The Oxidative Stress May be Induced by the Elevated Homocysteine in Schizophrenic Patients

The mechanisms of oxidative stress in schizophrenic patients are not fully understood. In the present study, we investigated the effect of elevated level of homocysteine (Hcys) on some parameters of oxidative stress, namely thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in plasma, the level of carbonyl groups in plasma proteins, as well as the amount of 3-nitrotyrosine in plasma proteins isolated from schizophrenic patients. Patients hospitalised in I and II Psychiatric Department of Medical University in Lodz, Poland were interviewed with special questionnaire (treatment, course of diseases, dyskinesis and other EPS). According to DSM-IV criteria all patients had diagnosis of paranoid type. They were treated with antipsychotic drugs (clozapine, risperidone, olanzapine). Mean time of schizophrenia duration was about 5 years. High-performance liquid chromatography was used to analyse the total level of homocysteine in plasma. Levels of carbonyl groups and 3-nitrotyrosine residues in plasma proteins were measured by ELISA and a competition ELISA, respectively. The lipid peroxidation in plasma was measured by the level of TBARS. Our results showed that in schizophrenic patients the amount of homocysteine in plasma was higher in comparison with the control group. We also observed a statistically increased level of biomarkers of oxidative/nitrative stress such as carbonyl groups or 3-nitrotyrosine in plasma proteins from schizophrenic patients. Moreover, our experiments indicate that the correlation between the increased amount of homocysteine and the oxidative stress exists. Considering the data presented in this study, we suggest that the elevated Hcys in schizophrenic patients may stimulate the oxidative stress.     

Effect of aronia on thiol levels in plasma of breast cancer patients

The various specific biomarkers of oxidative stress in plasma from patients with breast cancer, as well as biomarkers (the level of lipid hydroperoxides, conjugated dienes, thiobarbituric acid reactive substances) have been described. The aim of our present study was to evaluate the amount of low-molecular-weight thiols (which are physiological free radical scavengers) and establish the effects of the extract from A. melanocarpa on the amount of these thiols in plasma obtained from patients with invasive breast cancer, patients with benign breast diseases and from healthy volunteers. We observed in patients the higher amounts of homocysteine in plasma from patients in comparison to plasma from the control group; however the total level of glutathione, cysteine, cysteinylglycine, and the amount of thiols in reduced and oxidized forms was changed (e.g., in patients, the decrease of glutathione and cysteine reached about 50% of total values). Moreover, we showed that in the presence of the extract of A. melanocarpa (50 μg/mL, 5 min, 37°C), changes in amount of thiols in plasma from patients with invasive breast cancer and patients with benign breast diseases were significantly reduced in vitro. Considering the data presented in this study, we suggest that the extract from A. melanocarpa has an effect on thiol metabolism and the levels of all tested thiols observed in plasma obtained from breast cancer patients.     

Changes in plasma thiol levels induced by different phases of treatment in breast cancer; the role of commercial extract from black chokeberry

Different low-molecular-weight thiols, including glutathione, cysteine, and cysteinylglycine are physiological free radical scavengers. On the other hand, homocysteine may play a role as an oxidant. The aim of our present study was to establish in vitro the effects of the commercial extract of Aronia melanocarpa (Aronox^?) on the amount of selected low-molecular-weight thiols and the activity of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in plasma obtained from patients with invasive breast cancer during different phases of treatment [before or after the surgery and patients after different phases of chemotherapy (doxorubicin and cyclophosphamide)] and from healthy subjects. Patients were hospitalized in Department of Oncological Surgery and Department of Chemotherapy, Medical University of Lodz, Poland. The level of low-molecular-weight thiols was determined by high-performance liquid chromatography. We observed that in the presence of the Aronia extract changes in amount of thiols in plasma from breast cancer patients (at all tested groups) were significantly reduced. Our results showed that tested commercial extract reduced modifications of antioxidative enzymes activity in plasma from patients during different phases of treatment, but this effect was not statistical significant. Our results suggest that the Aronia extract supplementation in breast cancer patients has a beneficial effect on thiols concentration in plasma. Plasma, as reported in this work, could be used as an experimental model to evaluate the beneficial action of plant supplements, including phenolic extracts on thiols or other molecules during different phases of treatment.     

