - A process and a reagent kit for the examination of the metabolic state related to the carbohydrate and lipid metabolism of a human organism - Google Patents
A process and a reagent kit for the examination of the metabolic state related to the carbohydrate and lipid metabolism of a human organism Download PDFInfo
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- WO2007088446A2 WO2007088446A2 PCT/IB2007/000201 IB2007000201W WO2007088446A2 WO 2007088446 A2 WO2007088446 A2 WO 2007088446A2 IB 2007000201 W IB2007000201 W IB 2007000201W WO 2007088446 A2 WO2007088446 A2 WO 2007088446A2
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- Prior art keywords
- cells
- mitochondrial
- mitochondria
- nitric oxide
- insulin
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
Definitions
- the invention refers to a process and a reagent kit for the examination of the metabolic state related to the carbohydrate and lipid metabolism of a human organism i.e. for the detection of the healthy or ill state. Background of the invention
- the most frequent metabolic disease related to the carbohydrate and lipid metabolism of the human organism is diabetes mellitus the characteristic symptom of which includes high blood glucose level, however, in the background of the disease there is a complex disorder of the carbohydrate and lipid metabolism.
- diabetes mellitus the equilibrium of the metabolism that links up the continuous energy requirement of the organism with the periodical intake of food becomes overturned resulting in a limited adaptability of the metabolism.
- the change of the carbohydrate and lipid metabolic equilibrium is followed by a change of the level of key metabolites (e.g. glucose, triglyceride, cholesterols) and hormones that regulate the metabolism (e.g. insulin, adiponectin) leading to the development of secondary pathologic transformations.
- key metabolites e.g. glucose, triglyceride, cholesterols
- hormones that regulate the metabolism e.g. insulin, adiponectin
- a wide-spread pathological state related to the disorder of the carbohydrate and lipid metabolism of the human organism is a reduced insulin sensitivity i.e. insulin resistance.
- insulin resistance i.e. insulin resistance.
- the effect of insulin produced by the beta cells of the islets of Langerhans in the pancreas is lower than in healthy persons i.e. the patients have insulin resistance [Hinsworth, HP. and Kerr, R.B.: Insulin-sensitive and insulin-insensitive types of diabetes mellitus, Clin. Sci., 4, 119-152 (1939)].
- hyperinsulinaemia provides for normal glucose level and a somewhat reduced glucose tolerance.
- hyperglycaemia is developed [Beck-Nielsen, H. and Horten-Nielsen, O.: Insulin resistance, Diabetes Annual, 4, 565-591 (1998)].
- a secondary hyperinsulinaemia contributes to the development of further pathological states such as hyperlipidemia, hypertension and endothelial disfunction.
- Insulin resistance developed either due to genetic reasons or obesity - results in an increase of insulin production and is either a primary cause of several diseases or makes the patients susceptible to the development of such diseases [Bonnadonna, R. et al.: Obesity and insulin resistance in man: a dose response study, Metabolism, 39, 452-459 (1990)].
- These diseases include e.g. type Il diabetes mellitus, insulin resistance syndrome, diabetes in pregnancy, lipoatrophic diabetes, hypertension, Rabson-Mendenhall's syndrome as well as arteriosclerotic cardiovascular diseases.
- certain states or diseases e.g.
- insulin resistance develops as a secondary process. Insulin resistance may exist for a long time before the development of a metabolic or vascular disease indicating clinical symptoms.
- Insulin action and insulin resistance diseases involving defects in insulin receptors, signal transduction, and the glucose transport effector system, Am. J. Med., 105(4), 331-345 (1998)]. Consequently, there is an important medical requirement to detect insulin resistance in patients having normal fasting blood glucose levels. In the existing clinical practice, insulin sensitivity is estimated by the following principal methods:
- Insulin sensitivity can be concluded based on the fasting blood insulin, glucose, triglyceride and cholesterol levels as well as body mass index, waist and hip circumferences.
- HOMA homeostasis model assessment index: fasting insulin level (in mU/L) x fasting glucose level (in mmol/L)/22.5.
- GTT glucose tolerance test: the dynamics of blood glucose level following a peroral or intravenous glucose load indicates the consumption of glucose by the human organism.
- a drawback of the method resides in the fact that, in addition to insulin sensitivity, also insulin secretion has an influence on glucose tolerance [Yellow, R. S. and Berson, S.A.: Plasma insulin concentrations in non-diabetic and early diabetic subjects. Determination by a new sensitive immuno-assay technique, Diabetes, 9, 524-532 (1960)].
- Insulin glucose ,,clamp the test person is treated by a continuous infusion of insulin (in steady-state hyper- insulinaemia), while the glucose infusion rate that stabilizes a constant normal blood glucose level (i.e. euglycaemia) is determined to characterize insulin sensitivity.
- the hypo- glycaemic effect of insulin is compensated by the infusion of glucose. Due to hyperinsulinaemia, endogenic insulin production as well as glucose production of liver are reduced to a practically negligible level, thus, the glucose introduced by infusion will be used by the tissues under the influence of the insulin introduced by also infusion.
- Insulin sensitivity is expressed by the amount of glucose absorbed (in mg glucose/minute/kg body mass) (M value).
- this method is a professionally mainly accepted process for the determination of insulin sensitivity.
- the value of M is higher than 8 mg glucose/minute/kg body mass [DeForenzo, R. et al.: Glucose clamp technique: a method for quantifying insulin secretion and resistance, Am. J. Physiol., 273. E214-E223 (1979)].
- the method is much more accurate than the ones listed above, however, it is invasive, a stress for the test person, extremely time consuming and laboursome, consequently, expensive. It is absolutely not suitable for the mass screening of the population.
- the object of the invention is to provide a simple process for the examination of the metabolic state related to the carbohydrate and lipid metabolism of the human organism, and, especially, for the determination of insulin sensitivity.
- Leukocytes or white blood cells are involved in protecting the body against foreign substances.
- the major subdivisions thereof include lymphocytes (B-lymphocytes or B-cells and T- lymphocytes or T-cells), granulocytes, monocytes and macrophages. According to the invention, either all the leukocytes are tested or more than one subdivisions thereof are tested jointly or one subdivision is tested separately.
