The effect of class-specific protease inhibitors on the stabilization of B-type natriuretic peptide in human plasma.

Author(s): Belenky A, Smith A, Zhang B, Lin S, Despres N, Wu AH, Bluestein BI

Publication: Clin Chim Acta, 2004, Vol. 340, Page 163-72

PubMed ID: 14734208 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study examined the stability of BNP, assessed in plasma specimens spiked with synthetic BNP, after refrigerated storage as well as the preservation capacity of 8 different protease inhibitors.

   Summary of Findings:

   All protease inhibitors tested slowed down the proteolytic degradation of BNP. In the absence of inhibitors, 10% of the exogenously added BNP was detected after storage at 4 degrees C for 40 h and no BNP was detectable by 6 d. The addition of 50 ug/ml of antipain, leupeptin, PPACKI, PPACKII or DFP completely prevented the proteolysis of BNP for up to 6 d at 2-8 degrees C. Doses as low as 0.62 ug/ml allowed for 40% recovery after 6 days with antipain and leupeptin and 80% after 6 d with PPACKII. The generic serine protease inhibitors AEBSF and benzamidine allowed for 80% recovery of BNP at 6 d. Aprotinin was much less effective at preserving the BNP and only 20% of BNP was recoverable after 6 d. In conclusion antipain, leupeptin, PPACKI, PPACKII or DFP were highly effective while AEBSF and benzamidine were slightly less effective at blocking proteolysis of BNP.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Peptide	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	2-8 degrees C
   Storage	Storage duration	0 6 h 1 day 2 days 6 days
   Analyte Extraction and Purification	Protease inhibitor	No protease inhibitor added PPACKI PPACKII 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) Antipain Benzenecarboximidamide (benzamidine) Diisopropylfluorophosphate (DFP) Leupeptin

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2. Study Purpose

   The purpose of this study was to assess BNP stability in HSA and plasma specimens spiked with synthetic BNP then stored at 2-8 degrees C. The ability of PPACKII and DFP protease inhibitors to attenuate storage-induced BNP degradation was also accessed.

   Summary of Findings:

   HSA caused proteolysis of 85% of exogenously added BNP in only 3 days compared to 62% in plasma. After 4 weeks only 0.4% of the added BNP was detected in HSA and 1.4% in plasma. The addition of PPACKII or DFP allowed for more than 96% of the BNP in the HSA or plasma to be retained after 4 weeks of storage at 2-8 degrees C. The authors conclude that HSA contributes to BNP degradation, but that degradation can be prevented through the addition of PPACKII or DFP.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Peptide	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protease inhibitor	Diisopropylfluorophosphate (DFP) PPACKII
   Storage	Storage duration	0 3 days 1 week 2 weeks 4 weeks
   Storage	Storage temperature	2-8 degrees C
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum

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3. Study Purpose

   The effects of storage duration, temperature and addition of the protease inhibitor PPACKII on endogenous BNP levels in plasma were investigated.

   Summary of Findings:

   Storage of patient plasma at room temperature compared to 2-8 degrees C resulted in a greater loss of BNP for all time points examined. By 48 h, BNP levels decreased to 67% for room temperature specimens and 73% for those stored at 2-8 degrees C. Adding the protease inhibitor to specimens stored at 2-8 degrees C for 72 hours increased the detectable BNP from 67% to 100%. After storage for 6 days at 2-8 degrees C, 100% of BNP was retained when PPACK was added versus less than 10% when no protease inhibitor was added.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Peptide	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protease inhibitor	No protease inhibitor added PPACKII
   Storage	Storage duration	0 h 6 h 24 h 48 h 72 h 6 days
   Storage	Storage temperature	Room temperature 2-8 degrees C

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