Antithrombotic Effect and Mechanism of Radix Paeoniae Rubra.

The compounds of Radix Paeoniae Rubra (RPR) were isolated and identified by bioassay-guided method, and antithrombotic effects and mechanism were investigated by the acute blood stasis rat model. The RPR extract was evaluated by APTT, TT, PT, and FIB assays in vitro. Results indicated that RPR extract exhibited the anticoagulant activity. In order to find active compounds, six compounds were isolated and identified, and four compounds, paeoniflorin (Pae), pentagalloylglucose (Pen), albiflorin (Ali), and protocatechuic acid (Pro), exhibited the anticoagulant activity in vitro. Therefore, the antithrombosis effects of RPR extract and four active compounds were investigated in vivo by measuring whole blood viscosity (WBV), plasma viscosity (PV), APTT, PT, TT, and FIB. Meanwhile, the levels of TXB2, 6-Keto-PGF1α , eNOS, and ET-1 were detected. Results suggested that RPR extract and four active compounds had the inhibition effect on thrombus formation, and the antithrombotic effects were associated with the regulation of vascular endothelium active substance, activating blood flow and anticoagulation effect.

Comparison of UHPLC and HPLC methods for the assay of prostanoids: "are the methods equivalent in terms of accuracy and precision?".

BACKGROUND: As new methods are developed to increase efficiency and higher analytical performance, it is necessary to evaluate their quality in comparison to standard methods. To understand how the analytical performance changes between methods, it is common to compare the validation parameters; sensitivity, linearity, accuracy and precision. Here, we compare an UHPLC-UV method to the HPLC-UV method (reference method) for the simultaneous determination of seven prostanoids. Though the basic chromatography theory is the same for HPLC and UHPLC, the instrumentation has been modified to accommodate higher pressures, lower flow rates and smaller sample size. The differences in analytical instrumentation and procedures can give rise to method inequivalencies. Our approach evaluates the UHPLC and HPLC methods and poses the question: are the methods equivalent? To answer this question a statistical comparison of the analytical performance and method parameters is necessary. RESULTS: Statistical comparisons were performed using the t-test, F-test, regression analyses (ordinary linear regression and Deming regression) and Bland-Altman analyses. Statistical comparison of the results, suggested that the precision (amount of variability) is different (p < 0.05) for the HPLC and UHPLC methods. Whereas, the accuracy (method bias and the means) is similar (p > 0.05) for 8-isoprostane, 11-dehydro TXB2, PGE2 PGF(2α), PGD2 and 15-deoxy Δ¹²,¹4 PGJ2. DISCUSSION: Ordinary linear regression shows that the methods are well correlated for all compounds. The Deming regression, which assumes error in both the methods, suggests the existence of a proportional and constant bias for 11-dehydro TXB2 and only proportional bias for 8-isoprostane, PGF(2α), PGD2 and 15-deoxy Δ(12,14) PGJ2 between the two methods. According to Deming regression, the two methods are statistically similar for 6-keto PGF(1α) and PGE2. The Bland-Altman analyses indicate the two methods are commutable.

The effect of Ginkgo biloba in isolated ischemic/reperfused rat heart: a link between vitamin E preservation and prostaglandin biosynthesis.

The effect of Ginkgo biloba extract (EGb 761) was studied in rat hearts submitted to ischemia/reperfusion. Isolated hearts perfused in Langendorff mode were subjected to 60 minutes of global ischemia and 15 minutes of reperfusion. EGb 761 was administered by chronic or acute treatment: intra-peritoneal injections of 5 mg/Kg extract for 5 days, or 100 mg /L extract addition to the perfusion buffer, respectively. In hearts not treated with EGb 761, ischemia induced a 20% decrease in the concentration of membrane alpha-tocopherol. This effect was not worsened by reperfusion. alpha-tocopherol consumption was accompanied by about 650% increase in 6-ketoPGF1alpha release within 3 minutes of reperfusion. Moreover, ischemia induced activation of transcription factor NF-kappaB, as compared with the untreated group. In both chronic and acute treatment with EGb 761, heart concentration of alpha-tocopherol was completely spared during ischemia as much as after reperfusion, and a significant decrease of 6-ketoPGF1alpha release was observed at 3 minutes of reperfusion. Nuclear translocation of NF-kappaB was lowered during ischemia. EGb 761 might act as direct free radical scavenger or as tocopheryl radical recycler; in both cases sparing membrane vitamin E should affect phospholipase A2 activity. Finally, EGb 761, by lowering ROS produced during ischemia, challenges nuclear translocation of NF-kappaB.

Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone.

1. In human pulmonary vascular preparations, precontracted arteries were more sensitive to the relaxant effect of acetylcholine (ACh) than veins (pD(2) values: 7.25+/-0.08 (n=23) and 5.92+/-0.09 (n=25), respectively). Therefore, the role of prostacyclin (PGI(2)) was explored to examine whether this mediator may be responsible for the difference in relaxation. 2. In the presence of the cyclooxygenase (COX) inhibitor, indomethacin (INDO), the ACh relaxations were reduced in arteries but not in veins. On the contrary, an inhibitor (l-NOARG) of the nitric oxide synthase blocked preferentially the relaxation in veins. 3. A greater release of 6-keto-PGF(1alpha), the stable metabolite of PGI(2), was observed in arterial preparations than in venous preparations when stimulated with either ACh or arachidonic acid (AA). 4. Exogenous PGI(2) produced a reduced relaxant effect in the precontracted vein when compared with the artery. In the presence of the EP(1)-receptor antagonist AH6809, the PGI(2) relaxation of veins was similar to arteries. 5. In veins, AA (0.1 mm) produced a biphasic response, namely, a contraction peak (0.4-0.5 g) followed by a relaxation. These contractions in venous preparations were abolished either in the absence of endothelium or in the presence of INDO or an EP(1)-receptor antagonist (AH6809, SC19220). In the arterial preparations AA induced only relaxations. 6. In both vascular preparations, COX-1 but not the COX-2 protein was detected in microsomal preparations derived from homogenized tissues or freshly isolated endothelial cells. 7. The differential vasorelaxations induced by ACh may be explained, in part, by a more pronounced production and release of PGI(2) in human pulmonary arteries than in the veins. In addition, while PGI(2) induced relaxation by activation of IP-receptors in both types of vessels, a PGI(2) constrictor effect was responsible for masking the relaxation in the veins by activation of the EP(1)-receptor.

