[Comparison of prolactin, free T3 and free T4 levels in the follicular fluid of infertile women and healthy fertile oocyte donors]. / Srovnání hladin prolaktinu, fT3 a fT4 ve folikulární tekutine zen s poruchou plodnosti a zdravých plodných dárkyn oocytu

OBJECTIVE: Follicular fluid (FF) provides an important microenvironment for the development of oocytes. The biochemical composition of the FF plays critical role in the oocyte competence. The aim of our study was to compare the levels of selected hormones in FF of infertile women and healthy fertile oocyte donors. DESIGN: Retrospective comparative study. SETTING: Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague; Department of Gynecology and Obstetrics, Charles University and University Hospital in Pilsen; Institute of Reproductive Medicine and Endocrinology, IVF Centers Prof. Zech, Plzen. METHODS: Levels of prolactin, free T3 and free T4 hormones in the FF of 146 women were analyzed. We have analysed FF of 74 infertile patients (mean age 31 years, SD = 4.65) and 72 healthy fertile oocyte donors (mean age 26 years, SD = 4.44). Only blood free samples were studied after pooling of all FF samples each patient. Levels of hormones were determined using ECLIA method (Electro-Chemi-Luminiscent Immunoassay) on the Cobas e411. RESULTS: RESULTS showed statistically significantly higher levels of prolactin (p=0.0006) and free T4 hormone (p=0.0246) in FF of infertile women in comparison to the group of healthy fertile oocyte donors. CONCLUSION: Our study confirms the presence of prolactin and thyroid hormones in FF and it can be suggested that they play a key role in the regulation of reproductive processes. The study of FF from donors and their detailed comparison with infertile patients with various gynaecological causes of infertility has great value for better understanding of regulatory mechanisms of fertility.

[Comparison of selective oxidative stress parameters in the follicular fluid of infertile women and healthy fertile oocyte donors]. / Srovnání vybraných parametru oxidacního stresu ve folikulární tekutine u zen s poruchou plodnosti a u zdravých fertilních dárkyn oocytu

OBJECTIVE: Follicular fluid (FF) affects oocyte development and disruption of its homeostasis has a crucial effect on egg developmental potential. The aim of this study was to compare the levels of selected oxidative stress markers in the FF of women with impaired fertility and healthy fertile oocytes donors. DESIGN: A retrospective comparative study. SETTING: Faculty of Medicine in Pilsen, Charles University in Prague; Institute of Reproductive Medicine and Endocrinology, IVF Center Prof. Zech, Pilsen. METHODS: Levels of homocysteine (Hcy), malondialdehyde (MDA), glutathione peroxidase (GPx), total antioxidant capacity (AOK) and total protein (CB) were analyzed in the FF. We have analysed FF of 146 women - 74 infertile patients (mean age 31 years, SD = 4.65) and 72 healthy fertile oocyte donors (mean age 26 years, SD = 4.44). Only blood free samples were studied after pooling of all FF samples each patient. RESULTS: The study showed a statistically significantly higher Hcy levels (p < 0.0001) in the FF of healthy fertile women compared with impaired fertility group both - comparing the two groups regardless the age and in groups of the same age range (for the age group between 20 to 29 years isp = 0.0002, for the age group between 30 to 39 years is p < 0.0001). When divided into above age ranges we found statistically significantly higher levels of MDA in the control group aged 20 to 29 years compared to same age infertile patients (p = 0.0374) and statistically significantly higher AOK in infertile women between 30 to 39 years of age compared to same age control group (p = 0.0458). CONCLUSION: The presence or on the contrary the absence of prooxidant parameters in the FF has an important role in the ability of conception and subsequent embryo development.

Effect of Beer Consumption on Methylation and Redox Metabolism.

To investigate the influence of beer consumption on levels of homocysteine (HCY), vitamin B6, B12, folic acid (FA), dimethylglycine (DMG), betaine (BET) and other selected markers. One hundred and sixteen male volunteers were enrolled in the study. A one-month period of alcohol abstinence was followed by a one month when participants drank 830 mL of alcoholic beer every day. After that phase, one month of alcohol abstinence followed. At the beginning and after every phase blood samples were taken and analysed. Ninety-three participants completed the study. After the phase of alcohol consumption, uric acid (UA) (p<0.0001), antioxidative capacity (AOC) (p=0.02), superoxide dismutase (SOD) (0.025), glutathione reductase (GRH) (0.0001), total cholesterol (p<0.0001), HDL-cholesterol (p<0.0001), Apolipoprotein-AI (ApoAI) (p<0.0001), LDL-cholesterol (p<0.039) and Apolipoprotein B (ApoB) (p<0.009) increased, while vitamin B12 (p=0.0001) and fibrinogen (p<0.0001) decreased. Other tested parameters (DMG, BET, vitamin B6 and FA) did not show any significant changes. UA changes and changes in AOC were statistically significantly correlated (r=0.52, p<0.0001). HCY, DMG and BET levels did not show any statistically significant changes after beer consumption, whereas some markers of redox metabolism increased (UA, AOC, SOD and GRH). A statistically significant correlation denotes the dependence of UA and AOC changes in connection with beer consumption.

