Methionine and total homocysteine in hypertensive patients with renal excretory dysfunction.

The role of the kidneys in the metabolism and homeostasis of sulfur-containing amino acids is great, so the levels of methionine (Met), total homocysteine (tHcy) and their ratios can be of diagnostic value in chronic kidney disease (CKD), in a course of the arterial hypertension (AH). The aim of the study was to evaluate the Met/tHcy ratio in hypertensive patients with CKD. We used blood plasma of 76 patients aged 40-75 years with AH and the excretory dysfunction of the kidneys; subgroups: 1 - with proteinuria (n=37); 2 - without proteinuria with glomerular filtration rate (GFR) < 90 ml/min/1.73 m2 (n=39) and comparison group 3 - patients with AH without renal excretory dysfunction (n=28). Significantly lower Met levels were in subgroup 1. THcy levels were higher in subgroups 1 and 2 than in group 3. The Met/tHcy ratio revealed differences in subgroups 1 and 2 vs group 3. No differences were found in Arg and Lys levels. Positive correlations of the Met/tHcy ratio with the number of erythrocytes, but not with the level of hemoglobin, were revealed. In the ROC analysis, the cut-off points for the Met/tHcy ratio compared to group 3 were 3.08 for subgroup 1 and 3.36 for subgroup 2. With the progression of CKD, there is an increase in the levels of tHcy in the blood, and a decrease in the content of Met. A decrease in GFR, especially in a case with proteinuria, is accompanied by a decrease in the level of Met. The Met/tHcy ratio above 3.36 can be considered as the minimum of the balance between these sulfur-containing amino acids contents in a blood necessary for hypertensive patients with CKD.

Homoarginine test for evaluation of metabolic renal dysfunction.

Low plasma L-homoarginine (hArg) concentration is an independent predictor of adverse cardiovascular outcomes and overall mortality, as well as the progression of chronic kidney disease (CKD). The enzyme L-arginine:glycinamidinotransferase (AGAT, EC 2.1.4.1) acts in the mitochondrial membrane of the renal tubular epithelium, forming the precursor of creatine, guanidinoacetic acid, and additionnaly by-product hArg. As it was shown recently, there is a decreased level of hArg in the late stages of CKD, however, the the level of hArg in the early stages of CKD remained unexplored. The aim of this study was to determine the diagnostic threshold levels of hArg in the blood of patients with stages 1 and 2 of CKD. In patients with the initial stages of CKD (n = 44) at the age of 58 (45-67) years, compared with the group of donors of 55 (42-58) years (n = 30), a significant decrease of hArg level was found. In the subgroup with stage CKD 2, the cut-off point of 1.59 µM threshold was characterized by greater sensitivity and specificity than in the subgroup with stage CKD 1 with 1.66 µM threshold level of hArg. For the full group, the hArg cut-off threshold was 1.60 µM, which is about to 0.2 µM lower than the lower limit of the reference interval for healthy individuals. It can be assumed that even before the formation of symptoms of proteinuria and albuminuria, a significant part of individuals from population cohort develops a state of decreased AGAT activity, since the expression of this enzyme is associated with a certain regulatory feedback inhibition at the body level. As a result of the study, it can be noted that in patients with early stages of CKD in the age group 45-67 years, there is a disturbance of the kidneys metabolic function. These metabolic changes can be detected by testing the level of hArg.

[The evaluation of homoarginine and folic acid in patients with arterial hypertension.] / Оценка гомоаргинина и фолиевой кислоты у пациентов с артериальной гипертензией.

According to current data, a low level of folic acid (FA) contributes to the progression of arterial hypertension (AH), affecting the metabolism of cells that are involved in the vascular tone regulation, such as hypothalamic astrocytes of nervous tissue. It is also known that the level of FA in the nervous tissue and cerebrospinal fluid is 2-3 times higher than in plasma. There is another metabolic marker of cardiovascular diseases, the level of plasma homoarginine (hArg). The decrease in the level of plasma hArg is also known as a diagnostic sign. In our study, we established that in patients with AH (n = 60), the level of hArg was almost 2 times lower than in healthy individuals, and in 75% of cases the rate was below 1.80 µM. The insufficiency of FA taking into account its low level in plasma FA, as well as the level of total homocysteine (tHcy) higher than 10.9 µM, was observed in 78% of patients. HArg levels at values less than 1.80 µM corresponded to a statistically significant decrease in FA when its content was less than 13.5 nM. This relationship (r = 0.63, p = 0.020) appears in patients with AH, regardless of the number and severity of target organ damage (TOD). FA and hArg as metabolic markers exhibit various diagnostic capabilities when comparing subgroups of patients without TOD and with multiple TOD. Significant differences fared at an acceptable level (p = 0.007) only for the hArg levels, while for the FA concentrations there was only a trend to decrease. It is possible that metabolic disturbances in the central nervous system that are associated with the necessary to maintain high FA concentration contribute to the development of hypertensive status. The causal relationship of a parallel decrease in hArg and FA levels in patients with AH requires further research.