- lymphocytes The number of lymphocytes is estimated to be about 10 12 in the human body. After a maturation process, lymphocytes are transferred from the central lymphatic organ to the periphery. Lymphocyte cells that do not contact antigenes are destroyed after some days. Lymphocyte cells contacting antigenes are activated and differentiate to form effector and memory cells. Memory cells remain for years, even during the whole length of life in the vascular system, consequently, a part of the lymphocytes has a significant lifetime and is influenced by the metabolic environment of the human body. Therefore, lymphocytes are especially suitable for the examination of the metabolic state related to the carbohydrate and lipid metabolism of the human body. In the first place, T- lymphocytes or T-cells are responsible for immunity. According to the invention it is especially preferred to determine the number and characteristics of mitochondria that are present in the T-cells or the amount of nitric oxide produced in the T-cells.
- the mitochondrion is an essential organelle of the cell which occurs in varying number in the cytoplasm of every cell. That is the site of the cell's energy production. 98 % of the oxygen used by the human organism is applied by the mitochondria for energy production. Any damage of the mitochondria has a causal role in several diseases.
- the suitable mitochondrial number and function i.e. number and function of mitochondria
- the metabolic state related to the carbohydrate and lipid metabolism of the human organism can be well characterized by the mitochondrial number of leukocytes and/or one or more characteristic(s) describing the mitochondrial functions of leukocytes and/or the amount of nitric oxide produced in the cells/mitochondria of leukocytes.
- the mitochondrial number of leukocyte cells are determined in a manner known perse, preferably using a fluorescent dye that accumulates in the mitochondrion and determining the fluorescence.
- Preferred fluorescent dyes include the following ones:
- An especially preferred fluorescent dye for this purpose is MitoTracker Green FM Probe. Following a short incubation period at 37 0 C, this dye accumulates in the active mitochondria and exhibits a fluorescent emission at 516 nm following an excitation at 490 nm. Fluorescence is determined by a suitable apparatus e.g. FACS (fluorescence activated cell sorter). The intensity of fluorescence is proportional to the amount of the active mitochondria i.e. the number of mitochondria (mitochondrial number).
- adenosine triphosphate is produced using the energy of mitochondrial membrane potential (d ⁇ m ).
- mitochondrial membrane potential i.e. membrane potential of the mitochondria
- the principle of the determination of mitochondrial membrane potential is as follows: the leukocytes are incubated in the presence of a suitable fluorescent dye that accumulates in the active mitochondria. The accumulated fluorescent dye is present in either monomeric or aggregated form depending on the value of the mitochondrial membrane potential. After excitation, the two forms exhibit fluorescence of different wavelength.
- the value of mitochondrial membrane potential can be concluded based on the intensity of fluorescence of the aggregated form or the intensity ratio of the fluorescence of the two forms.
- Preferred fluorescence dyes for the determination of the mitochondrial membrane potential or the detectection of apoptosis include the following ones: JC-1 and JC-9: Dual-Emission Potential Sensitive Probes, Rhodamine 123,
- Rosamine and Rhodamine derivatives e.g. TMRM and TMRE, Reduced Rhodamine and Rosamine derivatives, Image-iT LIVE Mitochondrial Transition Pore Assay Kit for Fluorescence Microscopy,
- MitoProbe Transition Pore Assay Kit for Flow Cytometry SelectFX Alexa Fluor 488 Cytochrome Apoptosis Detection Kit, Antibodies against Mitochondrial Porin, Antibodies against Pyruvate Dehydrogenase.
- JC-1 manufactured by Molecular Probes. After incubation at 37 0 C for a short time, JC-1 accumulates in the active mitochondria. When the mitochondrial membrane potential is high, the dye forms an aggregate in the mitochondria. When the mitochondrial membrane potential is low, the dye remains in the monomeric form. Following an excitation at 490 nm, the wavelength of the fluorescent emission of the monomeric form is 530 nm while that of the fluorescent emission of the aggregated form is 590 nm. Again, fluorescence is determined by a suitable apparatus e.g. FACS (fluorescence activated cell sorter). The intensity of fluorescence is proportional to the membrane potential of mitochondria.
- FACS fluorescence activated cell sorter
- the metabolic state related to the carbohydrate and lipid metabolism of the mammalian organism can be also concluded based on the amount of ATP produced in the mitochondria.
- a reduction of glucose absoprtion stimulated by insulin as well as that of the ATP synthesis were observed in babies of parents having insulin resistance.
- This observation supported the assumption that intracellular fat and fatty acid that accumulated due to the reduced rate of mitochondrial ATP synthesis played a casual role in the development of insulin resistance [Petersen, K.F. et al.: Decreased insulin-stimulated ATP synthesis and phosphate transport in muscle of insulin- resistant offspring of type 2 diabetic parents, PIoS Med., 2(9), e233 (2005)].
- the ATP level of the cells can be determined by chemical methods or NMR (nuclear magnetic resonance) measurements in a manner known perse.
- the amount of mitochondrial DNA can be determined by PCR method in a manner known perse [Malakhova, L. et al.: The increase in mitochondrial DNA copy number in the tissues of gamma-irradiated mice, Cell. MoI. Biol. Lett., 10(4), 721-32 (2005)].
- the principle of the determination of the mitochondrial redox potential is that the value of the mitochondrial redox potential is proportional to the free radical production of mitochondria. When the value of mitochondrial redox potential is higher, there is an increase also in the production of free radicals.
- the production of free radicals in the mitochondria can be detected and determined by fluorescent dyes that are susceptible to free radicals. These dyes react with the free radical resulting in a change of the intensity or wavelength of the fluorescence of the dye. Other fluorescent dyes are susceptible to the redox state of the cells. In both cases, the chemical modification of fluorescent dyes results in a change of fluorescence.
- Preferred fluorescent dyes for these purposes include the following ones:
- calcium ions regulate the function of several enzymes that are present in the mitochondria.
- the calcium level is determined by using calcium dependent fluorescent dyes.
- Preferred fluorescent dyes for this purpose include the following ones: Rhod-2, Rhod-5N, Rhod-FF.
- nitric oxide Both function and genesis of mitochondria are regulated by nitric oxide. It is preferred to determine the amount of nitric oxide produced in the cells [Nisoli, E. et al.: Mitochondrial biogenesis as a cellular signaling framework, Biochemical Pharmacology, 67, 1 (2004); Nisoli, E.: Mitochondrial biogenesis in mammals: The role of endogenous nitric oxide, Science, 299, 896 (2003)].
- the basis of the determination is that a suitable fluorescent dye reacts with nitric oxide in a specific way and, after excitation, a fluorescent radiation is emitted by the formed product.
