Miho Yumoto

Effect of anticoagulants in colorimetric assay for basic carboxypeptidases.

Carboxypeptidases (CP) in plasma and sera serve as regulators of anaphylatoxins such as C3a and C5a. The activity of CP can be measured by determining hippuric acid after cleavage of the small synthetic substrate hippuryl‐l‐arginine. Although a colorimetric assay is convenient for determining hippuric acid generated by CP, we noticed that some anticoagulants, such as citrate, interfere with the color development of the reagents used. EDTA and heparin provide an appropriate value. EGTA used as anticoagulant also provides an appropriate value. Therefore, concentration of citrate in samples should be controlled to be constant for background subtraction.

Heat Stability of Carboxypeptidase R of Experimental Animals

Carboxypeptidase N (CPN) and carboxypeptidase R (CPR) are present in fresh serum, and cleave C‐terminal arginine or lysine residues from bioactive peptides such as anaphylatoxins and kinins resulting in regulation of peptide activity. Although CPN is present in the active form in plasma, CPR is generated from proCPR by trypsin‐like enzymes such as thrombin. CPR regulates not only inflammatory peptides but also restricts fibrinolysis. To elucidate the complex role of CPN and CPR in vivo, studies in animal models will be essential. CPR of guinea pig, rat and rabbit decayed at 37 C rapidly as in the case of human CPR. However, at 25 C, CPR of those species decayed to some extent, although human serum CPR did not decay within 60 min. In the presence of thrombin inhibitor, CPR in the sera of animals tested decayed more rapidly than CPR in serum without thrombin inhibitor suggesting that additional generation of CPR may have been prevented during decay evaluation. However, human serum CPR decayed more rapidly in the absence of thrombin inhibitor indicating that thrombin may accelerate the decay in human serum.

Sustained High-Efficiency Daily Diafiltration Using a Mediator-Adsorbing Membrane (SHEDD-fA) in the Treatment of Patients with Severe Sepsis

Sustained high-efficiency daily diafiltration using a mediator-adsorbing membrane (SHEDD-fA) is an effective, intensive modality for sepsis treatment. Here we describe the effectiveness of SHEDD-fA, which makes the best use of three principles: dialysis, filtration and adsorption, for mediator removal in the treatment of severe sepsis. SHEDD-fA was initiated after adequate fluid resuscitation and catecholamine support had been provided. A large (2.1 m(2)) polymethylmethacrylate membrane dialyzer was placed in the blood circuit. Operation conditions were as follows: blood flow rate 150 ml/min, filtration rate 1,500 ml/h (post-dilution), and dialysate flow rate 300-500 ml/min over 8-12 h daily. 55 consecutive patients with severe sepsis were studied. The following results were obtained: pressure catecholamine index significantly decreased at 3 h after initiation of septic shock, PaO(2)/F(IO2) significantly increased at 1 h after initiation of septic acute respiratory distress syndrome, a significant decrease in interleukin (IL)-6 level for 3 days was observed, and IL-6 was effectively adsorbed in one pass through the filter. The average sequential organ failure assessment score of patients was 10.1 and the mortality at 28 days was 16.4% (46 survived, 9 died). Because SHEDD-fA is an intensive and high-efficiency modality, removal of useful drugs or nutrients may be observed. Despite the fact that removal of useful substances cannot be ignored, we believe that an appropriate stage or timing can be identified so that we can avoid a vicious cycle and use blood purification with effective diffusion, filtration and adsorption. We demonstrate that SHEDD-fA may be an effective, intensive modality for the treatment of patients with severe sepsis and is a possible modality for cytokine modulation therapy.