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

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  • 1 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.
  • | 3 Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.
  • | 4 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66502.
  • | 5 PharmCATS Bioanalytical Services, 2005 Research Park Cir, Manhattan, KS 66502.
  • | 6 Department of Statistics, College of Liberal Arts and Sciences, Iowa State University, Ames, IA 50011.

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.

Contributor Notes

Dr. Coetzee's present address is Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011. Dr. Allen's present address is Unit for Laboratory Animal Medicine, Medical School, University of Michigan, Ann Arbor, MI 48109.

This manuscript represents a portion of a dissertation submitted by Dr. Mosher to the Kansas State University College of Veterinary Medicine as partial fulfillment of the requirements for a Doctor of Philosophy degree.

Supported by the USDA Agriculture and Food Research Initiative, National Institutes of Health, Merck Company Foundation, Merck Research Laboratories, and Merial Animal Health.

The authors thank Colleen Hill and Jordan Shelton for technical assistance.

Address correspondence to Dr. Coetzee (hcoetzee@iastate.edu).