- Preferred fluorescent dyes for the determination of biologically active nitric oxide include the following ones:
- Especially preferred fluorescent dyes for the determination of biologically active nitric monoxide include the DAF dyes (manufactured by Molecular Probes). Following incubation with the leukocytes for a short time, the dye acumulated in the cells react, after the split of the ester bond, with nitric oxide specifically. A fluorescent radiation is emitted by the reaction product at 516 nm after an excitation at 490 nm. Fluorescence is determined by a suitable apparatus e.g. FACS (fluorescence activated cell sorter). The intensity of fluorescence is proportional to the amount of nitric oxide.
- FACS fluorescence activated cell sorter
- Pathological metabolic states related to the carbohydrate and lipid metabolism of the human organism i.e. the ill states of the body include the following ones:
- the invention is based on the recognition that the mitochondrial number or mitochondrial function of the leukocyte cells separated from the blood as well as the amount of nitric oxide produced in the leukocyte cells reflects the healthy or pathological metabolic state related to the carbohydrate and lipid metabolism of the human organism, consequently, the healthy or ill state of the individual examined can be concluded based on the determination of the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells and/or the amount of nitric oxide produced in the leukocyte cells. These determinations can be carried out in a very simple way. Only one conventional blood sample has to be taken from the individual to be examined, and the laboratory tests can be performed in a relatively short time with acceptable expenses.
- the process of the invention can be used for screening the population that can be affected by a pathological metabolic state related to the carbohydrate or lipid metabolism, furthermore for following the effect of treatment in case of treated patients suffering from a disease related to a pathological state of carbohydrate or lipid metabolism.
- the process of the invention can be also suitable for monitoring the state in an acute energy producing crisis e.g. in heavy infections.
- the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells separated from the blood sample of the individual to be examined and/or the amount of nitric oxide produced in the leukocyte cells are determined in a manner known perse, and, based on the values obtained, the state of the individual examined is evaluated.
- All the leukocytes or one or more subdivisions thereof e.g. T-lymphocytes can be separated from the blood sample taken from the individual to be examined and the cells thereof are used for the determinations.
- all the leukocytes can be separated, however, in the fluorescent determination of mitochondrial number and/or of one or more mitochondrial characteristic(s) and/or of the amount of nitric oxide only that/those of a given subdivision e.g. T-lymphocytes are taken into consideration.
- the mitochondrial number and/or one or more mitochondrial characteristics) and/or the amount of nitric oxide can be determined in more than one leukocyte subdivisions, too.
- one, two, three or still more characteristic(s) of the leukocyte cells or subdivisions thereof can be determined, wherein the characteristics are selected from the group consisting of mitochondrial membrane potential, production of ATP, amount of mitochondrial DNA, mitochondrial redox state, amount of free radicals in the mitochondrion, and calcium level in the mitochondrion.
- the parameters are selected from the group consisting of mitochondrial number, mitochondrial membrane potential, production of ATP, amount of mitochondrial DNA, mitochondrial redox state, amount of free radicals in the mitochondrion, calcium level in the mitochondrion, and amount of nitric oxide produced in the cells tested.
- one or more further parameter(s) can be used for the examination of the metabolic state related to the carbohydrate or lipid metabolism of the individual.
- the invention includes a reagent kit for carrying out the process of the invention, said reagent kit containing at least two fluorescent dyes for the determination of the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells and/or the amount of nitric oxide produced in the leukocyte cells.
- the reagent kit of the invention contains at least two fluorescent dyes selected from the group consisting of MitoTracker Green for the determination of the number of mitochondria, JC-1 for the determination of mitochondrial membrane potential and DAF for the determination of the biologically active nitric oxide.
- Figure 1 indicates the relationship between mitochondrial number and amount of nitric oxide.
- Figure 2 shows the relationship between mitochondrial membrane potential and amount of nitric oxide.
- the group of healthy individuals consisted of persons having the following parameters:
- the group of insulin resistant individuals consisted of persons having a disorder of glucose metabolism based on the following criteria:
- BMI index above 27, fat content of the body: higher than 30 % (DEXA), insulin glucose ,,clamp" method: in case of empty stomach, under the influence of insulin infusion, the absorbed amount of glucose referred to the lean body mass (M value) is lower than 8 mg glucose/minute/kg body mass.
- the group of diabetic individuals consisted of patients suffering from type Il diabetes mellitus. The patients were not treated with an insulin sensitizing drug and a part of them had compensated glucose metabolism.
- One of the antecubital veins of the test persons was cannulated to allow the administration of glucose and insulin infusions.
- the vein of the hand was cannulated in a retrograde manner while maintaining the hand in a warm state to take blood samples from the vein.
- a dose of 40 U of human insulin (Actrapid HM, Novo Nordisk, Gentofte, Denmark) was administered in a logaritmically decreasing manner within 10 minutes, then insulin was administered at a rate of 120 mU/m 2 /minute for 360 minutes.
- Glucose production in the lever was inhibited by the hyperinsulinaemia developed in this way during the test.
- the rate of infusion containing 20 % of glucose was regulated to obtain a constant blood glucose level of 5.5 mM.
- the average glucose infusion rate maintaining the euglycaemic blood glucose level in the last 30 minutes of the euglycaemic period was considered as a measure of insulin sensitivity.
- the blood glucose level was determined from 10 ⁇ l of blood sample taken in every 5 minutes.
- a blood sample of 1 ml volume was taken into a test tube containing heparin from each individual taking part in the examination.
- the blood sample was layered onto Ficoll- Pague ® Plus (a branched chain hydrophil saccharose polymer produced by Amersham) gradient and leukocytes were separated by centrifuging at 2500 rpm at 4 0 C.
- the leukocyte layer accumulated at the boundary layer was removed, the cells were sedimented by centrifuging at 1500 rpm for 5 minutes and the pellet of cells was suspended in 1 ml of PBS (physiological saline containing phosphate buffer). 100 ⁇ l portions of cell suspension were employed for the incubation with a fluorescent dye.
- T- lymphocytes were identified by means of anti-CD3 antibody and the characteristics tested below were evaluated only in the population of T-lymphocytes.
- Tables I to III indicate that each of the characteristics of T-lymphocytes allows the identification of insulin resistant individuals and diabetic patients.
- the test groups can be identified still more clearly.
- the mitochondrial number was depicted as a function of the amount of nitric oxide. It is apparent that the squares referring to the group of healthy individuals are localized at higher values of both characteristics, separately from the also localized triangles that refer to the group of insulin resistant individuals and from the localized circles referring to the group of patients suffering from type Il diabetes mellitus.
- the mitochondrial membrane potential was shown as a function of the amount of nitric oxide. It is obvious that especially the group of insulin resistant individuals and the group of healthy individuals can be well identified.