[Homoarginine level and methionine-homocysteine balance in patients with ischemic heart disease.]

The level of homoarginine (hArg) in terms of prognostic significance may exceed the natriuretic peptides and other well-known markers according to the latest data about the progression of cardiovascular diseases. The lack of data on the association of hArg levels with levels of other metabolites makes it difficult to understand its role in the pathogenesis of cardiovascular diseases. Relationships of hArg and other amino acids, including methionine (Met) and total homocysteine (tHcy), and their ratio in patients with ischemic heart disease were evaluated. The study included 74 patients with coronary heart disease (57 men and 17 women) aged 62 (57 - 67) years before coronary artery bypass surgery and 27 healthy people of similar age. In patients, the level of hArg was almost 2 times lower (p < 0.05) than in healthy individuals and rates lower than 1.4 µM were in half of them. The statistically significant decrease (p = 0.0025) of the Met/tHcy ratio corresponded to a decrease in the level of hArg. This ratio did not correlate with glucose level or body mass index. Less statistical significance of hArg correlation with levels of Met or tHcy separately was observed. In the subgroup of patients with hAarg level above 2.1 µM, a lower incidence of myocardial infarction was noted. Thus, a low hArg level is associated with impaired metabolism of sulfur-containing amino acids involved in transmethylation reactions, in patients with ischemic heart disease. The Met/tHcy ratio, closely correlating with the level of hArg, apparently reveals a link between the reactions of creatine formation and transmethylation, highlighting a cohort of patients with the most profound and dangerous changes in tissue metabolism.

[The level of circulating humanin in patients with ischemic heart disease.]

At present, great interest is caused with evaluation of new markers in blood circulation for the estimation a tissue oxidative metabolism disturbance due to the presence of secondary mitochondrial dysfunction in patients with coronary heart disease. Сoronary heart disease is generally accompanied with a decline in mitochondrial respiration and represents the root cause of metabolic abnormalities in tissues. To gain insight into rate of decline of mitochondrial oxidative metabolism in tissues there were proposed humanin as a new marker. The content of humanin in compare with other markers of energy metabolism in 59 patients with coronary heart disease was studied. In the examined patients, a decrease in the level of humanin up to 250 ng/l was observed when compared with its level of 1110 (800 to 1500) ng/l in healthy individuals. In most of the patients increased level of lactic acid from 1.0 to 2.2 mM accompanied in 45 % cases with elevation of pyruvic acid concentration above 99.1 µM was observed. Also, it was found a significant decrease of homoarginine level down to 1.40 (1.0-2.0) versus 2.3 (1.8-3.1) µM in healthy individuals. We found an inverse correlation between the level of humanin and the age of patients (R = -0.35, p = 0.048). It can be concluded that patients with coronary heart disease are characterized by a lower level of humanin and homoarginine in the blood, as well as an increased content of lactic acid, indicators that are the criteria for inhibiting aerobic pathways and reducing mitochondriogenesis.

[The value of detection of homoarginin level in patients with ischemic heart disease under operations of myocardium re-vascularization.]

The homoarginine is proposed as a prognostic metabolic criterion of danger of stroke and myocardium infarction and evaluated in this capacity in a number of population studies. The actual study analyzed content of this cardio-metabolic marker and main amino acids in blood plasma of 59 patients (46 males and 13 females, aged 61,4 ± 6,2 years) wit6h ischemic heart disease send to planned operation of aortocoronary bypass surgery and also 30 healthy individuals aged 50,4 ± 9,2 years. The concentration of homoarginine and amino acids was detected using high-performance liquid chromatography with identification of ortho-phthalic derivative with fluorometric detecting using genuine developed technique. The examined patients had a significant decreasing of level of homoarginine in venous blood plasma. In contrast to healthy individuals, the patients were detected a positive dependence of level of homoarginine from content of level of lysine and arginine in blood plasma. These amino acids are substrates of reaction generating homoarginine catalyzed by argin ineglycineamidinotransferase. Thus, in patients' level of homoarginine in blood plasma depends both on activity of argininegly cineamidinotransferase in tissues and on content of lysine and arginine. The content of these amino acids in blood can abruptly increase at activation of coagulation and fibrinolysis. Consequently, it appears that increasing of bio-synthesis of homoarginine occurs in certain patients leveling its decreased level. In patients with cardio-vascular diseases at detection of homoarginine a level of arginine and lysine in blood is to be considered.

