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

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.

Stress and pain response after oligofructose induced-lameness in dairy heifers

“…Nonetheless, our results are discordant with the findings reported by Repenning et al [ 28 ], who did not find significant differences in the plasma substance P concentration of calves subjected to band castration and their respective controls. Although our results are similar to those of previous studies, several authors have mentioned that sampling time and handling procedures are critical to obtaining reliable substance P concentrations [ 28 22 ].…”

Substance P Is Upregulated in the Serum of Patients with Chronic Spontaneous Urticaria

“…In our hands there was a very robust and reproducible upregulation of SP in CSU patients from two independent centres as compared to healthy controls; we can therefore not explain this discrepancy. However, it has been shown before that bovine SP is markedly reduced in blood samples processed within 1 hour because of rapid degradation by enzymes (Mosher et al, 2014). To avoid degradation of SP, we have processed blood within 30 minutes and stored serum at À 80 1C.…”

A design of experiments concept for the minimization of nonspecific peptide adsorption in the mass spectrometric determination of substance P and related hemokinin‐1

“…The discrepancy between these failures and promising preclinical studies leads to the assumption that the roles of the tachykinins are not fully understood until now [5]. This lack of knowledge may be based on the high variability of SP blood levels caused by distinct analytical techniques and sample preparation methods [6][7][8]. The determination of SP and HK-1 blood levels are predominantly executed by immunoassays, which often lack the required selectivity to differentiate between SP and HK-1 due to crossreactivity [9,10].…”

“…Under these conditions, the benefit of using the optimized injection solvent composition in the most suitable container material was further evaluated in human plasma samples, as the presence of endogenous plasma matrix components could also affect adsorption processes. The degradation of the endogenous peptides was induced at ambient temperatures to generate blank plasma (donated by one male volunteer) [6]. Afterwards, 300 µL of fresh blank plasma was spiked with 1 ng/mL SP and HK-1.…”

“…This lack of knowledge may be based on the high variability of SP blood levels caused by distinct analytical techniques and sample preparation methods . The determination of SP and HK‐1 blood levels are predominantly executed by immunoassays, which often lack the required selectivity to differentiate between SP and HK‐1 due to cross‐reactivity .…”