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Abstract
The invention refers to a process for the examination of the metabolic state related to the carbohydrate and lipid metabolism of a human organism, thus, for the detection of the healthy or ill state. According to the invention, the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells separated from the blood sample of the individual to be examined and/or the amount of nitric oxide produced in the leucocyte cells are determined in a manner known per se, and, based on the values obtained, the state of the individual examined is evaluated.
Description
A process and a reagent kit for the examination of the metabolic state related to the carbohydrate and lipid metabolism of a human organism
Field of the invention
The invention refers to a process and a reagent kit for the examination of the metabolic state related to the carbohydrate and lipid metabolism of a human organism i.e. for the detection of the healthy or ill state. Background of the invention
The most frequent metabolic disease related to the carbohydrate and lipid metabolism of the human organism is diabetes mellitus the characteristic symptom of which includes high blood glucose level, however, in the background of the disease there is a complex disorder of the carbohydrate and lipid metabolism. In diabetes mellitus, the equilibrium of the metabolism that links up the continuous energy requirement of the organism with the periodical intake of food becomes overturned resulting in a limited adaptability of the metabolism. As a consequence, the change of the carbohydrate and lipid metabolic equilibrium is followed by a change of the level of key metabolites (e.g. glucose, triglyceride, cholesterols) and hormones that regulate the metabolism (e.g. insulin, adiponectin) leading to the development of secondary pathologic transformations.
A wide-spread pathological state related to the disorder of
the carbohydrate and lipid metabolism of the human organism is a reduced insulin sensitivity i.e. insulin resistance. In about 90 % of the patients suffering from diabetes mellitus, the effect of insulin produced by the beta cells of the islets of Langerhans in the pancreas (such as the increase of glucose uptake, inhibition of gluconeogenesis etc.) is lower than in healthy persons i.e. the patients have insulin resistance [Hinsworth, HP. and Kerr, R.B.: Insulin-sensitive and insulin-insensitive types of diabetes mellitus, Clin. Sci., 4, 119-152 (1939)]. In case of compensated insulin resistance, the insulin production in the pancreas is enhanced resulting in hyperinsulinaemia that provides for normal glucose level and a somewhat reduced glucose tolerance. In the decompensated phase of insulin resistance, the insulin production is not sufficient for the compensation of insulin resistance, therefore, hyperglycaemia is developed [Beck-Nielsen, H. and Horten-Nielsen, O.: Insulin resistance, Diabetes Annual, 4, 565-591 (1998)]. A secondary hyperinsulinaemia contributes to the development of further pathological states such as hyperlipidemia, hypertension and endothelial disfunction.
A part of the population has insulin resistance, nevertheless, the members thereof cannot be considered as diabetic patients since their blood glucose level is within the normal range. Insulin resistance - developed either due to genetic reasons or obesity - results in an increase of insulin production and is either a primary cause of several diseases or makes the patients susceptible to the development of such
diseases [Bonnadonna, R. et al.: Obesity and insulin resistance in man: a dose response study, Metabolism, 39, 452-459 (1990)]. These diseases include e.g. type Il diabetes mellitus, insulin resistance syndrome, diabetes in pregnancy, lipoatrophic diabetes, hypertension, Rabson-Mendenhall's syndrome as well as arteriosclerotic cardiovascular diseases. In case of certain states or diseases (e.g. obesity, pregnancy, stress, type I diabetes mellitus, hyperlipidemia, cirrhosis, hyperthyroidism, kidney insufficiency etc.), insulin resistance develops as a secondary process. Insulin resistance may exist for a long time before the development of a metabolic or vascular disease indicating clinical symptoms. Several observations support that an increase of the fasting blood glucose level and the development of reduced glucose tolerance are preceded by a significant necrosis of the beta cells of the islets of Langerhans. Thus, the development of diseases related to insulin resistance especially type Il diabetes mellitus could be more effectively prevented by starting the treatment of insulin resistance in a state when the blood glucose level has been still in the normal range. [Hunter, S.J. and Garvey, W.T.: Insulin action and insulin resistance: diseases involving defects in insulin receptors, signal transduction, and the glucose transport effector system, Am. J. Med., 105(4), 331-345 (1998)]. Consequently, there is an important medical requirement to detect insulin resistance in patients having normal fasting blood glucose levels. In the existing clinical practice, insulin sensitivity is
estimated by the following principal methods:
Insulin sensitivity can be concluded based on the fasting blood insulin, glucose, triglyceride and cholesterol levels as well as body mass index, waist and hip circumferences.
HOMA (homeostasis model assessment) index: fasting insulin level (in mU/L) x fasting glucose level (in mmol/L)/22.5.
GTT (glucose tolerance test): the dynamics of blood glucose level following a peroral or intravenous glucose load indicates the consumption of glucose by the human organism. A drawback of the method resides in the fact that, in addition to insulin sensitivity, also insulin secretion has an influence on glucose tolerance [Yellow, R. S. and Berson, S.A.: Plasma insulin concentrations in non-diabetic and early diabetic subjects. Determination by a new sensitive immuno-assay technique, Diabetes, 9, 524-532 (1960)].
The methods listed above are rather inexact, the results obtained correlate with each other only to a low degree, therefore, the final issue is not much better when even more tests are carried out simultaneously.
Insulin glucose ,,clamp" method: the test person is treated by a continuous infusion of insulin (in steady-state hyper- insulinaemia), while the glucose infusion rate that stabilizes a constant normal blood glucose level (i.e. euglycaemia) is determined to characterize insulin sensitivity. The hypo- glycaemic effect of insulin is compensated by the infusion of glucose. Due to hyperinsulinaemia, endogenic insulin production as well as glucose production of liver are reduced to
a practically negligible level, thus, the glucose introduced by infusion will be used by the tissues under the influence of the insulin introduced by also infusion. Insulin sensitivity is expressed by the amount of glucose absorbed (in mg glucose/minute/kg body mass) (M value). At present, this method is a professionally mainly accepted process for the determination of insulin sensitivity. The value of M is higher than 8 mg glucose/minute/kg body mass [DeForenzo, R. et al.: Glucose clamp technique: a method for quantifying insulin secretion and resistance, Am. J. Physiol., 273. E214-E223 (1979)]. The method is much more accurate than the ones listed above, however, it is invasive, a stress for the test person, extremely time consuming and laboursome, consequently, expensive. It is absolutely not suitable for the mass screening of the population.
The object of the invention is to provide a simple process for the examination of the metabolic state related to the carbohydrate and lipid metabolism of the human organism, and, especially, for the determination of insulin sensitivity. Summary of the invention
It has been found that the number and various characteristics of mitochondria that are present in leukocyte cells separated from a blood sample as well as the amount of nitric oxide produced in the leukocyte cells can be used for the determination of the metabolic state related to the carbohydrate and lipid metabolism of the human organism, consequently, for the detection of the healthy or ill state.