Mitochondrial dysfunction in kidney cortex and medulla of subtotally nephrectomized rats.

Five-sixths nephrectomy is a widely used experimental model of chronic kidney disease (CKD) that is associated with severe mitochondrial dysfunction of the remnant tissue. In this study, we assessed the effect of CKD on mitochondrial respiration separately in the rat kidney cortex and medulla 10 weeks after induction of CKD by subtotal 5/6 nephrectomy (SNX). Mitochondrial oxygen consumption was evaluated on mechanically permeabilized samples of kidney cortex and medulla using high-resolution respirometry and expressed per mg of tissue wet weight or IU citrate synthase (CS) activity. Mitochondrial respiration in the renal cortex of SNX rats was significantly reduced in all measured respiratory states if expressed per unit wet weight and remained lower if recalculated per IU citrate synthase activity, i.e. per mitochondrial mass. In contrast, the profound decrease in the activity of CS in SNX medulla resulted in significantly elevated respiratory states expressing the OXPHOS capacity when Complexes I and II or II only are provided with electrons, LEAK respiration after oligomycin injection, and Complex IV-linked oxygen consumption per unit CS activity suggesting compensatory hypermetabolic state in remaining functional mitochondria that is not sufficient to fully compensate for respiratory deficit expressed per tissue mass. The results document that CKD induced by 5/6 nephrectomy in the rat is likely to cause not only mitochondrial respiratory dysfunction (in the kidney cortex), but also adaptive changes in the medulla that tend to at least partially compensate for mitochondria loss.

Dynamic changes of high-sensitivity troponin T concentration during infancy: Clinical implications.

Cardiac troponin T determination plays a dominant role in diagnosis of myocardial pathologies. Despite generally accepted use of high-sensitive cardiac troponin T assays (hscTnT) and clearly defined cut-off limit in adults, the uncertainty persists in infants. The aim of this study was to assess plasmatic concentrations of hscTnT and describe sequential age-related dynamic changes of hscTnT in healthy infants and toddlers. Seventy-eight children (52 males/26 females) from Czech Republic aged 44 to 872 days (median, interquartile range 271; 126 to 486 days) were consecutively enrolled in the single-center, prospective observational study. Plasma concentrations of hscTnT were analyzed by the electrochemiluminescent method, age-related reference intervals were calculated using the polynominal regression model. Amongst the study population (n=78), the upper limit of hscTnT concentration defined as the 99th percentile was calculated. The 99th percentile with 95 % confidence interval at the end of 2nd, 3rd, 4th, 5th, 6th and 7th month of postnatal life were: 81 (40.6 to 63.6), 61 (36.0 to 55.3), 47 (31.9 to 48.3), 37 (28.1 to 42.3), 30 (24.7 to 37.2) and 25 (21.5 to 32.7) ng/l, respectively. Concentration of adults 99th percentile (14 ng/l) was achieved approximately at 1 year of postnatal life. Statistically significant negative correlation of hscTnT concentration with age (r=-0.81, p<0.001) was found. Significant gender differences were not found (p>0.07). The study revealed substantially increased reference intervals of hscTnT levels in infants when compared with adult population. Based on our preliminary results, the age-related interpretation of hscTnT plasmatic concentration is recommended.

Cardiovascular parameters in rat model of chronic renal failure induced by subtotal nephrectomy.

