[Determination of the trace levels of urinary fibrinopeptides by high-performance capillary electrophoresis].

OBJECTIVE: To establish a high-performance capillary electrophoresis (HPCE)-based method for detection of trace amount of urinary fibrinopeptide A and B (FPA and FPB, respectively) as the specific molecular markers of thrombus formation in vivo. METHODS: The HPCE system consisted of a 25 cm x 50 microm (inner diameter) coated capillary column, 0.1 mol/L phosphoric acid buffer (pH 2.5) and a UV-detector (wavelength at 190 nm). To improve the sensitivity and reproducibility, solid-phase extraction of FPA and FPB in the urine was performed using a Sep-pak C18 column, with a synthetical fibrinopeptide B-Tyr (FPB-Tyr) as the internal standard. RESULTS: With this HPCE method, optimal separations of FPA, FPB and FPB-Tyr was achieved within 16 min, with the migration time of 7.28 min, 14.31 min and 15.22 min, respectively. The adjusted peak area ratios of FPA or FPB and the internal standard showed good linearity with the corresponding concentrations of FPA or FPB spiked in the urine(R>0.99). Under the above chromatography conditions, the minimum detection concentration of FPA and FPB in untreated urine was 30 microg/L and 40 microg/L, respectively, and the assay precision and recovery of FPA and FPB were acceptable. CONCLUSION: The method we established is reliable and specific for separation and identification of fibrinopeptides and other bioactive peptides.

Urine and plasma levels of fibrinopeptide B in patients with deep vein thrombosis and pulmonary embolism.

BACKGROUND: Many patients with pulmonary thromboembolism remain undiagnosed, possibly because of the difficulty clinicians have in determining which patients merit work-up with accurate (but expensive) imaging techniques. OBJECTIVES: We present the first prospective clinical study of pulmonary embolism (PE) and deep venous thrombosis (DVT) detection using the FPBtot assay, which measures fibrinopeptide B and its first derivative, des-arginine fibrinopeptide B. METHODS: Twenty three patients with signs or symptoms of PE or DVT were enrolled in the study prior to the performance of definitive testing. Using a novel immunoassay, FPBtot levels were measured in urine and plasma samples from patients as well as from healthy controls. Urine and plasma FPBtot levels were compared to the diagnostic results, as blindly adjudicated by one of the investigators. Patients were excluded if they withdrew (n =1), had inconclusive diagnostic testing (n = 7), or did not give samples (n = 2 for urine, n = 3 for plasma). RESULTS: The mean FPBtot concentration in the urine of the 'DVT/PE positive' group was 78.4 +/- 35.2 ng/ml and 2.7 +/- 1.9 ng/ml in the 'DVT/PE negative' group (p = 0.03). The urine FPB(tot) concentrations in the 'DVT/PE negative' group were not significantly different from those in the healthy control group (2.2 +/- 0.4 ng/ml, p = 0.40). The area under the ROC curve for urine FPB(tot) concentrations was 97.3 +/- 3.8%, suggesting a high degree of diagnostic accuracy. Plasma FPB(tot) concentrations were not significantly different between groups. CONCLUSIONS: Urine FPBtot levels may help detect patients with PE and DVT.

Role of intrarenal coagulation and anticoagulant therapy in the progression of diabetic nephropathy.

The aim of the present study was to clarify the role of intrarenal coagulation in the progression of renal dysfunction and to assess the efficacy of anticoagulant therapy in diabetic nephropathy patients. Forty-one diabetic patients were divided into 2 groups: group 1 (G-1), 20 patients with nephropathy; and group 2 (G-2), 21 patients without nephropathy. The levels of fibrinopeptide A (FPA) and fibrinopeptide B beta 15-42 (FPB beta 15-42), fibrin/fibrinogen degradation products-D dimer (FDP-D dimer), and FDP-E products (FDP-E) and FDP, which are sensitive parameters of coagulation and fibrinolysis, were measured by radioimmunoassay, enzyme immunoassay (EIA), and latex photometric immunoassay, respectively, in both the blood and urine. The levels of urinary FPA, FDP-D, FDP-E, and FDP were found to be much higher in G-1 than in G-2. Significant relations were observed among the urinary levels of these four parameters. The renal function in all cases with higher levels of urinary parameters was severely deteriorated. Following heparin administration to these patients, marked reductions of the urinary FPA, FDP-D, and FDP-E and improvement of nephrotic syndrome were observed. The present data suggest that in diabetic nephropathy: (1) intrarenal coagulation is likely to occur and to induce progression of renal dysfunction; and (2) heparin therapy could be effective in diabetic nephropathy when the patients are selected according to the above parameters of coagulation and fibrinolysis.

In vitro stability of human fibrinopeptide B.

In anticipation of a future clinical application of plasma fibrinopeptide B (FPB) measurement, we studied the stability of FPB in an ultrafiltrate of normal plasma, normal urine and alkaline buffer by measuring the immunoreactivity of the peptide by FPB radioimmunoassay using anti FPB serum (R-29). FPB was unstable in an ultrafiltrate of plasma and urine and demonstrated a temperature dependent loss of activity. In plasma ultrafiltrate the loss of immunoreactivity was not significant during the first 24 hours, however, 92% of the peptide activity was lost at the end of seven days at 25 degrees C and 37 degrees C. The rate of FPB degradation in urine was comparable. The peptide was stable in an alkaline buffer (pH 8.5) at temperatures ranging from -10 degrees C to 37 degrees C or in plasma ultrafiltrate or urine when incubated at -10 degrees C. Treatment with carboxypeptidase B or leucine aminopeptidase for two hours at 37 degrees C (enzyme/substrate molar ratio of up to 1:100) did not cause a loss of FPB immunoreactivity. EDTA (1.0 mM) and Trasylol (500 units/ml) completely stabilized the peptide in a plasma ultrafiltrate.