Secretory Phospholipase A₂ Levels in Rat Small Bowel

Abstract

Preliminary studies on ischemia/reperfusion injury in transplanted small bowel grafts showed that secretory phospholipase A₂ (sPLA₂) may play a substantial role by breaking down membrane phospholipids. This study sought to determine the normal values of sPLA₂ in the rat small bowel as a function of site and length as a baseline for future studies. The entire small bowel of male Lewis rats (200 g) was flushed with normal saline to eliminate solid contents. In group 1, the entire small bowel was divided into 5-cm segments (numbered 1–9). which were snap frozen and processed the same day for sPLA₂. In group 2, a 25-cm segment of bowel (corresponding to segments 2–6 in group 1) was harvested from each animal, snap frozen, and immediately processed for sPLA₂. To assess the effect of bowel storage on enzyme content, group 3 and group 4 grafts were stored for 7 and 14 days, respectively, at -85°C prior to processing. All samples were homogenized in buffer, extracted with H₂SO₄, and assayed for sPLA₂ activity using [1–'⁴C]oleate-labeled autoclaved Escherichia coli as substrate. Results were analyzed statistically by ANOVA. sPLA₂ activity rose from 85.46 ± 14.46% hydrolysis/min fraction^-1 in segment 1, to 476.38 ±176.75% hydrolysis/min fraction^-1 in segment 9. The increase was linear and statistically significant (p <.0001). There was no significant difference in enzymatic activity between groups 2, 3, and 4. Group 2 activity was 263.02±43.74% hydrolysis/min fraction^-1. This value was not statistically different from the mathematically calculated mean of segments 2–6 in group 1 (237.75). The results show that (1) sPLA₂ activity increases predictably with distance from the ligament of Treitz, (2) storage at -85°C does not affect sPLA₂ activity, and (3) 25-cm grafts may be evaluated in toto with reproducible baseline enzyme activity. Given the variability of enzyme activity along the course of the rat small bowel, it is imperative that exact location be identified in any studies evaluating sPLA, activity.

Key Words:

• intestinal transplantation
• phospholipase A₂
• rat small bowel