Description of preferred embodiments
Leukocytes or white blood cells are involved in protecting the body against foreign substances. The major subdivisions thereof include lymphocytes (B-lymphocytes or B-cells and T- lymphocytes or T-cells), granulocytes, monocytes and macrophages. According to the invention, either all the leukocytes are tested or more than one subdivisions thereof are tested jointly or one subdivision is tested separately.
The number of lymphocytes is estimated to be about 1012 in the human body. After a maturation process, lymphocytes are transferred from the central lymphatic organ to the periphery. Lymphocyte cells that do not contact antigenes are destroyed after some days. Lymphocyte cells contacting antigenes are activated and differentiate to form effector and memory cells. Memory cells remain for years, even during the whole length of life in the vascular system, consequently, a part of the lymphocytes has a significant lifetime and is influenced by the metabolic environment of the human body. Therefore, lymphocytes are especially suitable for the examination of the metabolic state related to the carbohydrate and lipid metabolism of the human body. In the first place, T- lymphocytes or T-cells are responsible for immunity. According to the invention it is especially preferred to determine the number and characteristics of mitochondria that are present in the T-cells or the amount of nitric oxide produced in the T-cells.
Thus, in the description and claims, the expression Jeukocyte" or Jeukocyte cell" includes all the cells that can be
considered as a leukocyte, the cells of more than one subdivisions of leukocytes and the cells of one of the leukocyte subdivisions.
The mitochondrion is an essential organelle of the cell which occurs in varying number in the cytoplasm of every cell. That is the site of the cell's energy production. 98 % of the oxygen used by the human organism is applied by the mitochondria for energy production. Any damage of the mitochondria has a causal role in several diseases. Thus, the suitable mitochondrial number and function (i.e. number and function of mitochondria) are a principal condition in the normal life of the human organism.
It was found that the metabolic state related to the carbohydrate and lipid metabolism of the human organism can be well characterized by the mitochondrial number of leukocytes and/or one or more characteristic(s) describing the mitochondrial functions of leukocytes and/or the amount of nitric oxide produced in the cells/mitochondria of leukocytes.
The main characteristics that describe the mitochondrial functions include the following ones:
- mitochondrial membrane potential,
- production of ATP (adenosine triphosphate),
- amount of mitochondrial DNA,
- mitochondrial redox state,
- amount of free radicals in the mitochondrion,
- calcium level in the mitochondrion.
The mitochondrial number of leukocyte cells are determined
in a manner known perse, preferably using a fluorescent dye that accumulates in the mitochondrion and determining the fluorescence. Preferred fluorescent dyes include the following ones:
Orange-, Red- and Infrared-Fluorescent MitoTracker Dyes,
MitoTracker Green FM Probe,
MitoFluor Green Probe,
Long-Wavelength MitoFluor Red Probes,
Carbocyanines,
Styryl Dyes,
Nonyl Acridine Orange,
Carboxy SNARF-1 pH Indicator,
CoroNa Red Chloride,
Lucigenin,
Avidin Conjugates for staining mitochondria,
Antibodies to Mitochondrial Proteins.
An especially preferred fluorescent dye for this purpose is MitoTracker Green FM Probe. Following a short incubation period at 37 0C, this dye accumulates in the active mitochondria and exhibits a fluorescent emission at 516 nm following an excitation at 490 nm. Fluorescence is determined by a suitable apparatus e.g. FACS (fluorescence activated cell sorter). The intensity of fluorescence is proportional to the amount of the active mitochondria i.e. the number of mitochondria (mitochondrial number).
In the mitochondrion, adenosine triphosphate (ATP) is produced using the energy of mitochondrial membrane
potential (dΨm). Both the increase and decrease of the mitochondrial membrane potential (i.e. membrane potential of the mitochondria) are detrimental to the cell: the increase results in an enhancement of superoxide production while the decrease results in insufficient energy production leading to apoptosis. The principle of the determination of mitochondrial membrane potential is as follows: the leukocytes are incubated in the presence of a suitable fluorescent dye that accumulates in the active mitochondria. The accumulated fluorescent dye is present in either monomeric or aggregated form depending on the value of the mitochondrial membrane potential. After excitation, the two forms exhibit fluorescence of different wavelength. The value of mitochondrial membrane potential can be concluded based on the intensity of fluorescence of the aggregated form or the intensity ratio of the fluorescence of the two forms. Preferred fluorescence dyes for the determination of the mitochondrial membrane potential or the detectection of apoptosis include the following ones: JC-1 and JC-9: Dual-Emission Potential Sensitive Probes, Rhodamine 123,
Rosamine and Rhodamine derivatives e.g. TMRM and TMRE, Reduced Rhodamine and Rosamine derivatives, Image-iT LIVE Mitochondrial Transition Pore Assay Kit for Fluorescence Microscopy,
MitoProbe Transition Pore Assay Kit for Flow Cytometry, SelectFX Alexa Fluor 488 Cytochrome Apoptosis Detection Kit,
Antibodies against Mitochondrial Porin, Antibodies against Pyruvate Dehydrogenase.
An especially preferred fluorescent dye for the determination of mitochondrial membrane potential is JC-1 (manufactured by Molecular Probes). After incubation at 37 0C for a short time, JC-1 accumulates in the active mitochondria. When the mitochondrial membrane potential is high, the dye forms an aggregate in the mitochondria. When the mitochondrial membrane potential is low, the dye remains in the monomeric form. Following an excitation at 490 nm, the wavelength of the fluorescent emission of the monomeric form is 530 nm while that of the fluorescent emission of the aggregated form is 590 nm. Again, fluorescence is determined by a suitable apparatus e.g. FACS (fluorescence activated cell sorter). The intensity of fluorescence is proportional to the membrane potential of mitochondria.
The metabolic state related to the carbohydrate and lipid metabolism of the mammalian organism can be also concluded based on the amount of ATP produced in the mitochondria. As a matter of fact, a reduction of glucose absoprtion stimulated by insulin as well as that of the ATP synthesis were observed in babies of parents having insulin resistance. This observation supported the assumption that intracellular fat and fatty acid that accumulated due to the reduced rate of mitochondrial ATP synthesis played a casual role in the development of insulin resistance [Petersen, K.F. et al.: Decreased insulin-stimulated ATP synthesis and phosphate transport in muscle of insulin-
resistant offspring of type 2 diabetic parents, PIoS Med., 2(9), e233 (2005)]. The ATP level of the cells can be determined by chemical methods or NMR (nuclear magnetic resonance) measurements in a manner known perse.
