Effects of sample handling methods on substance P concentrations and immunoreactivity in bovine blood samples

Ruby A. Mosher Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Johann F. Coetzee Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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Portia S. Allen Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.

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James A. Havel PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66502.

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Gary R. Griffith PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66502.

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Chong Wang Department of Statistics, College of Liberal Arts and Sciences, Iowa State University, Ames, IA 50011.

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Abstract

Objective—To determine the effects of protease inhibitors and holding times and temperatures before processing on the stability of substance P in bovine blood samples.

Samples—Blood samples obtained from a healthy 6-month-old calf.

Procedures—Blood samples were dispensed into tubes containing exogenous substance P and 1 of 6 degradative enzyme inhibitor treatments: heparin, EDTA, EDTA with 1 of 2 concentrations of aprotinin, or EDTA with 1 of 2 concentrations of a commercially available protease inhibitor cocktail. Plasma was harvested immediately following collection or after 1, 3, 6, 12, or 24 hours of holding at ambient (20.3° to 25.4°C) or ice bath temperatures. Total substance P immunoreactivity was determined with an ELISA; concentrations of the substance P parent molecule, a metabolite composed of the 9 terminal amino acids, and a metabolite composed of the 5 terminal amino acids were determined with liquid chromatography–tandem mass spectrometry.

Results—Regarding blood samples processed immediately, no significant differences in substance P concentrations or immunoreactivity were detected among enzyme inhibitor treatments. In blood samples processed at 1 hour of holding, substance P parent molecule concentration was significantly lower for ambient temperature versus ice bath temperature holding conditions; aprotinin was the most effective inhibitor of substance P degradation at the ice bath temperature. The ELISA substance P immunoreactivity was typically lower for blood samples with heparin versus samples with other inhibitors processed at 1 hour of holding in either temperature condition.

Conclusions and Clinical Relevance—Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.

Abstract

Objective—To determine the effects of protease inhibitors and holding times and temperatures before processing on the stability of substance P in bovine blood samples.

Samples—Blood samples obtained from a healthy 6-month-old calf.

Procedures—Blood samples were dispensed into tubes containing exogenous substance P and 1 of 6 degradative enzyme inhibitor treatments: heparin, EDTA, EDTA with 1 of 2 concentrations of aprotinin, or EDTA with 1 of 2 concentrations of a commercially available protease inhibitor cocktail. Plasma was harvested immediately following collection or after 1, 3, 6, 12, or 24 hours of holding at ambient (20.3° to 25.4°C) or ice bath temperatures. Total substance P immunoreactivity was determined with an ELISA; concentrations of the substance P parent molecule, a metabolite composed of the 9 terminal amino acids, and a metabolite composed of the 5 terminal amino acids were determined with liquid chromatography–tandem mass spectrometry.

Results—Regarding blood samples processed immediately, no significant differences in substance P concentrations or immunoreactivity were detected among enzyme inhibitor treatments. In blood samples processed at 1 hour of holding, substance P parent molecule concentration was significantly lower for ambient temperature versus ice bath temperature holding conditions; aprotinin was the most effective inhibitor of substance P degradation at the ice bath temperature. The ELISA substance P immunoreactivity was typically lower for blood samples with heparin versus samples with other inhibitors processed at 1 hour of holding in either temperature condition.

Conclusions and Clinical Relevance—Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.

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