Eicosanoids measurements

Experiment I. In a time-course experiment, mucosal PGE production and phospholipid fatty acid profile were assessed at d 0,4,8,12, and 16 of dietary treatment in formula-fed and naturally reared piglets (n = 5 piglets per time per dietary treatment). Mucosal cells were scraped from proximal ends of the small intestine and frozen at -80°C for later lipid analysis. Lipids were extracted by a modified Folch procedure (15). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated by thin-layer chromatography (16), and fatty acids in each phospholipid fraction were analyzed by gas chromatography. For eicosanoid measures, fresh mucosal tissue was incubated in Kreb s Ringer bicarbonate buffer as described previously (17). PGE2 was extracted from the incubation media with ethyl acetate and quantified using a competitive enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). 

Production of eicosanoids begins after arachidonic acid is released from membrane phospholipid molecules by the enzyme phospholipase A2. The eicosanoids, which include the prostaglandins, thromboxanes, and leukotrienes (Figure 11 A), are extremely difficult to study because they are active for short periods (often measured in seconds or minutes). In addition, they are produced only in small amounts. 

We have evaluated the effect of black tea theaflavin derivatives on 12-0-tetra-decanoylphorbol-13-acetate induced skin inflammation in mice. The anti-inflammatory activities of theaflavin, theaflavin monogallates, and theaflavin digallate have been evaluated using this model. The effects of these compounds on ear edema (measured as ear punch weight) and levels of inflammatory mediators (cytokines and eicosanoids) have been evaluated. ... 

Morrow, J.D., Harris, T.M., and Roberts, L.J., Noncyclooxygenase oxidative formation of a series of novel prostaglandins analytical ramifications for measurement of eicosanoids. Anal Biochem 184 (1990) 1-10. 

A recent report by Parsons et a/." presented evidence for simultaneous elevation of complement fragments and measurable levels of endotoxin in plasma from patients with ARDS. Because complement activation by toxins and by other mechanisms" is thought to cause neutrophil and platelet activation" and subsequent generation of toxic oxygen species and eico-sanoids various strategies known to activate intravascular complement have been used to examine the role of eicosanoids in lung injury . To... 

Recently, Stenmark et al measured eicosanoids and PAF in the lung lavage fluid from neonatal calves exposed to two weeks of high-altitude hypoxia. Prostacyclin, thromboxane and PAF were increased and accompanied by... 

Endotoxin tolerance also results in altered eicosanoid metabolism. Plasma levels of iTxB2 and i6-keto-PGFio were measured after administration of endotoxin at normally lethal (15 mg kgand supralethal (50 mg kg doses In contrast to marked elevations of plasma iTxB2 and id-keto-PGFjgj after endotoxin administration in non-tolerant control rats, levels in tolerant rats were significantly reduced. Tolerant rats also demonstrated enhanced... 

SHR compared with normotensive WKY rats, whereas there was no significant difference at mo 12 of the hypertensive period. Our data indicate no correlation between the directly measured enzyme activity and the changes in hepatocyte n-3 FA composition (111). But the EFAbiosynthesis in isolated liver cells from SHR is not reflected in the low desat-urase activities observed in liver microsomes (see above). Thus, it is evident that mechanisms other than desaturation may play an important role in the control of the PUFA ratio in tissues. Storage, oxidation, eicosanoid, and other second-messenger biosynthesis, as well as the incorporation into a specific pool, have to be considered. 

Most experiments can be regarded as possible or impossible, and sensible or senseless. The chemically unstable eicosanoids, such as prostaglandin I2 (PGI2) and thromboxane A2 (TXA2) exist transiently. Is it possible, or sensible, to measure these unstable agents, and how do experimental conditions influence their measurements ... 

P6H2 aggregates PRP. Covalent binding represents an important alternative fate" for labile eicosanoids. Identification and measurement of eicosanoids in the "protein bound" compartment represents a principal problem now facing researchers in this field. 

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