The amount of mitochondrial DNA can be determined by PCR method in a manner known perse [Malakhova, L. et al.: The increase in mitochondrial DNA copy number in the tissues of gamma-irradiated mice, Cell. MoI. Biol. Lett., 10(4), 721-32 (2005)].
The principle of the determination of the mitochondrial redox potential is that the value of the mitochondrial redox potential is proportional to the free radical production of mitochondria. When the value of mitochondrial redox potential is higher, there is an increase also in the production of free radicals. The production of free radicals in the mitochondria can be detected and determined by fluorescent dyes that are susceptible to free radicals. These dyes react with the free radical resulting in a change of the intensity or wavelength of the fluorescence of the dye. Other fluorescent dyes are susceptible to the redox state of the cells. In both cases, the chemical modification of fluorescent dyes results in a change of fluorescence. Preferred fluorescent dyes for these purposes include the following ones:
Dichlorodihydrofluorescein diacetate and analogues thereof, Improved modifications of H2DCFDA,
Image-iT LIVE Green Reactive Oxygen Species Detection Kit, Aminophenylfluorescein and hydroxyphenylfluorescein,
Dihydrocalcein AM,
OxyBURST Green reagents,
Amine-Reactive OxyBURST Green reagents,
Dihydrorhodamine 123,
A Longer-Wavelength Reduced Rhodamine,
Reduced MitoTracker Probes,
Dihydroethidium (hydroethidine),
RedoxSensor Red CC-1 Stain,
Glutathiolation Detection with BioGee.
MitoSOX Red Mitochondrial Superoxide Indicator,
Fluorogenic Spin Trap.
A considerable amount of calcium ion are stored in the mitochondria. At the same time, calcium ions regulate the function of several enzymes that are present in the mitochondria. The calcium level is determined by using calcium dependent fluorescent dyes. Preferred fluorescent dyes for this purpose include the following ones: Rhod-2, Rhod-5N, Rhod-FF.
Both function and genesis of mitochondria are regulated by nitric oxide. It is preferred to determine the amount of nitric oxide produced in the cells [Nisoli, E. et al.: Mitochondrial biogenesis as a cellular signaling framework, Biochemical Pharmacology, 67, 1 (2004); Nisoli, E.: Mitochondrial biogenesis in mammals: The role of endogenous nitric oxide, Science, 299, 896 (2003)]. The basis of the determination is
that a suitable fluorescent dye reacts with nitric oxide in a specific way and, after excitation, a fluorescent radiation is emitted by the formed product. Preferred fluorescent dyes for the determination of biologically active nitric oxide include the following ones:
DAF-FM and DAF-FM diacetate,
2,3-Diaminonaphthalene,
1 ,2-Diaminoantraquinone,
NBD methylhydrazine,
Dichlorodihydrofluorescein diacetate,
Dihydrorhodamine 123,
Anti-nitrotyrosine antibody.
Especially preferred fluorescent dyes for the determination of biologically active nitric monoxide include the DAF dyes (manufactured by Molecular Probes). Following incubation with the leukocytes for a short time, the dye acumulated in the cells react, after the split of the ester bond, with nitric oxide specifically. A fluorescent radiation is emitted by the reaction product at 516 nm after an excitation at 490 nm. Fluorescence is determined by a suitable apparatus e.g. FACS (fluorescence activated cell sorter). The intensity of fluorescence is proportional to the amount of nitric oxide.
Pathological metabolic states related to the carbohydrate and lipid metabolism of the human organism i.e. the ill states of the body include the following ones:
- insulin resistance,
- insulin resistance syndrome,
- prediabetic state,
- type I diabetes mellitus,
- type Il diabetes mellitus,
- diabetes in pregnancy,
- glucose intolerance,
- lipoatrophic diabetes,
- hypertension,
- Rabson-Mendenhall's syndrome,
- arteriosclerotic caediovascular diseases,
- metabolic syndrome (existence of at least 3 symptoms from the group consisting of abdominal obesity, hyperinsulinaemia, glucose intolerance, insulin resistance, dislipidemia and hypertension) (the disease is also mentioned as metabolic X- syndrome or Raven's syndrome in the literature),
- hyperlipidemia.
The invention is based on the recognition that the mitochondrial number or mitochondrial function of the leukocyte cells separated from the blood as well as the amount of nitric oxide produced in the leukocyte cells reflects the healthy or pathological metabolic state related to the carbohydrate and lipid metabolism of the human organism, consequently, the healthy or ill state of the individual examined can be concluded based on the determination of the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells and/or the amount of nitric oxide produced in the leukocyte cells. These determinations can be carried out in a very simple way. Only one conventional blood
sample has to be taken from the individual to be examined, and the laboratory tests can be performed in a relatively short time with acceptable expenses. Therefore, the process of the invention can be used for screening the population that can be affected by a pathological metabolic state related to the carbohydrate or lipid metabolism, furthermore for following the effect of treatment in case of treated patients suffering from a disease related to a pathological state of carbohydrate or lipid metabolism. The process of the invention can be also suitable for monitoring the state in an acute energy producing crisis e.g. in heavy infections.
Thus, according to the process of the invention, the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells separated from the blood sample of the individual to be examined and/or the amount of nitric oxide produced in the leukocyte cells are determined in a manner known perse, and, based on the values obtained, the state of the individual examined is evaluated.
All the leukocytes or one or more subdivisions thereof e.g. T-lymphocytes can be separated from the blood sample taken from the individual to be examined and the cells thereof are used for the determinations. Alternatively, all the leukocytes can be separated, however, in the fluorescent determination of mitochondrial number and/or of one or more mitochondrial characteristic(s) and/or of the amount of nitric oxide only that/those of a given subdivision e.g. T-lymphocytes are taken into consideration. Of course, the mitochondrial number and/or
one or more mitochondrial characteristics) and/or the amount of nitric oxide can be determined in more than one leukocyte subdivisions, too.
According to the invention, one, two, three or still more characteristic(s) of the leukocyte cells or subdivisions thereof can be determined, wherein the characteristics are selected from the group consisting of mitochondrial membrane potential, production of ATP, amount of mitochondrial DNA, mitochondrial redox state, amount of free radicals in the mitochondrion, and calcium level in the mitochondrion.
