Measurement of plasma-derived substance P: biological, methodological, and statistical considerations

Abstract

The undecapeptide substance P (SP) is a member of the tachykinin family of neurotransmitters, which has a pivotal role in the regulation of inflammatory and immune responses. One of the major barriers to the study of the in vivo role of SP in a number of immune disorders is the accurate measurement of SP in fluids. This is reflected in the variability of reported SP levels in serum and plasma of humans in both healthy and diseased states. This study was initiated in order to identify sources of variability by the comparative evaluation of the influences of sample preparation and analytical detection methods on the measurement of SP in plasma. The results indicate that sample preparation (peptide extraction versus no extraction) and the choice of analytical method for SP quantitation may yield significantly different values and may contribute to the variability in SP values reported in the literature. These results further emphasize the need for careful consideration in the selection of methods for SP quantitation, as well as caution in the interpretation and comparison of data reported in the literature.

FIG. 1.

(a and b) Parallel evaluation of five extracted rhesus macaque plasma and unextracted human plasma samples for SP using two different SP antigen competition EIA kits. (c and d) Comparison of SP levels in 18 paired extracted and unextracted plasma samples (1:4 dilution prior to analysis) and the influence of sample dilution prior to analysis in rhesus macaque plasma using the Cayman SP EIA. (e and f) Influence of modification of the standard curve on SP values using the Cayman EIA and the Assay Designs EIA in the evaluation of unextracted human plasma at a 1:4 dilution. The upper and lower whiskers of the box-and-whisker plots are the maximum and minimum SP values. The first and third quartiles (25th and 75th percentiles) are represented by the lower and upper edges of the box, respectively. The mean is represented by the plus sign, and the median (50th percentile) is the line inside the box.

FIG. 2.

Evaluation of Cayman and Assay Designs SP standards in the other EIA using the manufacturer's recommended standard curves.