Failure patients (18 men and 14 women; age range, 34?4 years, mean age, 65611 years) hospitalized at Kyoto University Hospital. The primary causes of the heart failure were ischemic heart disease (n = 8), cardiomyopathy (n = 8), valvular heart disease (n = 7), pulmonary hypertension (n = 7) and others (n = 2), which were AKT inhibitor 2 diagnosed from the medical history, physical examination and chest radiographic, electrocar-Table 2. Effects of dilution on recovery rates with the proBNP and total BNP assay systems.Dilution magnitudeproBNP assay system Measured, pmol/L Recovery, 112 102 98 103 105total BNP assay system Measured, pmol/L 95 101 104 92 93 97 95 Recovery, 107 109 97 98 1031 2 5 10 20 5094 105 96 92 97 99doi:10.1371/journal.pone.0053233.tproBNP in Human PlasmaTable 3. Intra- and Inter-assay precision of the proBNP assay systems.Added proBNP concentration Measured concentration pmol/L pmol/L Mean Intra-assay (n = 5) 2.0 25 100 Inter-assay (n = 15) 2.0 25 100 doi:10.1371/journal.pone.0053233.t003 2.0 25 101 1.9 23 96 S.D. 0.2 1.3 5.5 0.1 1.7 6.CV 8.0 5.2 5.4 5.3 7.4 6.Bias 2.0 0.0 1.0 25.8 28.0 24.diographic, echocardiographic and/or cardiac catheterization findings. Patients with symptomatic heart failure were under medication, including angiotensin-converting-H 4065 custom synthesis enzyme inhibitors/ angiotensin-receptor blockers, digitalis and diuretics. The New York Heart Association (NYHA) functional classes were class I I (n = 19) and class III V (n = 13). Healthy subjects (61 men and 54 women; age range, 30?8 years, mean age, 50610 years) were selected based on their normal physical, laboratory, chest radiographic, electrocardiographic and 24272870 echocardiographic findings, and their BNP levels.(100 mg) and each fraction was analyzed using the total BNP and proBNP assay systems. Because recent studies have shown that glycosylated proBNP with a MW of about 30 K circulates in the plasma [7], we examined the gel filtration positions at which commercial recombinant proBNP and glycosylated proBNP, and synthetic BNP were eluted to determine which is the major molecular form of BNP in human plasma.Deglycosylation enzyme treatmentWe further analyzed the immunoreactive proBNP levels to determine whether immunoreactive proBNP in plasma is glycosylated. Eluate lyophilized after extraction on a Sep-Pak C18 column was dissolved in phosphate buffer and incubated with or without a cocktail of deglycosylation enzymes for 24 h at 37uC, as previously described [13]. The enzyme cocktail included Oglycosidase (Roche Diagnostic) and neuraminidase (Roche Diagnostics) at final concentrations of 4.25 and 42.5 mU/mL, respectively. These two enzymes were essential for the deglycosylation, and the enzyme concentrations and incubation period were selected based on the results of preliminary and previously reported studies [11,13,14]. We then lyophilized the sample again and dissolved it in 30 acetonitrile containing 0.1 TFA, after which it was subjected to gel-filtration HPLC as described above.Plasma samplesBlood samples were drawn into plastic syringes and quickly transferred to chilled tubes containing EDTA (1.5 mg/mL, blood) and aprotinin (500 U/mL blood) and centrifuged at 16006 g for 20 min at 4uC. The obtained plasma samples were stored at 280uC until assayed.Assay of plasma NT-proBNP levelsPlasma levels of NT-proBNP were measured using Elecsys proBNP II assay system (Roche Diagnostics, Basel, Switzerland).Gel filtration chromatographyPlasma samples were extracted using Sep-.Failure patients (18 men and 14 women; age range, 34?4 years, mean age, 65611 years) hospitalized at Kyoto University Hospital. The primary causes of the heart failure were ischemic heart disease (n = 8), cardiomyopathy (n = 8), valvular heart disease (n = 7), pulmonary hypertension (n = 7) and others (n = 2), which were diagnosed from the medical history, physical examination and chest radiographic, electrocar-Table 2. Effects of dilution on recovery rates with the proBNP and total BNP assay systems.Dilution magnitudeproBNP assay system Measured, pmol/L Recovery, 112 102 98 103 105total BNP assay system Measured, pmol/L 95 101 104 92 93 97 95 Recovery, 107 109 97 98 1031 2 5 10 20 5094 105 96 92 97 99doi:10.1371/journal.pone.0053233.tproBNP in Human PlasmaTable 3. Intra- and Inter-assay precision of the proBNP assay systems.Added proBNP concentration Measured concentration pmol/L pmol/L Mean Intra-assay (n = 5) 2.0 25 100 Inter-assay (n = 15) 2.0 25 100 doi:10.1371/journal.pone.0053233.t003 2.0 25 101 1.9 23 96 S.D. 0.2 1.3 5.5 0.1 1.7 6.CV 8.0 5.2 5.4 5.3 7.4 6.Bias 2.0 0.0 1.0 25.8 28.0 24.diographic, echocardiographic and/or cardiac catheterization findings. Patients with symptomatic heart failure were under medication, including angiotensin-converting-enzyme inhibitors/ angiotensin-receptor blockers, digitalis and diuretics. The New York Heart Association (NYHA) functional classes were class I I (n = 19) and class III V (n = 13). Healthy subjects (61 men and 54 women; age range, 30?8 years, mean age, 50610 years) were selected based on their normal physical, laboratory, chest radiographic, electrocardiographic and 24272870 echocardiographic findings, and their BNP levels.(100 mg) and each fraction was analyzed using the total BNP and proBNP assay systems. Because recent studies have shown that glycosylated proBNP with a MW of about 30 K circulates in the plasma [7], we examined the gel filtration positions at which commercial recombinant proBNP and glycosylated proBNP, and synthetic BNP were eluted to determine which is the major molecular form of BNP in human plasma.Deglycosylation enzyme treatmentWe further analyzed the immunoreactive proBNP levels to determine whether immunoreactive proBNP in plasma is glycosylated. Eluate lyophilized after extraction on a Sep-Pak C18 column was dissolved in phosphate buffer and incubated with or without a cocktail of deglycosylation enzymes for 24 h at 37uC, as previously described [13]. The enzyme cocktail included Oglycosidase (Roche Diagnostic) and neuraminidase (Roche Diagnostics) at final concentrations of 4.25 and 42.5 mU/mL, respectively. These two enzymes were essential for the deglycosylation, and the enzyme concentrations and incubation period were selected based on the results of preliminary and previously reported studies [11,13,14]. We then lyophilized the sample again and dissolved it in 30 acetonitrile containing 0.1 TFA, after which it was subjected to gel-filtration HPLC as described above.Plasma samplesBlood samples were drawn into plastic syringes and quickly transferred to chilled tubes containing EDTA (1.5 mg/mL, blood) and aprotinin (500 U/mL blood) and centrifuged at 16006 g for 20 min at 4uC. The obtained plasma samples were stored at 280uC until assayed.Assay of plasma NT-proBNP levelsPlasma levels of NT-proBNP were measured using Elecsys proBNP II assay system (Roche Diagnostics, Basel, Switzerland).Gel filtration chromatographyPlasma samples were extracted using Sep-.