Plasma MicroRNA Panel for Minimally Invasive Detection of Breast Cancer

Over the last few years, circulating microRNAs (miRNAs) have emerged as promising novel and minimally invasive markers for various diseases, including cancer. We already showed that certain miRNAs are deregulated in the plasma of breast cancer patients when compared to healthy women. Herein we have further explored their potential to serve as breast cancer early detection markers in blood plasma. Circulating miR-127-3p, miR-376a and miR-652, selected as candidates from a miRNA array-based screening, were found to be associated with breast cancer for the first time (n = 417). Further we validated our previously reported circulating miRNAs (miR-148b, miR-376c, miR-409-3p and miR-801) in an independent cohort (n = 210) as elevated in the plasma of breast cancer patients compared to healthy women. We described, for the first time in breast cancer, an over-representation of deregulated miRNAs (miR-127-3p, miR-376a, miR-376c and miR-409-3p) originating from the chromosome 14q32 region. The inclusion of patients with benign breast tumors enabled the observation that miR-148b, miR-652 and miR-801 levels are even elevated in the plasma of women with benign tumors when compared to healthy controls. Furthermore, an analysis of samples stratified by cancer stage demonstrated that miR-127-3p, miR-148b, miR-409-3p, miR-652 and miR-801 can detect also stage I or stage II breast cancer thus making them attractive candidates for early detection. Finally, ROC curve analysis showed that a panel of these seven circulating miRNAs has substantial diagnostic potential with an AUC of 0.81 for the detection of benign and malignant breast tumors, which further increased to 0.86 in younger women (up to 50 years of age).     

Laboratory diagnosis of the status of the hemostasis system

Activators of fibrinogen, prothrombin and protein C isolated from venoms of Agkistrodon halys halys and Echis multisquamatus may be used as a tool both for thrombosis investigations in the model systems and for diagnostic in the clinical practice. The complex of diagnostic tests developed on the base of these activators allows to characterize the haemostatic system at different pathologies and as well as to determine the unbalance between separate components of haemostasis. The tests are approved on plasma of patients with heart diseases, ulcers, nephrites, hestoses etc. The tests are sensitive, informative, easy to use and do not require an additional equipment. They have no analogues in Ukraine.     

Platelet - vessel wall interaction: role of blood clotting

Vascular damage initiates not only the adhesion and aggregation of blood platelets but also coagulation, which is of mixed (intrinsic and extrinsic) origin. Evidence is presented that thrombin, generated as a result of the injury, is a prerequisite for platelet aggregation. Platelets, after activation, in their turn promote coagulation. Prostaglandin I2 (PGI2 or prostacyclin) inhibits coagulation induced by damaged vascular tissue. This effect of PGI2 is mediated by the inhibition of platelets in their participation in the generation of factor Xa and thrombin. Dietary cod liver oil, by changing plasma coagulability, decreases the procoagulation activity of vessel walls, and arterial thrombosis. Another fish oil with similar effects on plasma coagulability and some other haemostatic parameters does not modify vessel wall-induced clotting, nor does it significantly lower arterial thrombosis tendency; this indicates the physiological relevance of vessel wall-induced clotting in arterial thrombus formation. Some evidence is also given for the importance of vessel wall-induced clotting in primary haemostasis.     

Blood platelet activation and increase in thrombin activity following a marathon race

To see whether strenuous prolonged exertion increases blood platelet activation and thrombin activity in healthy well-trained men, 16 male amateur runners (mean age 31,8) were studied. A marathon race (mean time 2 h 44 min 30 s) caused a significant increase in plasma beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), fibrinopetide A (FPA) and factor VIII (F VIII) activity. Sixty min after exertion beta-TG and F VIII activity were still significantly elevated. FPA continued to rise, reaching peak values 60 min after the run. 22 h after finishing the race F VIII activity was still significantly elevated. The study has demonstrated the great inter-individual variability of marathon race-induced haemostatic changes. The elevation of beta-TG varied from 42% to 156%, F VIII from 112% to 625%, and in three runners FPA reached more than 900% of its pre-exercise value. In some individuals the haemostatic changes observed could be potentially unfavourable for coronary heart disease prevention.     

The influence on coagulation of transdermal estrogen hormone replacement therapy as a preoperative preparation of the tissue before vaginal hysterectomy

In 32 postmenopausal patients who underwent vaginal hysterectomy due to the presence of uterine prolapse at the Department of Uro-gynaecology and Pelvic Floor Disorders in the Clinic of Gynaecology and Obstetrics, Medical School, Skopje in the period from 1st January 2002 to 1st January 2003, and who were preoperatively treated with transdermal estradiol 50 microg/day during 14 days the following parameters of the coagulating status were estimated: prothrombin time (PT) that is expressed in: absolute value, percentage and INR; activated partial thromboplastin time (aPTT Pathrombin SL); thrombin time and platelets number before and after hormone replacement therapy. After 14-day transdermal estrogen therapy, the parameters: PT, PT%, PT INR, aPTT Pathrombin SL didn't expressed significant changes, the thrombin time expressed significant extension, and the platelets expressed a significant decrease. According to our results, the transdermal estrogens might not have any influence on the hepatic synthesis of coagulating factors till the step of prothrombin formation. They might have an essential influence on the step of prothrombin transformation into thrombin, as well as on the process of megacaryocytes segregation into platelets.     