Chronic renal failure (CRF) is associated with high incidence of cardiovascular complications. To clarify pathogenesis of CRF numerous animal models have been developed. The aim of our work was to describe methodology of subtotal surgical renal ablation in rat and to characterize some biochemical and cardiovascular parameters of this animal model. Male rats underwent 5/6 surgical nephrectomy or sham operations in two steps. The following parameters were measured on day 10 and in week 10 after the surgery: plasma concentrations of creatinine and urea, blood pressure, resting heart rate, chronotropic response to atropine and metipranol, heart ventricles weight, contraction parameters and action potential duration in the left ventricle. Increased serum concentrations of creatinine and urea, decreased creatinine clearance, polyuria and alteration of the remnant kidney tissue were found in CRF rats. Changes in cardiovascular parameters identified after subtotal nephrectomy resembled alterations of cardiovascular system in uremic patients and included hypertension, elevated resting heart rate, diminished parasympathetic cardiac tone, hypertrophy of the left ventricle associated with weakened force of contraction, prolonged contraction and relaxation and shortening of action potential duration. These data suggest that the present model can be a useful tool in the study of CRF and its cardiovascular complications.

NGAL, albumin and cystatin C during cisplatin therapy.

Cisplatin is a commonly used chemotherapeutic drugs. It is known for its nephrotoxic side effects with an increased risk of acute kidney injury. Finding of clinically feasible cisplatin nephrotoxicity markers is of importance. In our study, we compared neutrophil gelatinase-associated lipocalin (NGAL) in serum and urine, the estimated glomerular filtration rate (based on serum cystatin C) and urine albumin as markers of nephrotoxicity. The study involved 11 men and 9 women (mean ± SD age 58.2±9.5 years) with different malignancies treated with cisplatin in four cycles of chemotherapy (I - IV). Samples 0-4 were taken before, immediately after, in 3, 6 and 24 hours after administering chemotherapy. We detected significant increase of ACR in Sample 2 (p=0.03) and decrease of eGFR in Sample 4 (p=0.03) up to 24 hours after cisplatin administration in the first chemotherapy cycle only. When cumulative effect of cisplatin was assessed, significantly increased values of urine albumin (vs cycle I) were found in Sample 0 (p=0.00058), 1 (p=0.00256), 2 (p=0.00456), 3 (p=0.00006) and 4 (p=0.00319) in cycles II to IV. We found a correlation between values of urine NGAL and urine albumin (r=0.68, p<0.0001). In conclusion, urine albumin was the only measured marker that consistently and statistically significantly increased after cisplatin containing chemotherapy cycles.

Norepinephrine turnover in the left ventricle of subtotally nephrectomized rats.

Increased activity of the sympathetic nervous system (SNS) has been proposed as a risk factor for increased cardiovascular mortality in patients with chronic kidney disease (CKD). Information on the activity of cardiac sympathetic innervation is non-homogeneous and incomplete. The aim of our study was to evaluate the tonic effect of SNS on heart rate, norepinephrine turnover and direct and indirect effects of norepinephrine in left ventricles of subtotally nephrectomized rats (SNX) in comparison with sham-operated animals (SHAM). Renal failure was verified by measuring serum creatinine and urea levels. SNX rats developed increased heart rates and blood pressure (BP). The increase in heart rate was not caused by sympathetic overactivity as the negative chronotropic effect of metipranolol did not differ between the SNX and SHAM animals. The positive inotropic effects of norepinephrine and tyramine on papillary muscle were not significantly different. Norepinephrine turnover was measured after the administration of tyrosine hydroxylase inhibitor, pargyline, tyramine, desipramine, and KCl induced depolarization. The absolute amount of released norepinephrine was comparable in both groups despite a significantly decreased norepinephrine concentration in the cardiac tissue of the SNX rats. We conclude that CKD associated with renal denervation in rats led to adaptive changes characterized by an increased reuptake and intracellular norepinephrine turnover which maintained normal reactivity of the heart to sympathetic stimulation.

Prognostic Value of MicroRNAs in Patients after Myocardial Infarction: A Substudy of PRAGUE-18.

