Prediction of Enzyme Catalysis by Computing Reaction Energy Barriers via Steered QM/MM Molecular Dynamics Simulations and Machine Learning.

The prediction of enzyme activity is one of the main challenges in catalysis. With computer-aided methods, it is possible to simulate the reaction mechanism at the atomic level. However, these methods are usually expensive if they are to be used on a large scale, as they are needed for protein engineering campaigns. To alleviate this situation, machine learning methods can help in the generation of predictive-decision models. Herein, we test different regression algorithms for the prediction of the reaction energy barrier of the rate-limiting step of the hydrolysis of mono-(2-hydroxyethyl)terephthalic acid by the MHETase ofIdeonella sakaiensis. As a training data set, we use steered quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulation snapshots and their corresponding pulling work values. We have explored three algorithms together with three chemical representations. As an outcome, our trained models are able to predict pulling works along the steered QM/MM MD simulations with a mean absolute error below 3 kcal mol-1 and a score value above 0.90. More challenging is the prediction of the energy maximum with a single geometry. Whereas the use of the initial snapshot of the QM/MM MD trajectory as input geometry yields a very poor prediction of the reaction energy barrier, the use of an intermediate snapshot of the former trajectory brings the score value above 0.40 with a low mean absolute error (ca. 3 kcal mol-1). Altogether, we have faced in this work some initial challenges of the final goal of getting an efficient workflow for the semiautomatic prediction of enzyme-catalyzed energy barriers and catalytic efficiencies.

Albumin of People with Diabetes Mellitus Is More Reduced at Low HbA1c.

Oxidative stress is involved in the development, progression, and complications of diabetes mellitus (DM). Oxidative modification of human serum albumin's cysteine-34 is a marker for oxidative stress-related pathological conditions. We aimed to evaluate the redox state of albumin in patients with DM to investigate possible correlations with age, diabetes duration, and disease control status. Plasma aliquots were collected from 52 participants (26 type 1 and 26 type 2 DM). Patients were divided into two groups according to their glycated hemoglobin levels less than or equal to and greater than 58 mmol/L. Albumin redox state was assessed with high-performance liquid chromatography by fractionating it into human mercaptalbumin (HMA) and human nonmercaptalbumin 1 and 2 (HNA1 and HNA2). Albumin redox fractions were differently related to the age of study participants. In age-matched T1DM and T2DM groups, the albumin redox state was essentially the same. Irreversibly oxidized HNA2 was positively correlated with diabetes duration, especially in the T1DM group. HNA was increased in people with an increased HbA1c (>58 mmol/mol). Our results support the hypothesis that oxidative stress plays a crucial role in DM pathogenesis and emphasize the importance of diabetes control on systemic oxidative burden.

Age progression from vicenarians (20-29 year) to nonagenarians (90-99 year) among a population pharmacokinetic/pharmacodynamic (PopPk-PD) covariate analysis of propofol-bispectral index (BIS) electroencephalography.

BACKGROUND: Pharmacokinetic/pharmacodynamic (PK/PD) modeling has made an enormous contribution to intravenous anesthesia. Because of their altered physiological, pharmacological and pathological aspects, titrating general anesthesia in the elderly is a challenging task. METHODS: Eighty patients were consecutively enrolled divided by decades from vicenarians (20-29 year) to nonagenarians (90-99 year) into eight groups. Using target controlled infusion (TCI) and electroencephalographic (EEG)-derived bispectral index (BIS) we set propofol plasma concentration (Cp) to gradually reach 3.5 µg mL-1 over 3.5-min. In each patient, we constructed a PK/PD model and conducted a population PK/PD (PopPK-PD) covariate analysis. RESULTS: Age was significant covariate for baseline BIS effect (E0), inhibitory propofol concentration at 50% BIS decline (IC50) and maximum BIS decline (Emax). First-order rate constant Ke0 of 0.47 min-1 in vicenarians (20-29 year) gradually increased with age-progression to 1.85 min-1 in nonagenarians (90-99 year). Simulation modelling showed that clinically recommended Cp of 3.5 µg mL-1 for 20-29 year BIS 50 should be reduced to 3.0 for 30-49 year, 2.5 for 50-69 year and 2.0 for 80-89 year. CONCLUSION: We quantified and graded EEG-BIS age-progression among different age groups divided by decades. We demonstrated deeper BIS values with decades' age progression. Our data has important implications for propofol dosing. The practical information for physicians in their daily clinical practice is using propofol Cp of 3.5 µg mL-1 might not yield BIS value of 50 in elderly patients. Our simulations showed that the recommended regimen of Cp 3.5 µg mL-1 for 20-29 year should be gradually decreased to 2.0 µg mL-1 for 80-89 year. CLINICAL TRIAL REGISTRY NUMBERS: European Community Clinical Trials Database EudraCT (http://eudract.emea.eu) initial trial registration number: 2011-002847-81, and subsequently registered at www.clinicaltrials.gov; trial registration number: NCT02585284. Xijing Hospital of Fourth Military Medical University ethics committee approval number 20110707-4.

