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

• Localización: American Journal of Veterinary Research, ISSN-e 1943-5681, ISSN 0002-9645, Nº. 3-75, 2014, págs. 109-116
• Idioma: inglés
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• Resumen

  • 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.