[The level of circulating PGC1a in cardiovascular disease].

The level of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1α) in human blood plasma was investigated. Samples of healthy individuals (n=34) and patients with cardiovascular diseases (n=110), including aortic aneurysm (n=69), aortic stenosis (n=25) and patients without aortic pathologies were analyzed. In patients the PGC1α concentration was higher than that in healthy persons, and tended to decrease with age. Elevated concentrations of lactic acid, total homocysteine and asymmetric dimethylarginine in the blood of patients suggested a parallel development of endothelial and secondary mitochondrial dysfunction. However, concentrations of lactic and pyruvic acids exceeding reference limit were associated with the decrease in the PGC1α level.

[The content of nitric oxide in blood plasma of health persons depending on age.]

The article considers results of study of total concentration of nitrites and nitrates (NOx) in blood plasma of healthy individuals residing in St. Petersburg. The samples from 58 healthy individuals were analyzed and out of them - 31 aged 18-25 years and 27 aged 48-63 years. The concentration of NOx in blood plasma was determined using Griess reagent after reduction of nitrates under effect of NADH-dependent recombinant nitrate reductase from Arabidopsis thaliana. The surplus of NADH preventing Griess reaction was removed at the expense of of enzyme reaction in presence of lactate dehydrogenase and sodium pyruvate. The analysis and its calibration was carried out using additions of solution of sodium nitrate to samples of blood plasma without deproteinization. The data was obtained concerning variation of content of NOx in healthy individuals of various age. In particular, it is demonstrated that the level of total content of nitrogen oxide in the group of elder age was reliably higher (p=0.0001) as compared with donors aged 18-25 years.

[The metabolic and protein markers of dysfunction of mitochondria in patients with cardio-vascular diseases.]

The article deals with study concerning content of cytochrome C (CytC) and protein PGC1α (1alpha-co-activator of gammareceptor, activating proliferation of peroxisomes) in blood of patients with pathology of left ventricular outflow tract conjointly with low-molecular metabolites characterizing dysfunction of mitochondria and endothelium. The samples of blood plasma were analyzed taking from healthy persons (n=34), patients with aortic aneurysm (n=69), aortic stenosis (n=25) and without pathology of aorta (n=16). In the group of patients with disturbed blood circulation increasing of level of metabolic markers of dysfunction of mitochondria (lactate, ratio lactate/pyruvate) and endothelium (asymmetric dimethylarginine, total homocysteine) was established. The occurrences of cells death were detected according to increasing of level of CytC in 17% 0f patients. The evaluation of level of intermediates of energy metabolism and simultaneously of protein PGC1α permits detecting at least three degrees of dysfunction of mitochondria depending on stage of its progression: 1) without lactoacidemia but with detection of PGC1α in blood, 2) with lactoacidemia up to 2.2 mM and increased level of PGC1α in blood (more than 61 ng/l), 3) with lactoacidemia and loss of cells capacity to transfer this protein in blood. The application of common metabolic indicators of disorder of function of mitochondria supplemented by determination of level of CytC and PGC1α in blood permits evaluating more comprehensively development of dysfunction of mitochondria in patients with disorders of blood circulation of various genesis.

[Arginine and lysine as products of basic carboxypeptidase activity associated with fibrinolysis].