Effects of Folic Acid and Homocysteine on the Morphogenesis of Mouse Cephalic Neural Crest Cells In Vitro

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies—a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exact mechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.     

Coagulation Changes during Presyncope and Recovery

Orthostatic stress activates the coagulation system. The extent of coagulation activation with full orthostatic load leading to presyncope is unknown. We examined in 7 healthy males whether presyncope, using a combination of head up tilt (HUT) and lower body negative pressure (LBNP), leads to coagulation changes as well as in the return to baseline during recovery. Coagulation responses (whole blood thrombelastometry, whole blood platelet aggregation, endogenous thrombin potential, markers of endothelial activation and thrombin generation), blood cell counts and plasma mass density (for volume changes) were measured before, during, and 20 min after the orthostatic stress. Maximum orthostatic load led to a 25% plasma volume loss. Blood cell counts, prothrombin levels, thrombin peak, endogenous thrombin potential, and tissue factor pathway inhibitor levels increased during the protocol, commensurable with hemoconcentration. The markers of endothelial activation (tissue factor, tissue plasminogen activator), and thrombin generation (F1+2, prothrombin fragments 1 and 2, and TAT, thrombin-antithrombin complex) increased to an extent far beyond the hemoconcentration effect. During recovery, the markers of endothelial activation returned to initial supine values, but F1+2 and TAT remained elevated, suggestive of increased coagulability. Our findings of increased coagulability at 20 min of recovery from presyncope may have greater clinical significance than short-term procoagulant changes observed during standing. While our experiments were conducted in healthy subjects, the observed hypercoagulability during graded orthostatic challenge, at presyncope and in recovery may be an important risk factor particularly for patients already at high risk for thromboembolic events (e.g. those with coronary heart disease, atherosclerosis or hypertensives).     

Diets enriched in (n-3) fatty acids affect rat coagulation factors dependent on vitamin K

The effects of dietary lipids on haemostasis were investigated in rats fed high fat diets enriched in saturated fatty acids (SAT), oleic acid (OLEIC), MaxEPA^® oil (MaxEPA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) and results were compared to those for rats fed standard chow (ST). Coagulant activities of factor IIc and factor VII-Xc were reduced by about 70 % in the MaxEPA group and 50 % in the EPA and DHA groups relative to the OLEIC, SAT and ST groups. Liver vitamin K levels were five times lower in the experimental groups than in the ST group, which would indicate an effect of high fat diets on vitamin K metabolism. However, only (n-3) fatty acids prolonged the prothrombin time. These components could act at the post-transiational modification level of vitamin K-dependent plasma clotting factors. The changes in haemostatic factors found in the MaxEPA group were counteracted by vitamin K supplementation.     

Effect of protein C activator on overall haemostasis potential in donor and hip arthroplasty patient plasma

A method of overall haemostasis potential (OHP) determination for quantitative and rapid estimation of coagulation-fibrinolysis balance in plasma has been presented. The method is based on the analysis of the absorbance at 350 nm vs. time curve, which records the clot formation and dissolution in plasma in the presence of thromboplastin and t-PA. Three parameters of coagulation system--time, rate of formation and maximal turbidity of the clot, and three parameters of fibrinolytic system--half-, full-time and maximal rate of the clot dissolution, and main integral parameter--the area under the curve that characterizes the size and time of the clot existence and expresses OHP of plasma, can be determined by this method. It was shown that OHP value of patients plasma was 3.8 times more than that of the donor plasma. It is in concordance with elevated level of Fg (4.25 mg/ml), soluble fibrin (50 microg/ml), D dimer (630 ng/ml) and insufficient decrease of APC activity (93%) in patients. AcPC, added to donor and patient plasma, reduced OHP value 1.6 and 3.7 times, correspondingly. AcPC increased amidase activity of thrombin and APC in the donor plasma, and did not change that of plasmin. These data indicated that the effect of AcPC on OHP is mediated by formation ofAPC that helps to reduce the level of inhibitors in the investigated plasma.     

