Parameters affecting substance P measurement in heart, lung, and skin

Abstract

Substance P (SP), a neuropeptide that is widely distributed both peripherally and centrally, mediates several pathophysiological processes. Among current assays for SP, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been most widely used. Several previous studies, mostly performed with nerve extracts or organ perfusates, determined that acidity of the extraction buffer as well as the number extractions performed constitute factors influencing accurate measurements. We used an ELISA protocol in this study to analyze methodological aspects of SP measurement in extracts from heart, skin, and lung. The extraction procedure had two steps, an acid extraction followed by a column extraction. We could effectively measure SP with extract from as little as 10 mg of tissue. For each tissue examined, different variables influenced the SP measured. For all tissues, the weight of tissue extracted was critical; the more tissue extracted, the lower the sensitivity of the assay. This problem could be overcome in skin by omitting the column extraction. When mechanical loses were considered (e.g., loss during extraction and SP retained by the column after elution), column extraction improved SP measurements only with lung tissue. The amount of SP remaining in the sample after the first extraction also varied among tissues. The first acid extraction effectively isolated 80% of total SP from skin. In contrast, the first extraction with lung tissue recovered only 58%. Because both acid and heat effectively release SP from nerve endings, this could reflect the presence of non-neuronal SP, especially in lung. High-dose capsaicin treatment, which depletes SP in nerve endings, caused 42% loss of SP in skin independent of amount of tissue extracted. Our results suggest that a second acid extraction of tissue should be performed and that column extraction is clearly detrimental with skin samples.