Identification of oxidation product of arbutin in mushroom tyrosinase assay system

Neurogenerative diseases, such as Parkinson’s disease, are associated, not only with the selective loss of dopamine (DA), but also with the accumulation of reactive catechol-aldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is formed as the immediate oxidation product of cytoplasmic DA by monoamine oxidase. DOPAL is well known to exhibit toxic effects on neuronal cells. Both catecholic and aldehyde groups seem to be associated with the neurotoxicity of DOPAL. However, the exact cause of toxicity caused by this compound remains unknown. Since the reactivity of DOPAL could be attributed to its immediate oxidation product, DOPAL-quinone, we examined the potential reactions of this toxic metabolite. The oxidation of DOPAL by mushroom tyrosinase at pH 5.3 produced conventional DOPAL-quinone, but oxidation at pH 7.4 produced the tautomeric quinone-methide, which gave rise to 3,4-dihydroxyphenylglycolaldehyde and 3,4-dihydroxybenzaldehyde as products through a series of reactions. When the oxidation reaction was performed in the presence of ascorbic acid, two additional products were detected, which were tentatively identified as the cyclized products, 5,6-dihydroxybenzofuran and 3,5,6-trihydroxybenzofuran. Physiological concentrations of Cu(II) ions could also cause the oxidation of DOPAL to DOPAL-quinone. DOPAL-quinone exhibited reactivity towards the cysteine residues of serum albumin. DOPAL-oligomer, the oxidation product of DOPAL, exhibited pro-oxidant activity oxidizing GSH to GSSG and producing hydrogen peroxide. These results indicate that DOPAL-quinone generates several toxic compounds that could augment the neurotoxicity of DOPAL.

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The Significance of TFPI in Clotting Assays – Gomparison and Combination with other Anticoagulants

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649603 ◽

1993 ◽

Vol 70 (03) ◽

pp. 448-453 ◽

Cited By ~ 12

Author(s):

Ole Nordfang ◽

Hanne I Kristensen ◽

Sanne Valentin ◽

Per Østergaard ◽

Johnny Wadt

Keyword(s):

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Antithrombin Iii ◽

Anticoagulant Activity ◽

Assay System ◽

Thrombin Inhibitor ◽

Clotting Time ◽

Normal Plasma ◽

Tissue Factor Pathway ◽

Plasma Heparin

SummaryThe anticoagulant activities of Tissue Factor Pathway Inhibitor (TFPI), heparin and hirudin were compared in intrinsic (APTT) and extrinsic (PT) activated clotting assays. In contrast to the thrombin inhibitor hirudin, heparin was 10 fold more potent in the APTT assay than in the PT assay, indicating that inhibition of intrinsic activation is important for the anticoagulant activity of heparin as measured in an APTT assay. TFPI was most potent in the PT assay and the effect of TFPI was most pronounced in the presence of other anticoagulants (heparin and hirudin). The activities of the two natural anticoagulants antithrombin III (ATIII) and TFPI were compared in a PT assay with very dilute tissue factor. In this assay system TFPI in normal plasma affected the clotting time more than ATIII in the plasma. However, when heparin was added ATIII was the major anticoagulant, but profound Prolongation of the clotting time was only seen when TFPI was also added. In an ATIII deficient plasma heparin did not augment the effect of TFPI, showing that the increased effect of TFPI in the presence of heparin is dependent on the anticoagulant activity of ATIII/heparin. The effect of TFPI at prolonged clotting times was also illustrated by the significant effect of blocking TFPI in the plasma from warfarin-treated patients. Thus TFPI is a major anticoagulant in normal plasma and the effect of TFPI is especially seen at prolonged clotting times.

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A Comparative Ex Vivo Study of Plasminogen Activators and Proteases for Fibrinolytic Activity and Side Effects in Rabbits

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649213 ◽

1977 ◽

Vol 37 (01) ◽

pp. 154-161 ◽

Cited By ~ 1

Author(s):

B. A Janik ◽

S. E Papaioannou

Keyword(s):

Side Effects ◽

Effective Dose ◽

Fibrinolytic Activity ◽

Ex Vivo ◽

Plasminogen Activators ◽

Assay System ◽

Coagulation Parameters ◽

Ex Vivo Assay

SummaryUrokinase, streptokinase, Brinase, trypsin, and SN 687, a bacterial exoprotease, have been evaluated in an ex vivo assay system. These enzymes were injected into rabbits and the fibrinolytic activity as well as other coagulation parameters were measured by in vitro techniques. Dose-response correlations have been made using the euglobulin lysis time as a measure of fibrinolytic activity and the 50% effective dose has been determined for each enzyme. Loading doses, equal to four times the 50% effective dose, were administered to monitor potential toxicity revealing that Brinase, trypsin, and SN 687 were very toxic at this concentration.Having established the 50% effective dose for each enzyme, further testing was conducted where relevant fibrinolytic and coagulation parameters were measured for up to two days following a 50% effective dose bolus injection of each enzyme. Our results have demonstrated that urokinase and streptokinase are plasminogen activators specifically activating the rabbit fibrinolytic system while Brinase, trypsin and SN 687 increase the general proteolytic activity in vivo.The advantages of this ex vivo assay system for evaluating relative fibrinolytic potencies and side effects for plasminogen activators and fibrinolytic proteases have been discussed.

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Quantitative Determination of Plasminogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654134 ◽

1970 ◽

Vol 23 (02) ◽

pp. 191-201 ◽

Cited By ~ 2

Author(s):

H. D Bruhn ◽

L Müller ◽

F Duckert

Keyword(s):

Quantitative Determination ◽

Plasminogen Activator ◽

Assay System ◽

Two Stage ◽

System Activation ◽

Frozen Plasma

SummaryA modification of the caseinolytic assay for plasminogen is described. This assay system is characterized by the following features :1. Urokinase is used as activator achieving a complete activation of the plasminogen whereas with streptokinase caseinolytically inactive plasminogen-activator complexes are formed.2. All incubation times are reduced to the minimum which is still compatible with accuracy.3. Results are expressed in percent of a standard of ten normal plasmas.4. In this two-stage assay-system (activation of plasminogen to plasmin, digestion of casein by plasmin) both stages proceed simultaneously in the same system, thus the plasmin formed is stabilized “in statu nascendi” by the casein.5. Several conditions (stability of plasminogen in frozen plasma, use of anticoagulants, reproducibility) are defined.

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