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Accurate evaluation of sialic acids in biological matrices for a more reliable assessment of their role
No full textAn efficient procedure for the contemporaneous qualitative and quantitative determination of the most important sialic acids (SA), Neu5Ac and Neu5Gc, present in a large number of glycoproteins and glycolipids has been developed.
The procedure represents the first protocol which uses isotope dilution liquid chromatography/tandem mass spectrometry without any sample derivatization, it involves the release of SA by acidic hydrolysis in presence of appropriate internal standards: the labeled analogues [1,2,3-13C3]-Neu5Ac and [1,2,3-13C3]-Neu5Gc. The use of isotopologous internal standards, considered the most appropriate in quantitative bioanalytical assays, compensates for variability in sample extraction, processing and analysis.
The accomplishment of this method, which allows the chromatographic separation of Neu5Ac and Neu5Gc, has required the preparation of [1,2,3-13C3]-Neu5Gc and a home-made LC-column, conceived considering the interactions of alkylboronic acids with neutral and acidic carbohydrates.
In order to confirm the reliability of this method, it was fully validated using fetuin as model glycoprotein and then applied to evaluate the presence of Neu5Ac and Neu5Gc in murine myoblasts (C2C12) and in human erythrocytes membranes of healthy subjects. Moreover, the levels of these SA were also determined in erythrocytes membranes of patients affected by diabetes or polycythemia vera.
Because of the presence of the isotopologous internal standards starting from the hydrolysis of the biological substrates and the absence of any derivatization of SA, the method has permitted to evaluate some important biological events not observable by detecting SA by means of the previous techniques. The methodology appears to be of utility to support progresses in the field of the sialic acids biology
Direct determination of sialic acids using a stationary phase modified with alkylboronic acid and isotope dilution HPLC-MS/MS
No full textDeterminazione diretta degli acidi N-Acetil e N-Glicolilneuraminico mediante HPLC/ESI-MS/MS
No full textInhibition of the platelet P2Y12 receptor for adenosine diphosphate does not impair the capacity of platelet to synthesize thromboxane A2
No full textAims Patients with acute coronary syndromes (ACSs) are treated with acetylsalicylic acid (ASA) and antagonists of the P2Y12 receptor (P2Y12R) for adenosine diphosphate (ADP). Based on the demonstration that P2Y12R antagonists inhibit thromboxane A2 (TxA2) production (target of ASA), it was surmised that ACS patients might be treated with P2Y12R antagonists only. However, this demonstration contrasts with the results of previous studies. The aim of this study was to test whether P2Y12R antagonists have off-target/indirect inhibitory effects on platelet TxA2 production.
Methods and results We studied 3 patients with inherited P2Y12R deficiency and 33 healthy subjects. Serum TxB2 (TxA2 metabolite) levels were similar in P2Y12R-deficient patients and healthy subjects and were not decreased by P2Y12R antagonists in vitro. Serum TxB2 levels did not decrease in 20 patients treated with prasugrel (10 mg q.i.d.) or placebo for 14 days. Arachidonic acid- and collagen-induced platelet aggregation (PA) and TxB2 production in platelet-rich plasma (PRP) of healthy subjects were inhibited in vitro by P2Y12R antagonists. However, P2Y12R antagonists did not inhibit TxB2 production when PA was prevented by avoiding the stirring of PRP in the aggregometer. The P2Y1 ADP-receptor antagonist MRS2500 had similar effects on PA and TxB2 production as P2Y12R antagonists. Acetylsalicylic acid inhibited TxB2 production more effectively than a P2Y12R antagonist; only the combination of ASA and a P2Y12R antagonist inhibited PA induced by high concentration of collagen.
Conclusion Inherited deficiency or pharmacological inhibition of P2Y12R does not affect the platelet capacity to synthesize TxA2. There is no pharmacological evidence that ACS patients may be safely treated with P2Y12R antagonists without ASA
Quantification of N-acetyl- and N-glycolylneuraminic acids by a stable isotope dilution assay using high-performance liquid chromatography–tandem mass spectrometry
No full textThe present report describes a method for the quantification of N-acetyl- and N-glycolylneuraminic acids without any derivatization, using their (13)C(3)-isotopologues as internal standards and a C(18) reversed-phase column modified by decylboronic acid which allows for the first time a complete chromatographic separation between the two analytes. The method is based on high-performance liquid chromatographic coupled with electrospray ion-trap mass spectrometry. The limit of quantification of the method is 0.1 mg/L(2.0 ng on column) for both analytes. The calibration curves are linear for both sialic acids over the range of 0.1-80mg/L (2.0-1600ng on column) with a correlation coefficient greater than 0.997. The proposed method was applied to the quantitative determination of sialic acids released from fetuin as a model of glycoproteins
Congenital defects of platelet function
No full textCongenital abnormalities of platelet function disorder (PFD) are associated with the heightened risk for bleeding. Typically, patients with PFDs have mucocutaneous bleeding of variable severity and excessive hemorrhage after surgery or trauma. The diagnostic laboratory assessment appropriate for the evaluation of suspected inherited PFD should be based on a two-step diagnostic strategy: the first step, based on screening tests, helps raising a diagnostic hypothesis, which should then be tested in the second step, which is based on the use of specific tests. The first step should include: complete blood cell count, examination of the peripheral blood smear, and assessment of platelet aggregation. Although light transmission aggregometry (LTA) is the most widely used platelet function test, it is relatively insensitive to defects of platelet secretion; for this reason, laboratory tests that measure platelet aggregation and secretion simultaneously, such as lumi-aggregometry, should be preferred to traditional LTA. The second step includes specific tests (e.g., flow cytometry, Western blotting, DNA analysis). Platelet transfusions should be used only to treat severe bleeding episodes. Recombinant Factor VIIa can be used in patients with severe bleeding episodes who do not respond to platelet transfusion because of alloimmunization. Fibrinolytic inhibitors or the vasopressin analogue desmopressin (DDAVP) should be used in all other circumstances
Effect of platelet count on platelet aggregation measured by impedance aggregometry (Multiplate(TM) analyser) and by light transmission aggregometry
No full textThe in vitro evaluation of platelet aggregation is useful to diagnose platelet function disorders [1, 2]; in some laboratories, the test is also used to monitor antiplatelet treatment [3]. Traditionally, platelet aggregation is measured by light transmission aggregometry (LTA), which measures the changes in transmission of a beam of light through a sample of platelet-rich plasma (PRP) or platelet suspensions in buffer, which occur when platelets change shape and aggregate upon stimulation. As this technique has some disadvantages [2], novel methods were introduced to measure platelet aggregation in vitro
Synthesis of unnatural homologues of deoxypyridinoline as possible internal standards in analytical detection of pyridinolinic cross links of collagen
No full textThree homologues of the collagen cross-links deoxypyridinoline, differing for the length of the side chain at the aromatic nitrogen, have been efficiently synthesized as possible internal standards in the quantitative analyses of pyridinolines. The first has a one carbon shorter N-chain, while other two have a one carbon and a two carbon longer N-chains. The stereogenic centers are introduced stereoselectively using Williams’ glycine template methodology and oxazinones to generate chirality and to suitably protect the aminoacid functionality during assembly of the pyridinoline nucleus
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