The chronic kidney disease epidemiology collaboration equation combining creatinine and cystatin C accurately assesses renal function in patients with cirrhosis.

BACKGROUND: Accurate measurement of renal function in cirrhotic patients is still challenging. To find the best test for the determination of the true glomerular filtration rate (GFR) in cirrhotic patients this study prospectively compared measured (m)GFR, the gold standard, with estimated (e)GFR using equations based on serum levels of creatinine and cystatin C. METHODS: GFR was measured by sinistrin clearance using the bolus method in 50 patients with cirrhosis (Child Turcotte Pugh score A, B and C) and 24 age-matched healthy subjects as controls. Measured (m)GFR was compared to eGFR using bias, accuracy 10 % and 30 %, as well as correlation coefficients. RESULTS: Creatinine-based equations generally overestimated GFR in patients with cirrhosis and showed a bias (average difference between mGFR and eGFR) of -40 (CG), -12 (MDRD) and -9 (CKD-EPI-Cr) ml/min/1.73 m(2). Cystatin C-based equations underestimated GFR, especially in patients with Child Turcotte Pugh score C (bias 17 ml/min/1.73 m(2)for CKD-EPI-CysC). Of these equations, the CKD-EPI equation that combines creatinine and cystatin C (CKD-EPI-Cr-CysC) showed a bias of 0.12 ml/min/1.73 m(2) as compared to measured GFR. CONCLUSIONS: The CKD-EPI equation that combines serum creatinine and cystatin C measurements shows the best performance for accurate estimation of GFR in cirrhosis, especially at advanced stages.

Caliper vs. Lipometer--Comparing Two Methods of Subcutaneous Body Fat Measurement by Bland-Altman Diagrams.

Skinfold Calipers are widely used to obtain subcutaneous adipose tissue thickness because of its non-invasive, simple and inexpensive technique. Nevertheless, Caliper skinfold thicknesses have the disadvantage of measuring compressed adipose tissue and double layers of skin, which might reduce the precision of these results. In contrast, the computerized optical device Lipometer was developed to permit a quick, precise and non-invasive determination of non-compressed mono layers of subcutaneous adipose tissue thickness. In the present paper we investigate the hypothesis that Caliper skinfold thicknesses are significantly different from subcutaneous adipose tissue thicknesses in mm, which can be measured by Lipometer. Caliper and Lipometer results were obtained from 371 Estonian boys aged between 9.0 and 12.8 years. Measurements were performed at six different body sites: triceps, biceps, upper back, upper abdomen, hip and front thigh. Caliper measurements were systematically higher than Lipometer results in a range between 1.2 mm (hip) and 11.08 mm (front thigh). The limits of agreement analysis provided intervals from 7.5 mm (biceps) up to 30.14 mm (front thigh). Comparing Caliper and Lipometer results very low measurement agreement was found. The two methods provided very poor interchangeability.

Optimum binary cut-off threshold of a diagnostic test: comparison of different methods using Monte Carlo technique.