Blood carboxypeptidases play an important role in the regulation of fibrinolysis. We have proposed here the method for the assay of blood carboxypeptidase activity associated with coagulation/fibrinolysis using the natural substrate fibrin and the detection of basic amino acids arginine and lysine as products in the conditions close to those in vivo. Plasma samples from 15 patients with arterial hypertension were investigated. The coagulation and subsequent fibrinolysis were initiated by addition of standard doses of thrombin and tissue plasminogen activator, respectively. Arginine and lysine concentrations before, during, and after completion of fibrinolysis were determined using HPLC. The parameters of fibrinolysis were evaluated by clot turbidity assay. Fibrinolysis led to a large and significant increase in concentrations of arginine and lysine in the incubation mixture by 101 and 81%, respectively. The duration of fibrinolysis initiation significantly correlated to the degree of increase of these amino acids: r(s) = -0.733 and -0.761 for arginine and lysine, respectively (p < 0.05). The rates of amino acids liberation during fibrinolysis demonstrate different pattern: arginine generation had two maximums: at the beginning of clot lysis and at his end, whereas the liberation of lysine occurred mainly at the middle of fibrinolysis. Thus, the carboxypeptidase activity associated with fibrinolysis can be considered as a local source of the essential aminoacids.

[The ultrafiltration at pre-analytical stage under detection of concentration of lactic acid in blood plasma].

The detection of concentration of lactic acid in blood plasma and other objects is especially applied to discover the mitochondria dysfunctions. The study was organized to analyze samplings of blood plasma and plasma ultra-filtrates taken from 80 healthy persons and 73 patients with activation of intravascular coagulation and fibrinolysis using lactate-oxidase test. The comparative analysis of results of detection of concentrations of lactic acid in blood plasma and its ultra-filtrate established that in 72% of cases the higher values of concentration of detecting lactic acid took place after procedure of ultra-filtration enabling separation of overwhelming quantity of protein. In accordance with accumulated experience in the field of clinical diagnostic practice the enzyme tests are to be applied to detect the concentration of lactic acid in blood plasma and other objects. The present study demonstrated the expediency of application of plasma ultra-filtrate to detect the concentration of lactic acid.

[Functional arginine-containing amino acid sequences in peptides and proteins].

L-arginine is a source of nitrogen oxide and plays a great role in a number of other biochemical processes. Functions and prospects for practical application of five groups of arginine-containing amino acid sequences and synthetic polyarginine sequences are considered. The physiological characteristics of well-known arginine-containing peptides, such as RGD peptides, kyotorphin, and tuftsin, are described in detail. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru

[The effects of divalent cations on the fibrin clot formation and its lysis].

Hemocoagulation is a Ca(2+)-dependent process, and we suppose that other bivalent cations (BC) can also influence this system. The effects of some BC (Ca2+, Fe2+, Zn2+, Cd2+, Co2+ and Ni2+) on thrombin-induced plasma clotting and clot lysis caused by streptokinase have been studied. All BC studied, except Fe2+, maintained the formation of fibrin clot followed by its lysis. In the presence of BC the coagulation part of the turbidimetry curve changed in a dose-dependen manner and was characterized by: (1) the decrease of the induction period which seems to be due to thrombin activation or accelerating fibrin self-association; (2) the elevation of the maximal optical density of the fibrin clot and the velocity of its achievement. The fibrinolytic part of the curve was characterized by the optical density downfall acceleration at low Co2+, Cd2+, Zn2+ and Ni2+ concentrations but marked delay of the process at BC higher concentrations. So, ions of Co2+, Cd2+, Zn2+ and Ni2+ but not Fe2+ significantly influence the dynamics of thrombin-induced clot formation and optical clot characteristics. At different ion concentrations BC may activate or inhibit fibrinolysis.

[Prophylaxis of thrombosis and thromboembolic complications in patients with domestic mechanical artificial heart valves (AHV) "Medinzh-2" in remote terms of observation]. / Profilaktika trombozov i tromboémbolicheskikh oslozhnenii u bol'nykh s otechestvennymi mekhanicheskimi iskusstvennymi klapanami serdtsa "MedInzh-2" v otdalennye sroki nabliudeniia.

The observation included 40 patients (20 men and 20 women) with the rheumatic heart diseases acquired at the age of (45.9 +/- 8.7) years after isolated prostheses of the aortal (n = 25) and mitral (n = 15) valves with bicuspid constructions of AHV "MedInzh-2" in remote terms in (2.52 +/- 0.48) years, I-II functional valve (NYHA). It was shown that only a permanent control not rarer than once a month and an adequate correction by means of anticoagulation and antiaggregation therapy using standard methods for patients with the bicuspid AHV made it possible to avoid the development of thromboses and thromboembolic complications during three years of observation.

[Turbidimetric analysis of fibrin polymerization in the plasma]. / Turbidimetricheskii analiz polimerizatsii fibrina v plazme krovi.

