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


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


METHODS
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
Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.
Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506.
Ruby A
RA
Mosher
Johann F
JF
Coetzee
Portia S
PS
Allen
James A
JA
Havel
Gary R
GR
Griffith
Chong
C
Wang
D000818:Animals; D001800:Blood Specimen Collection / Q000379:methods / Q000662:veterinary*; D002417:Cattle / Q000097:blood*; D002853:Chromatography, Liquid / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D006493:Heparin / Q000737:chemistry; D013373:Substance P / Q000097:blood* / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000662:veterinary; D013696:Temperature
D013373:Substance P; D006493:Heparin