Determination of a new rapid plasmin inhibitor in human blood by means of a plasmin specific tripeptide substrate.

A method for determination of antiplasmin activity is presented. Plasmin and plasma are incubated, and the remaining plasmin activity is measured spectrophotometrically by means of the plasmin specific tripeptide substrate H-d-Val-l-Leu-l-Lys-p-nitroanilide. The method is simple, rapid and easily automatized. By the immunoadsorption technique, and with the aid of purified substances it is shown that the measured activity is mainly due to a new antiplasmin [2,4] and possibly to some extent to alpha1-antitrypsin and C1-esterase inhibitor have no antiplasmin activity in the method. Heparin and epsilonaminocaproic acid interfered with the assay.     

Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase.

New chromogenic tripeptide substrates have been used for the determination of kallikreins and urokinase. The conditions have been optimized. It is possible to determine prekallikrein in plasma after activation with Cephotest. No significant loss in activity caused by plasma kallikrein inhibitors is observed at the dilutions used.     

A new sensitive and highly specific chromogenic peptide substrate for Factor Xa

Chromogenic tetrapeptide substrates for Factor X_a have been synthesized with the natural substrate, prothrombin, as a model. The substrate benzoyl-L-Ile-L-Glu-Gly-L-Arg-p-nitroanilide proved to be sensitive and highly specific for Factor X_a, allowing a rapid and convenient spectrophotometric method for the assay of Factor X_a activity. The optimal conditions with respect to pH, ionic strength and substrate concentration have been determined. This substrate has also been used for simple two stage methods for the determination of Factor X in plasma.     

Studies on the duration of local anaesthesia: a possible mechanism for the prolonging effect of dextran on the duration of infiltration anaesthesia

The mechanism of the prolonged effect of dextran on the duration of local anaesthesia has been studied. Using radio-active mepivacaine it was found that dextran prolonged the duration of infiltration anaesthesia in guinea-pigs by delaying the absorption of the local anaesthetic agent. Experiments in vitro indicate that molecular complexes between the local anaesthetic and dextran may be formed and it is assumed that the delayed absorption might be due to the formation of such molecular complexes. This hypothesis was strengthened by experiments in which dental infiltration anaesthesias were performed in healthy volunteers.     

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

Background

There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [⁸⁹Zr]Zr-oxine (8-hydroxyquinoline) and [⁸⁹Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers.

Procedures

Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [⁸⁹Zr]Zr-oxine or [⁸⁹Zr]Zr-DFO-NCS. The cellular retention of ⁸⁹Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining.

Results

The optimised synthesis of [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. ⁸⁹Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling.

Conclusions

Our study demonstrates that [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [⁸⁹Zr]Zr-oxine was superior to [⁸⁹Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.