Limit of detection: 80% B/B₀: 3.1 pg/ml · Sensitivity: 50% B/B₀: 15 pg/ml · Prostaglandin D₂ (PGD₂) is biosynthesized in the brain by a soluble, 26 kD glutathione-independent lipocalin-type PGD₂ synthase.¹ PGD₂ accumulates in the cerebrospinal fluid (CSF), where it induces physiologic sleep in rats and humans.² PGD₂ is also synthesized by mast cells and leukocytes by a cellular, myeloid-type, glutathione-dependent PGD synthase. This PGD₂ which is formed in the intracellular and vascular compartments is rapidly metabolized to 11β-PGF_2α.³ Thus, urinary measurements of PGD synthesis are most appropriately focused on the measurement of 11β-PGF_2α. Measurement of the parent eicosanoid PGD₂ is appropriate in the supernatants of cell cultures, where PGD₂ levels may reach several ng/ml, and in CSF, where concentrations of several hundred pg/ml have been measured.⁴ All studies of PGD₂ biosynthesis should take into consideration the chemical instability of PGD₂ and its rapid degradation in the presence of serum proteins such as albumin.⁵ PGD₂ also readily degrades in both acidic and basic media to give a variety of decomposition products. Similarly, antigenic protein conjugates of PGD₂, synthesized for the production of antisera, show considerable amounts of decomposition. Thus, the resulting antibody response is heterogeneous with poor specificity. This makes PGD₂ assay systems based on the parent compound unreliable and difficult to interpret. This PGD₂-MOX EIA is based on the conversion of PGD₂ to a stable MOX derivative. Treatment of the sample with methoxylamine hydrochloride (MOX HCl) converts PGD₂ into PGD₂-MOX, preventing its further chemical degradation. The antiserum used in the assay was developed using conjugates of this derivative and is very specific for PGD₂-MOX. The assay has been validated against stable isotope dilution GC/MS. Measurements using both techniques on identical samples showed a correlation coefficient of 0.97.

1 Urade, Y., Tanaka, T., Eguchi, N., et al. Structural and functional significance of cysteine residues of glutathione-independent prostaglandin D synthase. J Biol Chem 270 1422-1428 (1995).

2 Satoh, S., Matsumura, H., Nakajima, T., et al. Promotion of sleep by prostaglandin D₂ in rats made insomniac by pretreatment with para-chlorophenylalanine. Neurosci Res 21 41-50 (1995).

3 Roberts, L.J., and Sweetman, B.J. Metabolic fate of endogenously synthesized prostaglandin D₂ in a human female with mastocytosis. Prostaglandins 30 383-400 (1985).

4 Ujihara, M., Urade, Y., Eguchi, N., et al. Prostaglandin D₂ formation and characterization of its synthetases in various tissues of adult rats. Arch Biochem Biophys 260 521-531 (1988).

5 Fitzpatrick, F.A., and Wynalda, M.A. Albumin-catalyzed metabolism of prostaglandin D₂. Identification of products formed in vitro. J Biol Chem 258 11713-11718 (1983).

Kelly, R.W., Deam, S., Cameron, M.J., et al. Measurement by radioimmunoassay of prostaglandins as their methyl oximes. Prostaglandins Leukot Med 24 1-14 (1986).

Westcott, J.Y., Murphy, R.C., and Stenmark, K. Eicosanoids in human ventricular cerebrospinal fluid following severe brain injury. Prostaglandins 34 877-887 (1987).

Urade, Y., Tanaka, T., Eguchi, N., et al. Structural and functional significance of cysteine residues of glutathione-independent prostaglandin D synthase. J Biol Chem 270 1422-1428 (1995).

Ninomiya, H., Yu, X.Y., Hasegawa, S., et al. Endothelin-1 induces stimulation of prostaglandin synthesis in cells obtained from canine airways by bronchoalveolar lavage. Prostaglandins 43 401-411 (1992).

Ujihara, M., Urade, Y., Eguchi, N., et al. Prostaglandin D₂ formation and characterization of its synthetases in various tissues of adult rats. Arch Biochem Biophys 260 521-531 (1988).

Ko, F., Wu, C., Kuo, S., et al. YC-1, a novel activator of platelet guanylate cyclase. Blood 84 4226-4233 (1994).

Pradelles, P., Grassi, J., and Maclouf, J.A. Enzyme immunoassays of eicosanoids using acetylcholinesterase as label: An alternative to radioimmunoassay. Anal Chem 57 1170-1173 (1985).

Spitzer, J.A. Cytokine stimulation of nitric oxide formation and differential regulation in hepatocytes and nonparenchymal cells of endotoxemic rats. Hepatology 19 217-228 (1994).

Maclouf, J., Grassi, J., and Pradelles, P. Development of enzyme-immunoassay techniques for the measurement of eicosanoids. 355-364 (1987).

Tokugawa, Y., Kunishige, I., Kuboto, Y., et al. Lipocalin-type prostaglandin D synthase in human male reproductive organs and seminal plasma. Biol Reprod 58 600-607 (1998).

Maclouf, J., Corvazier, E., and Wang, Z. Development of a radioimmunoassay for prostaglandin D₂ using an antiserum against 11-methoxime prostaglandin D₂. Prostaglandins 31 123-132 (1986).

Ram, A., Pandey, H.P., Matsumura, H., et al. CSF levels of prostaglandins, especially the level of prostaglandin D₂, are correlated with increasing propensity towards sleep in rats. Brain Res 751 81-89 (1997).

Marshall, L.A., Bolognese, B., Winkler, J.D., et al. Depletion of human monocyte 85-kDa phospholipase A₂ does not alter leukotriene formation. J Biol Chem 272 759-765 (1997).

Hogaboam, C.M., Donigi-Gale, D., Shoupe, T.S., et al. Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds. Br J Pharmacol 105 87-92 (1992).

Maxey, K.M., Maddipati, K.R., and Birkmeier, J. Interference in enzyme immunoassays. J Clin Immunoassay 15 116-120 (1992).

Fitzpatrick, F.A., and Wynalda, M.A. Albumin-catalyzed metabolism of prostaglandin D₂. Identification of products formed in vitro. J Biol Chem 258 11713-11718 (1983).

Satoh, S., Matsumura, H., Nakajima, T., et al. Promotion of sleep by prostaglandin D₂ in rats made insomniac by pretreatment with para-chlorophenylalanine. Neurosci Res 21 41-50 (1995).

Pradelles, P., Grassi, J., and Maclouf, J. Enzyme immunoassays of eicosanoids using acetylcholinesterase. Methods Enzymol 187 24-34 (1990).

Roberts, L.J., and Sweetman, B.J. Metabolic fate of endogenously synthesized prostaglandin D₂ in a human female with mastocytosis. Prostaglandins 30 383-400 (1985).

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