Thus, it is preferred to determine one, two, three or still more parameters of the leukocyte cells or subdivisions thereof, wherein the parameters are selected from the group consisting of mitochondrial number, mitochondrial membrane potential, production of ATP, amount of mitochondrial DNA, mitochondrial redox state, amount of free radicals in the mitochondrion, calcium level in the mitochondrion, and amount of nitric oxide produced in the cells tested.
It is suitable to determine the mitochondrial number and/or mitochondrial membrane potential and/or production of ATP and/or amount of mitochondrial DNA and/or mitochondrial redox state and/or amount of free radicals in the mitochondrion and/or calcium level in the mitochondrion and/or amount of nitric oxide in the leukocyte cells or subdivisions thereof and, based on the values obtained, evaluate the state of the individual examined.
It is especially preferred to determine one, suitably two,
especially preferably three parameters from the group consisting of mitochondrial number, mitochondrial membrane potential and amount of nitric oxide in the cells tested. Of course, in addition to or instead of these parameters one or more further parameter(s) can be used for the examination of the metabolic state related to the carbohydrate or lipid metabolism of the individual.
It is practical to determine both mitochondrial number and mitochondrial membrane potential or both mitochondrial number and amount of nitric oxide or both mitochondrial membrane potential and amount of nitric oxide or the mitochondrial number, mitochondrial membrane potential, and the amount of nitric oxide in the leukocyte cells or subdivisions thereof.
Furthermore, the invention includes a reagent kit for carrying out the process of the invention, said reagent kit containing at least two fluorescent dyes for the determination of the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells and/or the amount of nitric oxide produced in the leukocyte cells. The reagent kit of the invention contains at least two fluorescent dyes selected from the group consisting of MitoTracker Green for the determination of the number of mitochondria, JC-1 for the determination of mitochondrial membrane potential and DAF for the determination of the biologically active nitric oxide.
The invention is further elucidated by means of the Example and the figures.
Figure 1 indicates the relationship between mitochondrial number and amount of nitric oxide.
Figure 2 shows the relationship between mitochondrial membrane potential and amount of nitric oxide.
Example
Comparative examination of healthy and insulin resistant individuals as well as patients suffering from type Il diabetes mellitus
A) Test individuals
25 to 60 years old volunteers of both sexes were drawn into the examination. In case of females, no hormonal effect having an influence on insulin resistance existed.
The group of healthy individuals consisted of persons having the following parameters:
- fasting glucose level: lower than 5.6 mM,
- HOMA index: lower than 2.5,
- ratio of waist and hip circumferences: lower than 0.95 in case of males and lower than 0.85 in case of females,
- BMI index: lower than 27,
- fat content of the body: lower than 30 % (DEXA).
The group of insulin resistant individuals consisted of persons having a disorder of glucose metabolism based on the following criteria:
- fasting glucose level: 5.6-7.0 mM,
- fasting glucose level: 7.0 mM or higher, however, the oral glucose tolerance test (OGTT) after 2 hours: lower than 11 mM of glucose,
HOMA index: above 2.5, ratio of waist and hip circumferences: higher than 0.95 in case of males and higher than 0.85 in case of females,
BMI index: above 27, fat content of the body: higher than 30 % (DEXA), insulin glucose ,,clamp" method: in case of empty stomach, under the influence of insulin infusion, the absorbed amount of glucose referred to the lean body mass (M value) is lower than 8 mg glucose/minute/kg body mass.
The group of diabetic individuals consisted of patients suffering from type Il diabetes mellitus. The patients were not treated with an insulin sensitizing drug and a part of them had compensated glucose metabolism.
Patients having the following disorders were not drawn into the examination:
- individuals suffering from type I diabetes mellitus,
- individuals suffering from type Il diabetes mellitus and treated with an insulin sensitizing drug,
- individuals having unregulated hypertension (systolic value: above 160 mm Hg, diastolic value above 95 mm Hg),
- individuals treated with drugs having an influence on the nitric oxide production of the body (e.g. molsidomine, sildenafil, tadalafil),
- individuals treated with drugs having an influence on the carbohydrate metabolism (glucocorticoids, thiazides),
- individuals having an acute inflammation process,
- pregnant women,
- individuals taking contraceptives or consuming alcohol or addictive drugs.
The following table contains the number of individuals participating in the test groups:
B) Insulin glucose ,,clamp" (hyperinsulinaemic euglycaemic clamp) test
One of the antecubital veins of the test persons was cannulated to allow the administration of glucose and insulin infusions. On the other side, the vein of the hand was cannulated in a retrograde manner while maintaining the hand in a warm state to take blood samples from the vein. A dose of 40 U of human insulin (Actrapid HM, Novo Nordisk, Gentofte, Denmark) was administered in a logaritmically decreasing manner within 10 minutes, then insulin was administered at a rate of 120 mU/m2/minute for 360 minutes. Glucose production in the lever was inhibited by the hyperinsulinaemia developed in this way during the test. The rate of infusion containing 20 % of glucose was regulated to obtain a constant blood glucose level of 5.5 mM. The average glucose infusion rate maintaining the euglycaemic blood glucose level in the last 30 minutes of the euglycaemic period was considered as a measure of
insulin sensitivity. The blood glucose level was determined from 10 μl of blood sample taken in every 5 minutes.
C) Tests according to the invention
Separation of leukocyte cells
A blood sample of 1 ml volume was taken into a test tube containing heparin from each individual taking part in the examination. The blood sample was layered onto Ficoll- Pague® Plus (a branched chain hydrophil saccharose polymer produced by Amersham) gradient and leukocytes were separated by centrifuging at 2500 rpm at 4 0C. The leukocyte layer accumulated at the boundary layer was removed, the cells were sedimented by centrifuging at 1500 rpm for 5 minutes and the pellet of cells was suspended in 1 ml of PBS (physiological saline containing phosphate buffer). 100 μl portions of cell suspension were employed for the incubation with a fluorescent dye. Within the leukocyte population, T- lymphocytes were identified by means of anti-CD3 antibody and the characteristics tested below were evaluated only in the population of T-lymphocytes.
Determination of the mitochondrial number
100 μl of cell suspension obtained as described above were incubated with 100 nM of MitoTracker Green fluorescent dye (manufactured by Molecular Probes) at 37 0C for 10 minutes. After an excitation with a light of 490 nm wavelength, the fluorescence of the dye accumulated in the active mitochondria was measured at 516 nm using a FACS apparatus. FACS analysis was carried out with a Becton Dickinson FACS Calibur
apparatus. Cell Analysis Software was used for the evaluation of the data obtained. The values of mitochondrial number for each test group are given in Table I expressed in fluorescence unit.
