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Background: A separator or barrier gel is a common component of serum and plasma collection tubes. Despite their advantages, the use of these tubes is not universally accepted, especially for therapeutic drug monitoring (TDM). The aim of this study was to evaluate whether the polyacrylester separator gel in Sarstedt S-Monovette\® tubes influences the concentration of 10 selected parameters (amikacin, vancomycin, valproic acid, acetaminophen, cortisol, free thyroxine, thyroid-stimulating hormone, transferrin, prealbumin and carcinoembryonic antigen) in a clinically significant way., Methods: Results from patient samples collected in plastic Sarstedt S-Monovette® tubes with separator gel were compared with those from plain serum sample tubes. Analytes were measured in both tubes on 4 consecutive days to study the influence of prolonged contact with the separator gel. Between analyses tubes were stored at 4°C. Stability was also evaluated over 72 h for each collection tube. When statistical differences were detected, the clinical significance was evaluated based on the total allowable error (TEa)., Results: On day 1 no statistically significant differences were observed between samples collected in Sarstedt S-Monovette® tubes with and without separator gel. Statistical differences were present from day 2 on, but were not clinically significant. All evaluated parameters were clinically stable over 72 h at 4°C based on TEa, except for transferrin en fT4., Conclusion: The separator gel in Sarstedt S-Monovette® tubes did not show statistically significant differences on the day of phlebotomy. Later on statistically significant differences appeared but except for the stability of fT4 and transferrin they all remained clinically insignificant., (Copyright © 2011 Elsevier B.V. All rights reserved.)

To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz-Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20-2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl-l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.

Background: Patients with hereditary mucocutaneous bleeding are difficult to diagnose and many of them fulfill the category of bleeders of unknown cause (BUC). The pathogenic role of hyperfibrinolysis has received little attention, despite the successful use of antifibrinolytic drugs in treating many of these patients. Theoretically, decreased plasma procarboxypeptidase U (proCPU) levels or lower carboxypeptidase U (CPU) stability would result in higher fibrinolytic activity and bleeding tendency., Methods: We analyzed plasma proCPU and the distribution of the Thr325Ile proCPU polymorphism in 193 patients with mucocutaneous bleeding of whom 116 were bleeders of unknown cause (BUC), and in 143 healthy, age and sex-matched controls., Results: ProCPU concentration was higher in women than in men, increased with age, and was significantly correlated with clot lysis time, platelet count, APTT, and PT. However, proCPU levels were unexpectedly higher in patients than in controls (968+/-134 vs. 923+/-147 U/L, p=0.004). The allele distribution of the Thr325Ile proCPU polymorphism was similar in both groups, with a low percentage of homozygous Ile/Ile., Conclusions: Our results indicate that the proCPU system is not of major importance in the bleeding pathogenesis of these patients. The higher proCPU levels in the patients may even modestly counteract the bleeding tendency.

Introduction: Thrombolytic therapy improves clinical outcome in patients with acute ischemic stroke but is compromised by symptomatic intracranial hemorrhage and an unpredictable therapeutic response. In vitro and in vivo data suggest that activation of procarboxypeptidase U (proCPU) inhibits fibrinolysis., Aims: To investigate whether the extent of proCPU activation is related to efficacy and safety of thrombolytic therapy in ischemic stroke patients., Methods: In twelve patients with ischemic stroke who were treated with intravenous (n=7) or intra-arterial (n=5) thrombolysis, venous blood samples were taken at different time points before, during and after thrombolytic therapy. ProCPU and carboxypeptidase U (CPU, TAFIa) plasma concentrations were determined by HPLC. The maximal CPU activity (CPU(max)) and the percentage of proCPU consumption during thrombolytic therapy were calculated. The efficacy and safety of the thrombolytic therapy were assessed by evolution of the clinical deficit, recanalisation, final infarct volume, thrombolysis-induced intracranial hemorrhage and mortality., Results: No correlations between CPU(max) or proCPU consumption and patient or stroke characteristics were found. However, CPU(max) is associated with evolution of the clinical deficit and achieved recanalisation. ProCPU consumption is related to the risk of intracranial hemorrhage, mortality and final infarct volume., Conclusions: Irrespective of patient and stroke characteristics, CPU(max) and proCPU consumption during thrombolytic treatment for ischemic stroke are parameters for therapeutic efficacy and safety. Further evaluation of the clinical applicability of these parameters and further investigation of the potential role for CPU inhibitors as adjunctive therapeutics during thrombolytic treatment may be of value.

Background: Measurement of procarboxypeptidase U (TAFI) in plasma by activity-based assays is complicated by the presence of plasma carboxypeptidase N (CPN). Accurate blank measurements, correcting for this interfering CPN activity, should therefore be performed. A selective CPU substrate will make proCPU determination much less time-consuming., Methods: We searched for selective and sensitive CPU substrates by kinetic screening of different Bz-Xaa-Arg (Xaa=a naturally occurring amino acid) substrates using a novel kinetic assay., Results: The presence of an aromatic amino acid (Phe, Tyr, Trp) resulted in a fairly high selectivity for CPU which was most pronounced with Bz-Trp-Arg showing a 56-fold higher k(cat)/K(m) value for CPU compared to CPN. Next we performed chemical modifications on the structure of those aromatic amino acids. This approach resulted in a fully selective CPU substrate with a 2.5-fold increase in k(cat) value compared to the commonly used Hip-Arg (Bz-Gly-Arg)., Discussion: We demonstrated significant differences in substrate specificity between CPU and CPN that were previously not fully appreciated. The selective CPU substrate presented in this paper will allow straightforward determination of proCPU in plasma in the future.

Background: Carboxypeptidase N is a plasma zinc metallocarboxypeptidase which is constitutively expressed in the liver and was identified as the enzyme responsible for inactivating bradykinin and kallidin by removing the C-terminal arginine. Because CPN can cleave the C-terminal arginine of C3a, C4a and C5a it is often referred to as anaphylatoxin inactivator. Markedly reduced levels of circulating CPN are associated with recurrent angioedema and abnormal cutaneous polymorphonuclear cell infiltration., Methods: In this paper we describe a fast kinetic coupled enzymatic assay for the sensitive measurement of carboxypeptidase N activities in serum samples. The assay makes use of the excellent CPN substrate Benzoyl-L-Alanyl-L-Arginine., Results: This novel assay is very fast, easy to perform and combines good reliability and reproducibility with excellent correlation with the HPLC-assisted assay (r=0.927; n=140)., Conclusion: The presented assay can be used for high throughput screening of this important regulator of inflammation in clinical plasma or serum samples.

Carboxypeptidase U (CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates in blood as an inactive zymogen, procarboxypeptidase U, which is activated during the process of coagulation and fibrinolysis. CPU has a very short half-life at 37 degrees C. Its intrinsic instability complicates the determination of kinetic parameters of different substrates using an endpoint method. We developed a fast kinetic assay for measuring continuously the release of the C-terminal arginine by CPU independent of the nature of the substrate peptide used, allowing us to perform substrate specificity studies of CPU. This method uses arginine kinase, pyruvate kinase, and lactate dehydrogenase as auxiliary enzymes. The CPU activities measured using this kinetic assay were in the range of 97-103% of those determined with our HPLC-assisted reference assay, and the obtained K(m) and k(cat) values for hippuryl-l-arginine and bradykinin were in good accordance with those described in the literature. As expected, no arginine cleaving was seen using dipeptides and peptide substrates with a proline in the penultimate position. The presented kinetic assay enables the fast screening of substrates with a C-terminal arginine and is a valuable new tool for the kinetic evaluation of both synthetic and physiological substrates of CPU.