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.

Highly Sensitive and Rapid Assay of Substance P and Streptolysin O in Human Serum Using Immuno-liposomes and Gramicidin Channels

“…14 However, endogenous SP in human plasma and serum is present in free and bound states; SP (MW 1347.6) reversibly and nonspecifically binds to proteins with high-molecular (> 400000 Da) and intermediate-molecular (58000 Da) mass. [26][27][28] Hence, the extraction procedure is likely to eliminate SP bound to proteins, leading to the underestimates of the total SP amount in the biological fluids. This may be the reason for a considerable variation in the reported levels of SP with or without extraction steps.…”

“…This may be the reason for a considerable variation in the reported levels of SP with or without extraction steps. 26,27 In addition, not only SP, 9 but also total amount of serum and plasma proteins, vary under physiological and pathological conditions. 29,30 RIA and EIA are highly sensitive methods that provide the possibility of measuring SP levels without any extraction steps.…”

“…The dilution of serum or plasma samples increases the ratio of the probe antibody to serum or plasma proteins, allowing effective dissociation of nonspecifically bound SP from proteins. 26 However, RIA and EIA necessitate time-consuming procedures. Furthermore, the assay has to be highly sensitivity, because the SP levels are lowered by dilution.…”

Novel Method for Determination of Substance P Levels in Unextracted Human Plasma by Using Acidification

“…Such extractions should be considered carefully since most are designed for the enrichment of low-molecular-mass peptides that can potentially exclude peptides nonspecifically bound to high-molecular-mass plasma or serum proteins. Such procedures may lead to underestimates of the total quantity of SP (4,6).…”

“…3, linear regression analysis revealed excellent correlations of SP values derived from either standard curve, yielding an R 2 of 0.978. Difficulty in the accurate measurement of SP in plasma is attributed, in part, to reversible nonspecific binding of SP to plasma proteins (4,6). This study was undertaken to develop a gentle nonchromatographic, liquid-phase procedure that permits the dissociation of SP from plasma proteins though limited hydrogen ion pressure, allowing for the capture of SP by a specific antibody of higher avidity after a return to more physiologic conditions.…”