BACKGROUND: Using Monte Carlo simulations, we compare different methods (maximizing Youden index, maximizing mutual information, and logistic regression) for their ability to determine optimum binary cut-off thresholds for a ratio-scaled diagnostic test variable. Special attention is given to the stability and precision of the results in dependence on the distributional characteristics as well as the pre-test probabilities of the diagnostic categories in the test population. METHODS: Fictitious data sets of a ratio-scaled diagnostic test with different distributional characteristics are generated for 50, 100 and 200 fictitious "individuals" with systematic variation of pre-test probabilities of two diagnostic categories. For each data set, optimum binary cut-off limits are determined employing different methods. Based on these optimum cut-off thresholds, sensitivities and specificities are calculated for the respective data sets. Mean values and SD of these variables are computed for 1000 repetitions each. RESULTS: Optimizations of cut-off limits using Youden index and logistic regression-derived likelihood ratio functions with correct adaption for pre-test probabilities both yield reasonably stable results, being nearly independent from pre-test probabilities actually used. Maximizing mutual information yields cut-off levels decreasing with increasing pre-test probability of disease. The most precise results (in terms of the smallest SD) are usually seen for the likelihood ratio method. With this parametric method, however, cut-off values show a significant positive bias and, hence, specificities are usually slightly higher, and sensitivities are consequently slightly lower than with the two non-parametric methods. CONCLUSIONS: In terms of stability and bias, Youden index is best suited for determining optimal cut-off limits of a diagnostic variable. The results of Youden method and likelihood ratio method are surprisingly insensitive against distributional differences as well as pre-test probabilities of the two diagnostic categories. As an additional bonus of the parametric procedure, transfer of the likelihood ratio functions, obtained from logistic regression analysis, to other diagnostic scenarios with different pre-test probabilities is straightforward.

Glomerular filtration rate (GFR) determination via individual kinetics of the inulin-like polyfructosan sinistrin versus creatinine-based population-derived regression formulae.

BACKGROUND: In renal patients estimation of GFR is routinely done by means of population-based formulae using serum creatinine levels. For GFR determination in the creatinine-blind regions or in cases of reno-hepatic syndrome as well as in critical cases of live kidney donors individualized measurements of GFR (mGFR) employing the kinetics of exogenous filtration markers such as the inulin-like polyfructosan sinistrin are necessary. The goal of this study is to compare mGFR values with the eGFR values gained by the Modification of Diet in Renal Disease (MDRD4) and Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) formulae. METHODS: In 170 subjects comprising persons with normal renal function or with various stages of kidney diseases (CKD 1-4) GFR was measured by application of intravenous bolus of sinistrin and assessment of temporal plasma concentration profiles by means of pharmacokinetic methods (mGFR). Comparisons of mGFR with MDRD4- and CKD-EPI-derived eGFR values were performed by means of linear regression and Bland-Altman analyses. RESULTS: Reasonable agreement of mGFR and eGFR values was observed in patients with poor renal function [GFR below 60 (ml/min)/1.73 m²]. In cases of normal or mildly impaired renal function, GFR determination by MDRD4 or CKD-EPI tends to underestimate GFR. Notably, there is practically no difference between the two eGFR methods. CONCLUSIONS: For routine purposes or for epidemiological studies in cases of poor renal function eGFR methods are generally reliable. But in creatinine-blind ranges [GFR above 60 (ml/min)/1.73 m²] eGFR values are unreliable and should be replaced by clinically and physiologically suitable methods for mGFR determination. CONSORT: http://www.consort-statement.org/index.aspx?o=1190.

The weak spots of saliva buffering tests.

Saliva buffering test is in need of improvements. This article illustrates the most commonly used saliva buffering capacity tests and its major problems. Starting with Ericsson and his laboratory buffer capacity test and all the way to Kitasako a lot of issues are to release. The aim of this paper is to put saliva buffering tests up to serious discussion.

Determination of binding characteristics as a measure for effective albumin using different methods.