BACKGROUND: The evaluation of the long-term risk of major adverse cardiovascular events and cardiac death in patients after acute myocardial infarction (AMI) is an established clinical process. Laboratory markers may significantly help with the risk stratification of these patients. Our objective was to find the relation of selected microRNAs to the standard markers of AMI and determine if these microRNAs can be used to identify patients at increased risk. METHODS: Selected microRNAs (miR-1, miR-133a, and miR-499) were measured in a cohort of 122 patients from the PRAGUE-18 study (ticagrelor vs. prasugrel in AMI treated with primary percutaneous coronary intervention (pPCI)). The cohort was split into two subgroups: 116 patients who did not die (survivors) and 6 patients who died (nonsurvivors) during the 365-day period after AMI. Plasma levels of selected circulating miRNAs were then assessed in combination with high-sensitivity cardiac troponin T (hsTnT) and N-terminal probrain natriuretic peptide (NT-proBNP). RESULTS: miR-1, miR-133a, and miR-499 correlated positively with NT-proBNP and hsTnT 24 hours after admission and negatively with left ventricular ejection fraction (LVEF). Both miR-1 and miR-133a positively correlated with hsTnT at admission. Median relative levels of all selected miRNAs were higher in the subgroup of nonsurvivors (N = 6) in comparison with survivors (N = 116), but the difference did not reach statistical significance. All patients in the nonsurvivor subgroup had miR-499 and NT-proBNP levels above the cut-off values (891.5 ng/L for NT-proBNP and 0.088 for miR-499), whereas in the survivor subgroup, only 28.4% of patients were above the cut-off values (p = 0.001). CONCLUSIONS: Statistically significant correlation was found between miR-1, miR-133a, and miR-499 and hsTnT, NT-proBNP, and LVEF. In addition, this analysis suggests that plasma levels of circulating miR-499 could contribute to the identification of patients at increased risk of death during the first year after AMI, especially when combined with NT-proBNP levels.

How to interpret elevated plasmatic level of high-sensitive troponin T in newborns and infants?

Research and clinical implications on novel cardiac biomarkers has intensified significantly in the past few years. The high-sensitive troponin T (hscTnT) assay plays a dominant role in diagnostic algorithm regarding myocardial injury in adults. Despite generally accepted use of hscTnT there are no data about physiological concentrations and cut-off limits in neonates and infants to date. The aim of this study is to assess hscTnT levels in healthy newborns and infants. Consecutively 454 healthy full termed newborns and 40 healthy infants were enrolled in the study. Samples of cord or venous blood were drawn and tested for hscTnT concentrations with high-sensitive TnT assay (Roche Cobas e602 immunochemical analyzer). The 97.5 percentile of hscTnT concentration was assessed and correlation analysis was performed in neonates. Two hundred and thirteen samples (47 %) were excluded due to blood hemolysis of various degrees in neonates. Finally, the group of 241 healthy newborns was statistically analyzed. The median concentration of hscTnT was 38.2 ng/ml, 97.5 percentile reached 83.0 ng/l (confidential interval 74.1 to 106.9 ng/l). HscTnT concentrations were statistically decreased in hemolytic samples when compared to non-hemolytic samples (34.3 ng/l [26.7 to 42.0 ng/l] and 37.1 ng/l [30.5 to 47.9 ng/l], respectively, p=0.003). Elevated plasma concentrations of hscTnT decreased to adult level within six months. This study has confirmed the higher reference levels of hscTnT in neonates and young infants when compared with adult population. Many extracardiac factors as hemolysis and age may affect the hscTnT level. Based on presented results, a careful clinical interpretation of hscTnT is recommended.

Effect of white wine consumption on oxidative stress markers and homocysteine levels.

The aim of this study was to assess the influence of regular daily consumption of white wine on oxidative stress and cardiovascular risk markers. Forty-two healthy male volunteers consumed 375 ml of white wine daily. Each participant provided three venous blood samples (before wine consumption, following the wine consumption period and again a month later). Levels of superoxide dismutase, glutathione peroxidase, reduced glutathione, total antioxidant capacity, total cholesterol, HDL-cholesterol, apolipoprotein A I, apolipoprotein B, triglycerides, paraoxonase 1, C-reactive protein, homocysteine, thiobarbituric acid reactive substances (TBARS) and advanced oxidation protein products (AOPP) were measured. Immediately following the month of white wine consumption there was a significant increase in HDL-cholesterol (p<0.0001), paraoxonase 1 (p<0.001), glutathione peroxidase (p<0.001) and reduced glutathione (p<0.01) levels, a decrease in superoxide dismutase activities (p<0.0001), and a decrease in oxidation protein products (p<0.001) and TBARS (p<0.05) concentrations. However, there was also a clear increase in homocysteine (p<0.0001) after a month of white wine consumption. The results of our non-placebo controlled trial suggest that regular daily white wine consumption is associated not only with both antioxidative and antiatherogenic effects but also with a potentially proatherogenic increase of homocysteine concentrations.

[Asymmetric dimethylarginine--a novel cardiovascular risk factor]. / Asymetrický dimetylarginin--nový rizikový faktor kardiovaskulárních onemocnení.