Determination of the mitochondrial membrane potential 100 μl of cell suspension obtained as described above were incubated with 5 μM of JC-1 fluorescent dye (manufactured by Molecular Probes) at 37 0C for 10 minutes. After an excitation with a light of 490 nm wavelength, the fluorescence of the dye accumulated in the active mitochondria was measured at 530 nm (monomeric form) and 590 nm (aggregated form), respectively, using a FACS apparatus. FACS analysis was carried out with a Becton Dickinson FACS Calibur apparatus. Cell Analysis Software was used for the evaluation of the data obtained. The values of mitochondrial membrane potential for each test group are given in Table Il expressed in fluorescence unit.
Determination of the amount of biologically active nitric oxide
100 μl of cell suspension obtained as described above were incubated with 10 μM of DAF fluorescent dye (manufactured by Molecular Probes) at 37 0C for 10 minutes. After an excitation with a light of 490 nm wavelength, the fluorescence of the dye accumulated in the active mitochondria was measured at 516 nm using a FACS apparatus. FACS analysis was carried out with a Becton Dickinson FACS Calibur apparatus. Cell Analysis Software was used for the evaluation
of the data obtained. The values of mitochondrial number for each test group are given in Table III expressed in fluorescence unit.
Table I Mitochondrial number in the test groups
ANOVA p<0,01.
From Table 1 it can be seen that in healthy individuals the mitochondrial number of the T-lymphocytes was nearly twice as high as that in insulin resistant individuals and more than twice as high as that in diabetic patients.
Table Il Mitochondrial membrane potential in the test groups
ANOVA p<0,05.
From Table Il it can be seen that in healthy individuals the mitochondrial membrane potential was more than three times as high as that in insulin resistant individuals and more than twice as high as in diabetic patients.
Table III
Nitric oxide production in T-lymphocyte cells in the test groups
ANOVA p<0,01.
From Table III it can be seen that in healthy individuals
the nitric oxide production by the T-cells was over 12 times higher than that in insulin resistant individuals and 3.6 times higher than that in diabetic patients.
The data of Tables I to III indicate that each of the characteristics of T-lymphocytes allows the identification of insulin resistant individuals and diabetic patients.
When two of the characteristics of T-lymphocytes are considered simultaneously, the test groups can be identified still more clearly. In Figure 1 , the mitochondrial number was depicted as a function of the amount of nitric oxide. It is apparent that the squares referring to the group of healthy individuals are localized at higher values of both characteristics, separately from the also localized triangles that refer to the group of insulin resistant individuals and from the localized circles referring to the group of patients suffering from type Il diabetes mellitus.
In Figure 2, the mitochondrial membrane potential was shown as a function of the amount of nitric oxide. It is obvious that especially the group of insulin resistant individuals and the group of healthy individuals can be well identified.
Of course, it can be also preferred to consider three characteristics simultaneously.
Claims
1. A process for the examination of the metabolic state related to the carbohydrate and lipid metabolism of a human organism, for the detection of the healthy or ill state in which the number and/or one or more characteristics of the mitochondria that are present in the leukocyte cells separated from the blood sample of the individual to be examined and/or the amount of nitric oxide produced in the leukocyte cells are determined in a manner known perse, and, based on the values obtained, the state of the individual examined is evaluated.
2. A process of Claim 1 in which the rate of any insulin resistance is determined.
3. A process of Claim 1 or 2 in which the number of mitochondria and/or the mitochondrial membrane potential and/or the amount of biologically active nitric oxide are determined in T-lymphocyte cells.
4. A reagent kit for carrying out the process of Claim 1 characterized by containing at least two fluorescent dyes for the determination of the number and/or one or more characteristics of the mitochondria that are present in the leucocyte cells and/or the amount of nitric oxide produced in the leucocyte cells.
5. A reagent kit of Claim 4 characterized by containing at least two fluorescent dyes selected from the group consisting of MitoTracker Green for the determination of the number of mitochondria, JC-1 for the determination of mitochondrial membrane potential and DAF for the determination of the biologically active nitric oxide.
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| CN111579763A (en) * | 2020-04-09 | 2020-08-25 | 北京博瑞世安科技有限公司 | Method for detecting respiratory function of leucocyte mitochondria and method for detecting kidney yin deficiency |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2021619C1 (en) * | 1991-11-25 | 1994-10-15 | Ирина Иосифовна Влох | Method of insulin-resistance diagnosis in patients with depressive states |
| US6232078B1 (en) * | 1997-02-21 | 2001-05-15 | Hong-Kyu Lee | Method for diagnosing preclinical diabetes by quantification of mitochondrial DNA in peripheral blood |
| US20030235812A1 (en) * | 1999-04-30 | 2003-12-25 | Mitokor | Indicators of altered mitochondrial function in predictive methods for determining risk of type 2 diabetes mellitus |
-
2006
- 2006-02-02 HU HU0600082A patent/HUP0600082A2/en not_active Application Discontinuation
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2021619C1 (en) * | 1991-11-25 | 1994-10-15 | Ирина Иосифовна Влох | Method of insulin-resistance diagnosis in patients with depressive states |
| US6232078B1 (en) * | 1997-02-21 | 2001-05-15 | Hong-Kyu Lee | Method for diagnosing preclinical diabetes by quantification of mitochondrial DNA in peripheral blood |
| US20030235812A1 (en) * | 1999-04-30 | 2003-12-25 | Mitokor | Indicators of altered mitochondrial function in predictive methods for determining risk of type 2 diabetes mellitus |
Non-Patent Citations (2)
| Title |
|---|
| DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; June 2006 (2006-06), KOLONICS ATTILA ET AL: "Mitochondrial status of lymphocytes shows correlation with metabolic state" XP009087496 Database accession no. PREV200600669552 & DIABETES, vol. 55, no. Suppl. 1, June 2006 (2006-06), pages A143-A144, 66TH ANNUAL MEETING OF THE AMERICAN-DIABETES-ASSOCIATION; WASHINGTON, DC, USA; JUNE 09 -13, 2006 ISSN: 0012-1797 * |
| NAGY GYORGY ET AL: "Nitric oxide-dependent mitochondrial biogenesis generates Ca2+ signaling profile of lupus T cells" JOURNAL OF IMMUNOLOGY, vol. 173, no. 6, 15 September 2004 (2004-09-15), pages 3676-3683, XP002444870 ISSN: 0022-1767 * |
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| CN111579763A (en) * | 2020-04-09 | 2020-08-25 | 北京博瑞世安科技有限公司 | Method for detecting respiratory function of leucocyte mitochondria and method for detecting kidney yin deficiency |
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