BACKGROUND & AIMS: Transport functions of albumin are of clinical and pharmacological interest and are determined by albumin's properties like posttranslational modifications or bound ligands. Both are affected in pathological conditions and in therapeutic grade albumin solutions. The term effective albumin concentration was introduced as a measure of functionally intact albumin. Our aim was to evaluate the impact of ligands and modifications with different approaches as a measure of effective albumin. APPROACH & RESULTS: We used a spin labelled fatty acid and dansylsarcosine to characterize binding properties of albumin i) prepared from plasma of patients and healthy control donors, ii) measured directly out of plasma, iii) research grade albumin, iv) in vitro modified albumin, and v) therapeutic infusion solutions before and after removal of stabilizers. Bilirubin is the main determinant for binding function in patients' albumin. In in vitro prepared albumin bound fatty acids correlated with impaired binding. Human nonmercaptalbumin1, not human nonmercaptalbumin2, showed reduced binding properties. Binding and transport function of therapeutic albumin was severely impaired and restored by filtration. Glycation of research grade albumin had no effect on the binding of dansylsarcosine and only a minor effect on fatty acid binding. CONCLUSIONS: Our results suggest that effective albumin -in terms of binding properties- is primarily determined by bound ligands and only to a minor extent by posttranslational modifications. Characterizing albumin directly from plasma better reflects the physiological situation whereas in the case of therapeutic grade albumin stabilizers should be removed to make its binding properties accessible.

Metformin Impedes Oxidation of LDL In Vitro.

Metformin is the most commonly prescribed glucose-lowering drug for the treatment of type 2 diabetes. The aim of this study was to investigate whether metformin is capable of impeding the oxidation of LDL, a crucial step in the development of endothelial dysfunction and atherosclerosis. LDL was oxidized by addition of CuCl2 in the presence of increasing concentrations of metformin. The extent of LDL oxidation was assessed by measuring lipid hydroperoxide and malondialdehyde concentrations, relative electrophoretic mobilities, and oxidation-specific immune epitopes. Cytotoxicity of oxLDL in the vascular endothelial cell line EA.hy926 was assessed using the alamarBlue viability test. Quantum chemical calculations were performed to determine free energies of reactions between metformin and radicals typical for lipid oxidation. Metformin concentration-dependently impeded the formation of lipid hydroperoxides, malondialdehyde, and oxidation-specific immune epitopes when oxidation of LDL was initiated by addition of Cu2+. The cytotoxicity of oxLDL was reduced when it was obtained under increasing concentrations of metformin. The quantum chemical calculations revealed that only the reaction of metformin with hydroxyl radicals is exergonic, whereas the reactions with hydroperoxyl radicals or superoxide radical anions are endergonic. Metformin, beside its glucose-lowering effect, might be a suitable agent to impede the development of atherosclerosis and associated CVD. This is due to its capability to impede LDL oxidation, most likely by scavenging hydroxyl radicals.

The assessment of GFR after orthotopic liver transplantation using cystatin C and creatinine-based equations.

The measurement of kidney function after orthotopic liver transplantation (OLT) is still a clinical challenge. Cystatin C (CysC) has been proposed as a more accurate marker of renal function than serum creatinine (sCr). The aim of this study was to evaluate sCr- and CysC-based equations including the Chronic kidney disease (CKD)-EPI to determine renal function in liver transplant recipients. CysC and sCr were measured in 49 patients 24 months after OLT. The glomerular filtration rate (GFR) was calculated using the MDRD 4, the Cockroft-Gault, Hoek, Larsson, and the CKD-EPI equations based on sCr and/or CysC. As reference method, inulin clearance (IC) was estimated. Bias, precision, and accuracy of each equation were assessed and compared with respect to IC. Forty-five percent had a GFR < 60 ml/min/1.73 m(2) according to the IC. The Larsson, the Hoek and the CKD-EPI-CysC formula identified the highest percentage of patients with CKD correctly (88%, 88%, and 84%, respectively). The sCr-based equations showed less bias than CysC-based formulas with a similar precision. All CysC-based equations were superior as compared with sCr-based equations in the assessment of renal function in patients with an IC < 60 ml/min/1.73 m(2).

Location matters: Overlooked ethnic-geographic effect in China and Austria on propofol/cisatracurium sex differences among a population pharmacokinetic/pharmacodynamic (PopPK/PD) covariate analysis in men, women, and one transgender subject.

