ABSTRACT
There have been a number of recent advances in catalysis assays applicable for screening biocatalyst libraries in high-throughput format. These include instrumental assays such as high-performance liquid chromatography, mass spectrometry, capillary electrophoresis and IR-thermography, reagent-based assays producing spectroscopic signals (UV/VIS or fluorescence) in response to reaction progress, and assays based on fluorogenic or chromogenic substrates. These fluorogenic substrates enable the assaying of a variety of enzymes in enantioselective and stereoselective manner, including alcohol dehydrogenases, aldolases, lipases, amidases, epoxide hydrolases and phosphatases.
Subject(s)
Catalysis , Combinatorial Chemistry Techniques/methods , Enzymes/metabolism , Selection, Genetic , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Chromatography, High Pressure Liquid/methods , Chromogenic Compounds/chemistry , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/trends , Electrophoresis, Capillary/methods , Enzyme-Linked Immunosorbent Assay/methods , Enzymes/genetics , Fluorometry/methods , Gene Library , Mass Spectrometry/methods , Substrate SpecificityABSTRACT
High levels of factor VIII (FVIII) but not von Willebrand factor (vWF) are known to increase the risk for venous thromboembolism. Whether high FVIII levels originate from hereditary defects or from acquired conditions remains unanswered. The objective of our study was to investigate whether there is evidence for familial clustering of elevated FVIII levels in families in which >/=1 member has been affected by a thromboembolic event and had reproducibly high FVIII levels. We investigated FVIII levels in 361 patients with previous venous thromboembolism. FVIII levels were measured by a chromogenic assay; the cutoff value was defined as the 98th percentile of FVIII plasma levels of 266 blood donors. vWF levels were determined by an enzyme immunoassay. After exclusion of known causes of FVIII elevation, such as the acute thrombotic event itself; inflammation; malignancy; liver, renal, or vascular disease; surgery; or pregnancy, we included 17 patients with unexplained, reproducibly high FVIII levels. The investigation was also extended to these patients' relatives. Multiple regressive analysis of blood donors and asymptomatic family members showed that the affiliation with a family in which 1 member suffered from venous thromboembolism and had reproducibly high FVIII levels is the second most important predictor for FVIII levels. Familial clustering was analyzed by the Houwing-Duistermaat familial aggregation test. After adjustment for the influence of age, sex, blood group, and vWF, FVIII levels were significantly (P:=0.038) clustered within families. In conclusion, FVIII levels seem to be familially determined in families in which a member showed high FVIII levels after previous venous thromboembolism.
Subject(s)
Factor VIII/genetics , Family , Thromboembolism/blood , Venous Thrombosis/blood , Adult , Aged , Blood Donors/statistics & numerical data , Cluster Analysis , Factor VIII/analysis , Female , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Regression Analysis , Thromboembolism/epidemiology , Thromboembolism/genetics , Thrombophilia/blood , Thrombophilia/epidemiology , Thrombophilia/genetics , Venous Thrombosis/epidemiology , Venous Thrombosis/geneticsABSTRACT
Recent progress in high-throughput enzyme assays includes new examples of fluorogenic and chromogenic substrates, fluorescence resonance energy transfer substrates, and applications of the pH and pM indicator methods. Recent developments of Horeau's pseudo-enantiomer derivatisation method to screen enantioselectivities in high-throughput have also been reported.
