PP086. The modern search for possible predictors of preeclampsia in the first trimester of pregnancy (preliminary study).

INTRODUCTION: Preeclampsia is a major complication affecting at least 3-4% of all pregnancies and is globally responsible for approximately 50,000 maternal deaths annually. Currently the main cause of preeclampsia is a shallow placentation, with abnormal invasion of cytotrophoblasts and incomplete remodeling of placenta-supplying maternal uterine spiral arteries. However, up to 20weeks, these processes are asymptomatic, although they are accompanied by the release of various macromolecules in the bloodstream of the mother, which are potential biomarkers of disease, needed to detect. Much progress has been made in recent years towards first-trimester models using combined factors that will be of realistic use in clinical practice.The ability to predict the most severe forms of preeclampsia would allow closer surveillance and earlier intervention to improve pregnancy outcomes. OBJECTIVES: To determine the significance of measuring the concentration of PlGF and sFlt-1 and their ratio in 11-13 weeks of pregnancy as early markers of preeclampsia. METHODS: The study included 85 pregnant women in 11-13 weeks with subsequent uncomplicated pregnancy and 11 patients which developed various hypertensive disorders, four of them have developed severe (n=2) and mild (n=2) preeclampsia later. Concentrations of markers were determined on automated analyzers Cobas E411 (Hoffmann La Roche) using test kits Elecsys PlGF and Elecsys sFlt-1. RESULTS: In the first trimester the concentration of sFlt-1 in healthy pregnant women was 1618,0±18,2pg/ml, PlGF - 47,5±3,5pg/ml, the sFlt-1/PLGF ratio - 35,9±3,2. In the second trimester parameters were as follows: sFlt-1 - 1898,0±29,8pg/ml, PlGF - 208,0±11,0pg/ml, the sFlt-1/PLGF ratio-11,2±2,6. Two patients with severe preeclampsia had sFlt-1 concentration at 11-12 weeks of 2185 and 14923pg/ml, PlGF - 12,8 and 61.3pg/ml, the ratio sFlt-1/PLGF - 170,7 and 243.4. In two patients with mild preeclampsia, these figures were in 1700 and 2312pg/ml, 38.2 and 37,6pg/ml, 44,4 and 61,4, respectively. CONCLUSION: The optimal prediction biomarker does probably not exist. Our preliminary results confirm the feasibility of determining concentrations of sFlt-1 and PlGF, and their ratio in the 1st trimester of pregnancy to assess the risk of preeclampsia in order to reduce the frequency of obstetric complications and perinatal loss. Effective prediction of PE can be achieved at 11-13weeks' gestation. Further studies on the development and implementation a modified integrated screening program for early diagnosis of PE at 11-14weeks of gestation should be based on the assessment of maternal factors, biophysical, biochemical, and molecular genetic markers are needed. Better individualised patient prediction will allow us to target existing prevention, as it is, and to also develop new treatments in future.

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies.

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

The crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-dependent, acyl-CoA-condensing enzyme.

8-Amino-7-oxononanoate synthase (or 8-amino-7-ketopelargonate synthase; EC 2.3.1.47; AONS) catalyses the decarboxylative condensation of l-alanine and pimeloyl-CoA in the first committed step of biotin biosynthesis. We have cloned, over-expressed and purified AONS from Escherichia coli and determined the crystal structures of the apo and PLP-bound forms of the enzyme. The protein is a symmetrical homodimer with a tertiary structure and active site organisation similar to, but distinct from, those of other PLP-dependent enzymes whose three-dimensional structures are known. The critical PLP-binding lysine of AONS is located at the end of a deep cleft that allows access of the pantothenate arm of pimeloyl-CoA. A cluster of positively charged residues at the entrance to this cleft forms a putative diphosphate binding site for CoA. The structure of E. coli AONS enables identification of the key residues of the PLP-binding site and thus provides a framework with which to understand the biochemical mechanism, which is similar to that catalysed by 5-aminolevulinate synthase and two other alpha-oxoamine synthases. Although AONS has a low overall sequence similarity with the catalytic domains of other alpha-oxoamine synthases, the structure reveals the regions of significant identity to be functionally important. This suggests that the organisation of the conserved catalytic residues in the active site is similar for all enzymes of this sub-class of PLP-dependent enzymes and they share a common mechanism. Knowledge of the three-dimensional structure of AONS will enable characterisation of the structural features of this enzyme sub-family that are responsible for this important type of reaction.