A quick literature search using "sex/gender" vs. the commonly used hypnotic propofol or neuromuscular-blocking agent cisatracurium will reveal numerous contradictory sex difference publications depending on the ethnic-geographic location of where these studies were conducted. We induced anesthesia with cisatracurium besylate (GlaxoSmithKline) 100 µg kg-1 administered exactly 1 minute following propofol (AstraZeneca) 2 mg kg-1 . In 20 male and 20 female ethnic Han-Chinese test set patients (Xi'an China), and in similar ethnic white Austrian validation set patients (Graz Austria), we quantified propofol/cisatracurium pharmacodynamic parameters namely propofol onset time, lag time, plasma concentrations (Cp ) at loss-of-behavioral response (LOBR) using bispectral index (BIS); cisatracurium onset time, lag time, and Cp at T1 % (first twitch of train-of-four) complete twitch suppression using mechanomyography (MMG). Serial arterial blood samples were collected for population pharmacokinetic (PopPK) analysis of all demographic and biological covariates (region, sex, age, weight, and height) versus volumes of distribution and clearances pharmacokinetic parameters. In Chinese women (but not in white women), propofol Cp at LOBR was 33.60% lower than men and cisatracurium Cp at T1 % complete twitch suppression was 21.49% lower than men, a clear pharmacodynamic assertion. Region and weight were significant PopPK covariates. We demonstrated that sex differences are influenced by ethnic-geographic location as only in Chinese women (but not in white women) propofol Cp at LOBR and cisatracurium Cp at T1 % complete twitch suppression were lower than in men. When defining sex differences, ethnic-geographic location should be taken into consideration as a predictive factor for optimizing propofol/cisatracurium initial loading recommended dosages.

Only Subclinical Alterations in the Haemostatic System of People with Diabetes after COVID-19 Vaccination.

People with diabetes have an increased risk of experiencing adverse COVID-19 outcomes. COVID-19 vaccination is, therefore, highly recommended. However, people with diabetes have an inherently elevated risk of thrombotic events and the impact of the vaccination on the coagulation system in this patient population remains to be elucidated. The aim of this study was to investigate the impact of COVID-19 vaccination on the haemostatic system in people with type 1 or type 2 diabetes. We evaluated the effects of COVID-19 vaccination (BioNTech Pfizer, Moderna, AstraZeneca) on standard coagulation parameters, whole blood coagulation (Thrombelastometry), platelet function (impedance aggregation), and thrombin generation (calibrated automated thrombography) in people with type 1 diabetes mellitus (n = 41) and type 2 diabetes mellitus (n = 37). Blood sampling points were prior to vaccination and two weeks after the respective vaccination. Thrombelastometry measurements indicated moderately increased clot formation post-vaccination in people with type 1, as well as with type 2, diabetes: "Clot formation times" were significantly shorter, and both "maximum clot firmness" and "alpha angles" were significantly higher, as compared to the respective pre-vaccination values. Therefore, TEM parameters were not altered after vaccination in patients receiving ASA. Moreover, platelet aggregation was enhanced in people with type 1 diabetes, and plasma levels of D-Dimer were increased in people with type 2 diabetes, following COVID-19 vaccination. All other standard coagulation parameters, as well as thrombin generation, were not affected by the vaccination. The coagulation responses of people with diabetes to COVID-19 vaccination were only subclinical and comparable to those observed in healthy individuals. Our findings suggest that people with diabetes do not face an increased activation of the coagulation post-vaccination.

Geographic differences in the target-controlled infusion estimated concentration of propofol: bispectral index response curves.