The turbidimetrical assay of thrombin-induced plasma coagulation provides a possibility to estimate both stages of fibrinogen-fibrin conversion. The initial one, which proceeds without any change of turbidity, reflects the process of protofibril formation, and the second stage of lateral aggregation, is characterized by the rise of turbidity. The influence of heparin, alga (Laminaria digitata) aqueons extracts, and collagenase on the indices of the turbidity-time curve has been studied. It was established that the alga extracts possessed the powerful heparin-like anticoagulant activity. The both agents influenced the first stage of the turbidity-time curve, suppressing protofibril formation, which reflects the thrombin inhibition. Nevertheless, they differed in their mode of dose-dependence. While the time of protofibril formation was direct proportional to the alga extract concentration, it was rising more intensively with heparin dose elevation. Plasma pre incubation with alga extract or heparin did not influence their action. Treatment with plasma collagenase changed only the second stage of the coagulation curve. It inhibited the process of protofibril lateral aggregation in the direct proportional manner. It must be due to fibrin digestion by the enzyme. We propose that fibrin cleavage by collagenase occurred out of the thrombin action sites, because the velocity of protofibril accumulation stayed unchanged. Our data illustrate the usefulness of the turbidimetrical analysis in the studies of the agents' action mechanisms on blood coagulation, in conditions close to physiological ones.

[Turbidimetric express method in the assessment of the hemostasis parameters in patients with prosthetic bileaflet heart valves]. / Turbidimetricheskii ékspress-metod v otsenke parametrov gemostaza u bol'nykh s iskusstvennymi dvustvorchatymi klapanami serdtsa.

Thirty patients of medium (47 +/- 3) years of age with prosthetic bileaflet heart valves without any specific complications of the I-II functional classes and with the postoperative period after mitral (11) and aortal (19) valve replacements during (2.52 +/- 0.48) years were studied. The defects of coagulation and fibrinolytic systems were found in 9 patients with high prothrombin index (93.21 +/- 9.8)% and low INR (0.9-1.7). The activation of fibrinolysis, low platelets, hypofibrinogenemia, D-dimer which are the risk factors of thrombogenic complications were found in these patients. The updated turbo-dynamic express-method which helps to activate fibrinolysis in short terms and to make its correction is proposed.

The study of pathogenetic basis of asthma using biostatistic models.

In 50 patients with asthma we have determined skin tests on dust and pollen allergens, forced expiratory volume for one second (FEV1) before and after subcutaneous injections of epinephrine standard dose, adrenoreactivity by adrenodependent cyclic AMP levels and glycogenolysis, cyclic AMP phosphodiesterase activity, levels of eosinophils in blood (in all 28 characteristic features). Factor and correlation analysis were carried out by electronic computer. In addition, in 121 patients we have determined adrenodependent glycogenolysis in leucocyte suspension incubated with various allergens the content of diene conjugates and total antioxidative activity of plasma. It is concluded that atopic and nonatopic forms of asthma are diseases of different nature in their constitutional origin, pathogenic agents of environment and cellular and subcellular mechanisms of their development. Moreover, the pathogenetic role of allergen-induced lipid peroxidation that correlates with lymphocyte adrenoreactivity is shown.

[Disorders of cellular regulation systems as a basis for changed bronchial reactivity in bronchial asthma]. / Narusheniia sistem kletochnoi reguliatsii kak osnova izmenennoi reaktivnosti bronkhov pri bronkhial'noi astme.

Leukosuspensions from 217 patients with bronchial asthma and 73 normal subjects were examined for alterations in the levels of cAMP and cGMP under the effect of incubation with adrenaline, propranolol, acetylcholine. A study was also made of the activity of cAMP PDE and glycogen metabolism. Simultaneous measurement of the basal and stimulated levels of cyclic nucleotides and their ratios revealed a decrease in cell adrenoreactivity and a rise in cholinoreactivity. Variation of the beta-adrenoblocking action of propranolol typical for the atopic mechanism of bronchial asthma was demonstrated. Comparison of the adrenodependent glycogenolysis and alterations in the cAMP level, analysis of the action of analgin on these characteristics allowed the conclusion about the secondary nature of alterations in cAMP metabolism dependent on the features of prostaglandin metabolism. A significant decrease in the adrenodependent glycogenolysis was revealed in lymphocytes, with that decrease induced by drug, dust and pollen allergens. It was demonstrated that in verified viral infection, cAMP PDE was significantly reduced.