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Possible proteolyzation of circulating betatrophin in human serum
Update time:2016-08-01 22:35:14   【 Font: Large  Medium Small

OBJECTIVES: Betatrophin (BTH) is a secreted peptide which stimulates beta cell proliferation in mouse and rat models. However, the results of previous clinical investigations on BTH levels in human serum are inconsistent. We hypothesized that circulating BTH could be proteolyzed in human serum and investigated whether circulating BTH levels change when blood samples are collected with or without protease inhibitors.

METHODS: We obtained 3 sets of fasting blood samples collected into both a serum tube and a P800 tube (Becton Dickinson, NJ, USA) which contained a protease inhibitor cocktail from each healthy subject (n=10) and incubated each set at room temperature for different time intervals (0, 3 hours and 6 h), and afterwards frozen below -20°C until the assay. We also withdrew fasting blood samples into the same set of tubes from T2DM patients (n = 20). Circulating BTH levels were determined using two commercial ELISA kits: N-terminal kits based on antibodies against the N-terminus of BTH which recognize (Wuhan Eiaab Science, Wuhan, China, Catalogue No.E11644 h), and C-terminal kits based on antibodies against the C-terminus of BTH (Phoenix Pharmaceuticals, CA, USA, Catalogue No.EK-051-55).
RESULTS: Both N-terminal and C-terminal ELISA kits showed linear regression (r = 0.99) when measuring a human recombinant betatrophin. BTH levels in healthy subjects decreased in a time-dependent manner when assayed by Cterminal kits, but remained constant over time when assayed by N-terminal kits. Both ELISA kits demonstrated significant differences between BTH levels in serum tubes and in P800 tubes (P < 0.05, Wilcoxon test). The BTH levels in P800 tubes were much higher than those in serum tubes (31.91 – 12.38 ng/mL vs 1.89 – 1.70 ng/mL, by C-terminal ELISA kits; 599.44 – 510.46 pg/mL vs 181.01 – 45.48 pg/mL, by N-terminal ELISA kits). A significant correlation between BTH levels in serum tubes and in P800 tubes was found when measured by N-terminal kits (Spearman’s rank-order correlation; r = 0.70, P < 0.001), but not when measured by C-terminal kits (r = 0.21, P = 0.35).

CONCLUSION: Our results provide new evidences on the proteolytic regulation of BTH, suggesting the existence of a protease for BTH in human serum. The discrepancy between serum and protease inhibited plasma levels of circulating BTH must be taken into consideration in interpreting results of these commercial ELISA kits. Further studies are needed to elucidate the proteolytic regulation of BTH.

 

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Source:Journal of Diabetes Investigation      by S. Tokumoto, Y. Hamamoto, E. Okamura, et al
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