PURPOSE: Variability in drug responses could result from both genetic and environmental factors. Thus, drug effect could depend on geographic location, although regional variation is not generally acknowledged as a basis for stratification. There is evidence that the pharmacokinetic set developed in a European population for the target-controlled infusion (TCI) of propofol does not apply in Chinese patients; however, we are not aware of previous studies comparing the estimated concentration-bispectral index (BIS) response of Caucasian patients in Europe with that of Chinese patients in China. METHODS: The Diprifusor™ TCI pump, incorporating the pharmacokinetic model proposed by Marsh et al., was applied to 30 Caucasian patients in Austria and 30 Chinese patients in China. The estimated plasma concentration (C(p)) of propofol for the two groups was set at 1 µg·mL(-1) and increased by 1 µg·mL(-1) every minute to gradually reach 5 µg·mL(-1) after 5 min. The BIS values were fitted against the estimated C(p) and the predicted effect-site concentration (C(e)) in a sigmoid E(max) model. RESULTS: The sigmoid E(max) curves were shifted significantly to the left in the Chinese group compared with the Austrian group. After 5 min, the BIS value in the Chinese group was lower than in the Austrian group (mean ± standard deviation [SD], 47.2 ± 3.6 vs 63.6 ± 5.4, respectively; P = 0.0006). The estimated C(p) at loss of consciousness (LOC), predicted C(e) at LOC, and time to LOC, were lower in the Chinese group than in the Austrian group (3.3 ± 0.8 µg·mL(-1), 1.6 ± 0.4 µg·mL(-1), 2.8 ± 0.6 min, respectively, vs 4.6 ± 2.8 µg·mL(-1), 2.4 ± 1.5 µg·mL(-1), 3.9 ± 0.5 min, respectively; P < 0.0001). CONCLUSION: When propofol is given using the same TCI protocol, Chinese patients in China lost consciousness faster and at a lower estimated plasma concentration than Caucasians in Austria. Larger studies are needed to map geographically appropriate TCI infusion models.

Propofol-Bispectral Index (BIS) Electroencephalography (EEG) Pharmacokinetic-Pharmacodynamic Model in Patients With Post-Cerebral Hemorrhage Hydrocephalus.

Background. Titrating hypnotic agents for patients who suffer from a cerebral insult is a challenging task. To date there is no real gold standard to precisely quantify electroencephalography (EEG) response in a fashion that could be utilized for patients with post-cerebral hemorrhage hydrocephaly. While we must administer "as per usual" analgesics for noxious stimuli, we have to administer the hypnotic agents more "sparingly" due to lack of objective monitoring. Methods. We compared 15 adult post-cerebral hemorrhage hydrocephalus patients undergoing ventriculo-peritoneal shunt placement with 15 controls matched for gender and approximate age. We set propofol target controlled infusion estimated plasma concentrations (Cp) to gradually reach 4 µg/mL over 4 minutes. To precisely quantify post-cerebral hemorrhage mental dysfunction, we used electronically retrieved bispectral index (BIS) and propofol Cp data points to create individual inhibitory monophasic mathematical model for each patient that incorporates an independent hysteresis "lag" function. Results. In post-cerebral hemorrhage patients Cp-BIS curve, C50 (propofol concentration associated with inhibitory 50% BIS response) cutoff point was significantly shifted to the left by 39%. Whereas before infusion and at stable propofol 4 µg/mL aneurismal surgical sides ipsilateral (75 ± 13, 25 ± 9) and contralateral (73 ± 15, 27 ± 9) mean ± SD BIS values were significantly lower than ipsilateral (95 ± 3, 46 ± 12) and contralateral (94 ± 3, 46 ± 12) matched controls. Conclusions. Using BIS as surrogate marker of propofol hypnotic effect, BIS monitoring in patients with post-cerebral hemorrhage hydrocephaly showed a pattern of change and trend that was similar albeit 39% significantly lower than subjects without.

Progress of medical students after open admission or admission based on knowledge tests.