Syncytiotrophoblast microvesicles released from pre-eclampsia placentae exhibit increased tissue factor activity

Background

Pre-eclampsia is a complication of pregnancy associated with activation of coagulation. It is caused by the placenta, which sheds increased amounts of syncytiotrophoblast microvesicles (STBM) into the maternal circulation. We hypothesized that STBM could contribute to the haemostatic activation observed in pre-eclampsia.

Methodology/Principal Findings

STBM were collected by perfusion of the maternal side of placentae from healthy pregnant women and women with pre-eclampsia at caesarean section. Calibrated automated thrombography was used to assess thrombin generation triggered by STBM-borne tissue factor in platelet poor plasma (PPP). No thrombin was detected in PPP alone but the addition of STBM initiated thrombin generation in 14/16 cases. Pre-eclampsia STBM significantly shortened the lag time (LagT, P = 0.01) and time to peak thrombin generation (TTP, P = 0.005) when compared to normal STBM. Blockade of tissue factor eliminated thrombin generation, while inhibition of tissue factor pathway inhibitor significantly shortened LagT (p = 0.01) and TTP (P<0.0001), with a concomitant increase in endogenous thrombin potential.

Conclusions/Significance

STBM triggered thrombin generation in normal plasma in a tissue factor dependent manner, indicating that TF activity is expressed by STBM. This is more pronounced in STBM shed from pre-eclampsia placentae. As more STBM are shed in pre-eclampsia these observations give insight into the disordered haemostasis observed in this condition.     

Haemostasis during transvesical prostatic adenomectomy. A controlled trial on the effect of drugs with antifibrinolytic and thrombin-like activities.

Blood loss, plasma fibrinolytic activity, prothrombin, P.T.T., and fibrinogen plasma levels were measured in 30 patients subjected to transvesical adenomectomy. These parameters were evaluated during 7 consecutive days after the operation. Ten patients received the antifibrinolytic substance (AMCHA), ten patients received Bothrops Jararaca venom extract, and ten patients served as controls. The results show that the coagulating fraction of Bothrops Jararaca snake venom reduces intraoperative bleeding without influencing the haemostatic balance. Postoperative haematuria could be reduced by antifibrinolytic agents.     

Microangiopathic hemolytic anemia in patients with diseases of the hematopoietic and lymphatic systems

Microangiopathic haemolytic anaemia was diagnosed in the course of haematopoietic and lymphatic disorders such as chronic granulocytic leukemia, chronic myelofibrosis, chronic lymphatic leukemia, Osler's disease, chronic monocytic leukemia, and lymphoplasmocytic lymphoma, in 11 patients (6 women and 5 men) aged between 33 and 81 years (mean age 58.8 years) treated at the Haematological Out-Patient Clinic of the Postgraduate Medical Education Centre within 1977-1987. The following laboratory tests were carried out: 1) morphology of the peripheral blood and bone marrow, especially some haematological parameters concerning erythrocytes and blood platelets; 2) biochemical tests reflecting erythrocytes disintegration; 3) haemostasis. All examined patients suffered from haemolytic anaemia of various degree with characteristic changes in erythrocyte shape (helmets, tear-drops etc.). Haemolytic origin of anaemia was confirmed by the increased LDH activity. In the majority of patients no compensative stimulation of haematopoiesis (reticulocytosis, red blood cells hyperproliferation in bone marrow) was seen. Clinical symptoms of haemostatic disorders such as haemorrhagic diathesis and vein thrombosis were diagnosed in 50% of the patients. Blood platelet counts ranged from markedly decreased to significantly increased. Bone marrow smears did not show increased number of megacariocytes. Bleeding time was prolonged in the majority of examined patients while prothrombin index--decreased). Abnormal fibrinogen levels (decreased or increased) were found in the majority of patients with fibrin degradation products. Microangiopathic haemolytic anaemia in these patients differ from the typical Moschowitz's disease clinically probably due to the lack of compensative stimulation of erythropoiesis and lower thrombocytopenia.     

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.     

