The urine samples were collected and 3-ml aliquots were transferred to special tubes containing 0.75 ml of an aqueous anticoagulant solution. The anticoagulant solution contained the following (all values are final concentrations): sodium azide, 0.2 mg/ml; sodium citrate, 22 mmol; porcine interstitial heparin (Sigma H7005), 200 U/ml; and aprotonin (Sigma A-1153), 0.2 trypsin inhibitory units (TIU)/ml. All the samples were frozen within 20 min of collection at — 70°C. buy cheap antibiotics
Before assaying, the samples were tested with dipstick. The heme-positive samples were treated with bentonite to remove fibrinogen contamination. All samples were treated with phosphate buffer (concentration, 25 mmol) to bring the pH to 7.5; a urine aliquot of 450 til was treated with 50 jxl of the buffer. The FPA concentration (ng/ml) was divided by urine creatinine concentration (measured by alkaline picrate method) to correct for the changes in urine concentration. Thus, the final result was expressed in nanograms of uFPA per milligram of creatinine (ng/mg).
The assay calibration curve was derived by using standard samples with known concentration of synthetic human 16-amino-acid peptide. The calibration standards were prepared with the same addition of phosphate buffer as was used for patient samples.