CONTEXT: Although admission to university in Austria is generally open for applicants who have successfully completed secondary school, in some areas of study, including human medicine and dentistry, the selection of students by additional criteria has become legally possible as a result of a decision by the European Court in 2005. We studied the impact of this important change on the temporal pattern of medical students' progress through the study programme. METHODS: All 2532 regular students admitted to the diploma programme in human medicine at the Medical University of Graz during the academic years 2002/03-2007/08 were included in the analysis. Non-parametric and semi-parametric survival analysis techniques were employed to compare the time required to complete the first two study semesters (first part of the curriculum) before and after the implementation of admission tests. Temporal patterns of dropout before this goal was achieved were also investigated. Sex, age and nationality of students were assessed as potential confounding variables. RESULTS: The cumulative probability of study success was dramatically better in selected students versus those who were admitted openly (P < 0.0001). Whereas only 20.1-26.4% of openly admitted students completed the first two study semesters within the scheduled time of 1 year, this percentage rose to 75.6-91.9% for those selected by admission tests. Similarly, the cumulative probability for dropping out of study was also significantly lower in selected students (P < 0.0001). By univariate as well as multivariate techniques, student nationality, age and sex were also identified as partly significant, albeit weak, predictors. DISCUSSION: The analysis convincingly demonstrates that, by contrast with open admission, performance-based selection of medical students significantly raises the probability of successful study progress. Additionally, the proportion of dropouts is significantly reduced. Thus, admission tests save considerable costs, in terms of both student time and public resources.

Menstrual Phase Affects Coagulation and Hematological Parameters during Central Hypovolemia.

BACKGROUND: It has been reported that women have a higher number of heart attacks in the "follicular phase" of the menstrual cycle. We, therefore, tested the hypothesis that women in the follicular phase exhibit higher coagulability. As lower body negative pressure (LBNP) has been used previously to assess coagulation changes in whole blood (WB) samples in men and women, effects of menstrual phase on coagulation was assessed during LBNP. METHODS: Seven women, all healthy young participants, with no histories of thrombotic disorders and not on medications, were tested in two phases of the menstrual cycle (early follicular (EF) and mid-luteal (ML)). LBNP was commenced at -10 mmHg and increased by -10 mmHg every 5 min until a maximum of -40 mmHg. Recovery up to 10 min was also monitored. Blood samples were collected at baseline, at end of LBNP, and at end of recovery. Hemostatic profiling included comparing the effects of LBNP on coagulation values in both phases of the menstrual cycle using standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and markers of thrombin formation. RESULTS: LBNP led to coagulation activation determined in both plasma and WB samples. During both phases, coagulation was affected during LBNP, as reflected in their decreased partial thromboplastin time (PTT) and elevated coagulation factor VIII FVIII, F1 + 2, and thrombin-antithrombin (TAT) levels. Additionally, during the ML phase, greater PT [%] and shorter time to peak (ttPeak) values (implying faster maximum thrombin formation) suggest that women in the ML phase are relatively hypercoagulable compared to the early follicular phase. CONCLUSIONS: These results suggest that thrombosis occurs more during the midluteal phase, a finding with substantial medical implications.

Coagulation Changes during Central Hypovolemia across Seasons.

Lower body negative pressure (LBNP) application simulates hemorrhage. We investigated how seasons affect coagulation values at rest and during LBNP. Healthy participants were tested in cold (November-April) and warm (May-October) months. Following a 30-min supine period, LBNP was started at -10 mmHg and increased by -10 mmHg every five minutes until a maximum of -40 mmHg. Recovery was for 10 min. Blood was collected at baseline, end of LBNP, and end of recovery. Hemostatic profiling included standard coagulation tests, calibrated automated thrombogram, thrombelastometry, impedance aggregometry, and thrombin formation markers. Seven men (25.0 ± 3.6 years, 79.7 ± 7.8 kg weight, 182.4 ± 3.3 cm height, and 23.8 ± 2.3 kg/m2 BMI) and six women (25.0 ± 2.4 years, 61.0 ± 8.4 kg weight, 167 ± 4.7 cm height, and 21.8 ± 2.4 kg/m2 BMI) participated. Baseline levels of prothrombin (FII), tissue factor (TF) and markers for thrombin generation F1+2 and the thrombin/antithrombin complex (TAT) were higher during summer. Factor VIII, prothrombin fragment 1+2 (F1+2), TAT and the coagulation time showed significant increases during LBNP in both seasons. Some calibrated automated thrombography variables (Calibrated automated thrombography (CAT): lag, time to peak (ttPeak), peak) shifted in a procoagulant direction during LBNP in summer. Red blood cell counts (RBC), hemoglobin and white blood cell counts (WBC) decreased during LBNP. LBNP application reduced prothrombin time in winter and activated partial thromboplastin time in summer. Greater levels of FII, TF, F1+2, and TAT-a more pronounced LBNP-induced procoagulative effect, especially in CAT parameters (lag time (LT), Peak, ttPeak, Velindex)-were seen in summer. These results could have substantial medical implications.

