Effect of age on leukotriene B4 production in guinea pig whole blood.
We evaluated the effect of age on eicosanoid production in guinea pig blood. Heparinized blood from 7-10-day, 6-week, or 6-month-old guinea pigs was incubated with 150 microM arachidonic acid (AA) for 5 min, followed by stimulation with A23187 (20 micrograms/mL) for an additional 10 min at 37 degrees. The reaction was terminated by centrifugation, and the production of plasma leukotriene (LT) B4 and C4, prostaglandin E2 (PGE2), and thromboxane B2 (TXB2) was determined by enzyme-linked immunoassay (ELISA). LTC4, PGE2, and TXB2 formation were unaffected by age. In marked contrast, production of LTB4 was increased 4- to 5-fold as age increased from 7-10 days (9.51 +/- 2.07 ng/mL) or 6 weeks (8.83 +/- 1.81 ng/mL) to 6 months (40.57 +/- 9.66 ng/mL). To determine the effect of age on the total eicosanoid product profile, blood was stimulated in the presence of [14C]AA, and plasma metabolites were separated by reverse-phase high pressure liquid chromatography (RP-HPLC) and quantitated using on-line radiochemical detection. In addition to increased LTB4 production, a modest increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was also observed in the 6-month-old animals. Previous studies have demonstrated interference of 12-HETE in the immunoassay of LTB4. Therefore, to validate the authenticity of the plasma leukotriene ELISA measurements, samples were precipitated with methanol and fractionated by RP-HPLC. The fractions co-eluting with [3H]LTB4 or [3H]LTC4 were dried under vacuum and reconstituted in ELISA buffer, and leukotrienes were quantitated. As seen previously, following HPLC purification LTB4 production remained significantly elevated in the 6-month-old guinea pigs, whereas LTC4 production was unaffected by age. To further document the selectivity of this effect on LTB4 production, we evaluated the effect of increasing age on cyclooxygenase or phospholipase A2 (PLA2) activity. Neither cyclooxygenase nor PLA2 activity was elevated as animals matured. In conclusion, the capacity of whole blood to produce LTB4, but not LTC4, TXB2, or PGE2, was elevated markedly in older animals.
Spaethe
S M
SM
Department of Pulmonary Research, Lilly Research Laboratories, Indianapolis, IN 46285.
Scaife
C L
CL
Pechous
P A
PA
VanAlstyne
E L
EL
D000367:Age Factors; D000818:Animals; D016718:Arachidonic Acid / Q000097:blood; D001772:Blood Cell Count; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D006168:Guinea Pigs; D007975:Leukotriene B4 / Q000096:biosynthesis* / Q000097:blood; D008297:Male; D010741:Phospholipases A / Q000097:blood; D054467:Phospholipases A2; D011451:Prostaglandin-Endoperoxide Synthases / Q000097:blood
D007975:Leukotriene B4; D016718:Arachidonic Acid; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2