Protein Carboxyl Methylation in Synaptic Membrane of Rat Brain: The Possible Presence of Adenosine-Bound S-Adenosyl-L-Homocysteine Hydrolase in the Membrane

The effects of some neurotransmitters, adenosine (Ad), and homocysteine (Hcys) on protein carboxyl methylation in synaptic plasma membranes from rat cerebral cortex were examined. Neither any of the neurotransmitters nor Ad had a detectable effect. Incubation of membrane with DL-Hcys alone (5 X 10(-5) M), the combination of both Ad (5 X 10(-5)) and DL-Hcys (5 X 10(-5)), or S-adenosyl-L-homocysteine (SAH) (1 X 10(-6)) strongly decreased the methyl ester formation. The inhibitory effect of the combination of both compounds may be interpreted in terms of the increased SAH concentration due to the presence of SAH hydrolase in the membrane. The inhibitory effect of Hcys alone was blocked by preincubation with Ad deaminase or Neplanocin A, a potent inhibitor of SAH hydrolase, suggesting the presence of Ad-bound SAH hydrolase in the synaptic membrane. Ad-bound SAH hydrolase activity estimated by the inhibition of methylation in the presence of Hcys was located in the membrane fractions including synaptosomes, myelin, and microsomes (about 70%), but the SAH hydrolase activity estimated on the basis of the inhibitory effect of the combination of both Ad and Hcys was localized exclusively in the soluble fraction (about 90%). The distribution of the latter activity is coincident with that of SAH hydrolase reported to date. Incubation of the synaptic membrane with Hcys markedly increased the SAH concentration. The stimulatory effect of Hcys alone was blocked by Ad deaminase.     

Homocysteine concentrations in a German cohort of 500 individuals: Reference ranges and determinants of plasma levels in healthy children and their parents

Summary. Elevated plasma homocysteine is a risk factor for cardiovascular disease and a sensitive marker of inadequate vitamin B12 and folate status. We studied 257 pupils (120 boys, 137 girls, aged 6–17 years) and their parents (88 males, 172 females, aged 26–50 years). Our measurements were part of a national Bavarian health and nutrition examination survey evaluating cardiovascular risk factors. A mild hyperhomocysteinemia (Hcys >15 μmol/l) occurred in 7% of the adults, but in none of the children. Men had significantly higher Hcys levels than women (p < 0.0001), boys and girls had comparable concentrations. For adults and children, Hcys correlated inversely with vitamin B12 and folate and positively with the lean body mass and creatinine in serum, but not with cystatin C. Genetic and nutritional factors are determinants of Hcys metabolism. The correlation of Hcys and serum creatinine is dependent on the metabolic link between Hcys production and creatine synthesis. Received July 4, 1999 Accepted May 22, 2000     

高凝状态对非小细胞肺癌患者深静脉血栓形成的影响

目的:探讨高凝状态对非小细胞肺癌患者深静脉血栓形成的影响,为临床治疗提供依据。方法:选择2010年5月至2013年6月在我院接受治疗的非小细胞肺癌患者195例,记录患者姓名、年龄、性别、病理类型、手术、临床分期、放化疗、伴随疾病、身体状况评分(eastern cooperative oncology group,ECOG评分)及是否发生血栓等指标。调查入选患者的高凝状态指标,包括血小板(platelet,PLT)、纤维蛋白原(fibrinogen,Fib)、血浆凝血酶原时间(plasma prothrombin time,PT)、凝血酶时间(thrombin time,TT)、活化部分凝血活酶时间(activated coagulation time of whole blood,APTT)和D-二聚体(D-dimer)。结果:195例非小细胞肺癌患者中,PLT高于正常上限的有42例(21.5%),FIB升高的有78例(40%),TT延长的有12例(6.2%),PT缩短的有45例(23.1%),D-D升高的有60例(30.8%),APTT缩短的有15例(7.7%)。两项指标异常的有62例(31.8%),FIB和D-D升高的有32例(16.4%);三项指标异常的有27例(13.8%),PLT、FIB及D-D升高的有15例(7.7%);四项指标异常的有5例(2.6%),PLT、FIB及D-D升高,而PT缩短的有5例(2.6%)。所有患者各凝血指标中位值PLT为219×109/L,FIB为3.6 g/L,TT为13.4 s,PT为10.8 s,D-D为0.341 mg/L,APTT为29.7 s。结论:高凝状态对非小细胞肺癌患者深静脉血栓形成具有重要的促进作用,应受到临床的重视。     

Rat prothrombin: Purification,characterization, and activation

Prothrombin has been purified from rat plasma and its properties compared to prothrombin isolated from other species. The molecular weight, amino acid composition, and amino-terminal sequence of rat prothrombin are similar to human and bovine prothrombin. Rat prothrombin binds to phospholipid in the presence of calcium ions, and calcium-binding measurements indicate that it may bind somewhat more calcium than does bovine prothrombin. The proteolytic cleavage of purified rat prothrombin by Factor X_a or thrombin yields the same peptides that are formed from similar proteolysis of bovine prothrombin. Factor V and phospholipid were shown to enhance the rate of Factor X_a and calcium ion generation of thrombin from rat prothrombin.