Gender differences in albumin and ascorbic acid in the vitreous antioxidant system.

Ascorbic acid is present at high concentrations in the vitreous and plays a central role in vitreous redox chemistry. Albumin is the main protein in the vitreous with antioxidant properties and occurs in different oxidation states, which can be used as redox indicators, but have not been studied in the vitreous. This study, therefore, addressed the vitreous redox state of cysteine-34 of albumin in relation to the ascorbic acid content, which has been suggested to exert a main function in detoxifying reactive oxygen in the vitreous. A total of 58 vitreous samples obtained from patients undergoing vitrectomy were analyzed for (i) human mercaptalbumin (HMA), the reduced thiol form; (ii) human non-mercaptalbumin1 (HNA1), a reversible oxidative modification with a disulfide at cysteine-34; and (iii) human non-mercaptalbumin2 (HNA2), a non-reversibly (highly) oxidized form of albumin; as well as (iv) ascorbic acid concentrations, to study possible relations. In addition, blood samples were taken to compare albumin redox state between plasma and the vitreous. Vitreous albumin showed greater variability in the redox state of cysteine-34 and a shift to the oxidized fractions compared to plasma albumin (P < 0.001). A strong positive relation was observed between the vitreous ascorbic acid concentrations and the reversibly oxidized form, HNA1 (P < 0.001), and a negative relation with the reduced form, HMA. Positive relations between ascorbic acid and HNA1 in the vitreous were stronger in men than in women. In contrast to HMA and HNA1, there was a distinct gender difference noted for the irreversibly oxidized form, HNA2. While males showed a positive relation between the vitreous ascorbic acid concentrations and HNA2, there was no correlation found with HNA2 in females. Our results support the view that ascorbic acid, by decreasing either directly or indirectly the concentrations of molecular oxygen, generates hydrogen peroxide, and that thiols, including HMA, are acting as antioxidants. This study for the first time provides evidence that vitreous albumin can be used as a marker molecule for the appearance of reactive oxygen species in the vitreous of patients undergoing vitrectomy. Moreover, it can be shown that there are gender differences in vitreous ascorbic acid and albumin concentrations as well as in oxidation state of vitreous albumin.

Mortality in dialysis patients with cinacalcet use: A large observational registry study.

BACKGROUND: Secondary hyperparathyroidism (sHPT) is associated with higher mortality in dialysis patients. The calcimimetic cinacalcet reduces intact parathyroid hormone (iPTH) in dialysis patients. The randomized controlled EVOLVE trial failed to unequivocally prove survival advantage of cinacalcet in dialysis patients. However, recent post hoc analyses suggested a benefit in subgroups of dialysis patients. Large observational cohort studies may represent an option to better determine such subgroups. METHODS: Data from the nationwide Austrian registry of dialysis patients between January 2004 and December 2009 were analyzed with follow-up until December 2010. All-cause and cardiovascular mortality analyses were performed using the Kaplan-Meier and Cox proportional hazards regression. To reduce confounding effects a propensity score (PS) based method (matching by stratification) was used for group comparison. RESULTS: The cohort included 7983 dialysis patients, 1572 (19.7%) were prescribed cinacalcet. During a median follow-up of 2.7years, 3574 (44.8%) patients died, including 1342 (16.8%) deaths from cardiovascular causes. Survival analyses in the PS-matched study population (n=6109) showed lower all-cause mortality for cinacalcet-treated as compared to untreated patients only in subsets characterized by younger age, low prevalence of diabetes, iPTH levels between 300 and 599pg/mL, concomitant therapy with vitamin D and phosphate binders. CONCLUSIONS: Our data suggest that a subgroup of dialysis patients, namely those with moderate sHPT, younger age and without diabetes benefit from cinacalcet with reduced overall and cardiovascular mortality. These findings may help to identify populations for further controlled trials and may allow a more individualized sHPT treatment using cinacalcet in specific patient subgroups.