Understanding metabolism of nitric oxide (NO), signal molecule releasing from endothelial cells and influencing vascular tone, belongs to the most remarkable knowledge of last ten years. NO increases vascular tone, inhibits adhesion of monocytes and leukocytes to the vascular endothelium and reduces atherogenic process. Low NO level is one of pathogenic factors starting cardiovascular diseases. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of enzyme NO synthase, enzyme catalyzing NO production from arginine. This article gives a brief overview of contemporary state of the relation between ADMA and cardiovascular diseases. Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilatation. In several prospective studies, ADMA evolved as a marker of cardiovascular risk. In the first chapters is described state of the art of biosynthesis, degradation and excretion of ADMA in connection with endothelial dysfunction, coronary artery disease, chronic heart failure, cardiovascular risk in haemodialysis patients, diabetes mellitus, hypertension, lipid metabolism disorders and intensive care unit treatment. Next chapters shortly summarize methods of ADMA detection and their applications. In conclusion clinical relevance of measurement of ADMA levels as a marker of endothelial dysfunction is discussed. Future research tasks of ADMA lead to prospective studies with different types of patients and also healthy population. Moreover ADMA is becoming a goal for pharmacotherapeutic intervention to improve endothelium-dependent vascular function in subjects with high ADMA levels.

Betaine increases the butyrylcholinesterase activity in rat plasma.

The physiological function of butyrylcholinesterase (EC 3.1.1.8, BChE) is not clearly understood, but a role was suggested in the fat utilization process, resulting in positive correlation between plasma triglyceride (TG) levels and BChE activity. Consequently we tested the hypothesis that regular intake of betaine, a natural compound intervening in the liver TG metabolism could influence the BChE activity. The BChE activity was estimated spectrophotometrically in plasma of rats fed with betaine enriched standard (B) or high-fat diet (HFB). The results confirmed decreased TG plasma levels after betaine treatment independently on the type of diet (0.15+/-0.03 (B) vs. 0.27+/-0.08 (control) mmol/l; p=0.003 and 0.13+/-0.03 (HFB) vs. 0.27+/-0.08 (control) mmol/l; p=0.005). The BChE activity increased significantly with betaine administration, however the change was more distinct in the HFB group (0.84+/-0.34 (HFB) vs. 0.22+/-0.04 (control) O.D./min/mg; p<0.001 and 0.41+/-0.11 (B) vs. 0.22+/-0.04 (control) O.D./min/mg; p=0.001). In conclusion, betaine intake led to elevated BChE activity in plasma and this effect was potentiated by the HF diet. Since betaine is in general used as a supplement in the treatment of liver diseases accompanied by TG overload, its impact on the BChE activity in the role of the liver function marker should be taken into account.

Markers of oxidative stress in diabetic mothers and their infants during delivery.

Oxidative stress is probably a pathophysiological process leading to disadvantageous outcomes in diabetic pregnancies. We aimed to map a complex of potential markers of oxidative stress in this condition. Diabetic mothers had significantly higher concentrations of thiobarbituric acid reactive substances in the plasma [TBARS] both before (p<0.0001) and after (p<0.001) delivery and also their newborns showed higher values of TBARS (p<0.0001) in comparison with the control group. Diabetic mothers also showed lower concentrations of reduced glutathione in erythrocytes [GSH] both before (p<0.05) and after (p<0.01) delivery and their infants also had lower levels of GSH (p<0.0001). We found a lower total antioxidative capacity of plasma [AOC] before delivery (p<0.05) in the diabetic group in comparison with the control group. Newborns of diabetic mothers had higher plasmatic concentrations of apolipoproteine B [apo B] (p<0.05), higher erythrocyte glutathione peroxidase [GPx] activity (p<0.05) and lower pH (p<0.001) in the umbilical cord blood, when compared with infants of control non-diabetic mothers. We conclude that pregestational and gestational diabetes mellitus represent increased oxidative stress for both mother and her infant. TBARS in plasma are a valuable marker of oxidative stress in this condition. Disruption of glutathione peroxidase/glutathione pattern can be involved in pathophysiology of enhanced oxidative stress in diabetic pregnancies.

Two cases of spuriously elevated cerebrospinal glucose concentration.

We present two cases of spuriously high cerebrospinal fluid glucose concentration with approximately normal blood glucose concentrations. The cause of these abnormal findings was the use of inappropriate collection tubes during the pre-analytical phase. Whilst no patient harm was identified following this error, significant time and effort were expended by both laboratory and clinical staff to explain the cause of these findings.