Purification and characterization of recombinant human prostacyclin synthase.

Prostacyclin synthase (PGIS), which catalyzes the conversion of prostaglandin (PG) H(2) to prostacyclin (PGI(2)), is a member of the cytochrome P-450 (P450) superfamily, CYP8A1. To study the enzymatic and protein characteristics of human PGIS, the enzyme was overexpressed in Spodoptera frugiperda 21 (Sf21) cells using the baculovirus expression system. PGIS was expressed in the microsomes of the infected Sf21 cells after culture in 5 microg/ml hematin-supplemented medium for 72 h. The holoenzyme was isolated from the solubilized microsomal fraction by calcium phosphate gel absorption and purified to homogeneity by DEAE-Sepharose and hydroxyapatite column chromatography. The K(m) and V(max) values of the purified human PGIS for PGH(2) were 30 microM and 15 micromol/min/mg of protein at 24 degrees C, respectively. The optical absorption and EPR spectra of the enzyme revealed the characteristics of a low-spin form of P450 in the oxidized state. The carbon monoxide-reduced difference spectrum, however, exhibited a peak at 418 nm rather than 450 nm. The addition of a PGH(2) analogue, U46619, to the enzyme produced an oxygen-ligand type of the difference spectrum with maximum absorption at 407 nm and minimum absorption at 430 nm. Treatment with another PGH(2) analogue, U44069, produced a peak at 387 nm and a trough at 432 nm in the spectrum (Type I), while treatment with tranylcypromine, a PGIS inhibitor, produced a peak at 434 nm and a trough at 412 nm (Type II). A Cys441His mutant of the enzyme possessed no heme-binding ability or enzyme activity. Thus, we succeeded in obtaining a sufficient amount of the purified recombinant human PGIS from infected insect cells for spectral analyses that has high specific activity and the characteristics of a P450, indicating substrate specificity.

Destabilase complexes--natural liposome produced by medicinal leeches Hirudo medicinalis.

Electrophoretic analysis of destabilase preparation demonstrates the presence of protein combinations with MW 12.3, 25 and 50 kD. Fraction (MW 12.3 D) is a monomer of destabilase aggregation having properties of micellar proteins and represents a stable lipid-protein complex, where the role of lipid component is played by the stable analogue of prostacyclin (MW 391 D). The synthesis of a low molecular fraction of destabilase is fulfilled with bacteria--symbiont of leeches Aeromonas hydrophila. When the destabilase (MW 12.3 kD) contacts with blood a process of complexe formation is triggered with hirudin and blood plasma kallikrein inhibitor, forming a stable 'destabilase complex' (DC; MW 25 kD), possessing also a high aggregation capacity. Polymer forms of the destabilase complex form a liposome changing its spatial orientation depending on the nature of the solvent. Such structural organization provides a high stability of DC components and a rapid penetration through cellular membranes (transmembrane transfer) and it also provides prophylactic antithrombotic action in the case of peroral application to animals, due to the blockade of vascular platelets (inhibition of platelet aggregation by prostacyclin analogue) and plasmic (inhibition of thrombin activity and blood plasma kallikrein) links of the hemostasis process. Destabilase fraction with MW 50 kD is a dimer of the destabilase complex. As a result of DC destruction (liposome), hirudin, prostacycline analogue and blood plasma kallikrein inhibitor are released.

Relationship between exchangeable body sodium and urinary 6-keto-prostaglandin F1 alpha excretion in normal man.

The renal prostaglandins are involved in the regulation of sodium balance. In the present study exchangeable body sodium (NaE) and the urinary excretion of the stable metabolite of prostacyclin, 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) were determined simultaneously in 10 hospitalized healthy individuals. NaE was 1461 +/- 107 mmol/m2 body surface area, or 98.5 +/- 6.9% when expressed as percent of the normal value assessed on the basis of measurements in 54 control subjects. The excretion of 6-k-PGF1 alpha amounted to 68.3 +/- 39.2 ng/4 hr. Statistical evaluation revealed significant correlation between NaE and PGF1 alpha excretion (r = 0.642; p less than 0.05) and between the serum Na concentration and the urinary excretion of 6-k-PGF1 alpha (r = 0.865; p less than 0.001). The obtained results indicate that urinary 6-k-PGF1 alpha excretion, hence the renal synthesis of prostacyclin, are regulated, among other factors, by body sodium stores. The increased production of prostacyclin with expanding sodium space might be regarded as a compensatory response contributing to the renal elimination of excess sodium from the body. The signal to this response could be the serum Na concentration.

Use of radio-gas chromatography and tritium label to optimize derivatization procedures.

A sensitive determination of gas chromatographic peak yields by a new radio-gas chromatograph equipped with a synchronized accumulating radioisotope detector is described. Peak yields could be easily determined by the radio-gas chromatograph using [3H]hexadecane or [3H]androstenedione as a standard substance. The usefulness of the determination of peak yields was demonstrated by optimizing the derivatization conditions of 6-keto-prostaglandin F1 alpha.