Thomas D. Eller

Extraction of thromboxane B2 from urine using an immobilized antibody column for subsequent analysis by gas chromatography-mass spectrometry

This paper describes an antibody affinity (immunoaffinity) column which, in one step, extracts and sufficiently purifies urinary thromboxane B2 (TXB2) for quantitative analysis by high resolution gas chromatography-negative ion chemical ionization-selected ion monitoring-mass spectrometry (HRGC-NICI-SIM-MS). Polyclonal TXB2 antibody from rabbit was partially purified using immobilized Staphylococcus aureus Protein A. The purified IgG fraction was then immobilized using an N-hydroxysuccimidyl silica gel. The resulting matrix bound 570 ng TXB2 per ml of gel. TXB2 was quantitatively eluted with acetonitrile-water (19:1). Columns constructed from the gel could be used repeatedly since binding capacity was reconstituted using 0.01 M phosphate buffer (pH 7.4) with no apparent loss of activity. Using these columns, urinary TXB2 was sufficiently purified in one step such that in subsequent analysis by HRGC-NICI-SIM-MS interference free chromatograms were observed.

Protective effects of trans-13-APT, a thromboxane receptor antagonist, in endotoxemia

The effect of the thromhoxane A2prosta-glandin H2 (TxA2PGH2) receptor antagonist trans- 712-(p-hydroxyphenethylamino)-cyclopentyl]-heptanoic acid Trans-13-APT) on certain pathogenic sequelae of endotoxic shock and associated changes in araehidonic acid metabolism in the rat was investigated. trans-13-APT. an analog of 13-aprostanoic acid, was synthesized and found lo block human platelet aggregation induced by the thromboxane mimetic U46619. Pretreatment with iraus-13-APT did not significantly alter the elevations in plasma immunoreactive (i) TxB, or iPGE, 0.5 or 4 h after the intravenous administration of Salmonella enterilidis endotoxin. However, in the trans-13-APT-pretreated group, 4 h after administration of the endotoxin. plasma i6-keto-PGI was significantly (p < 0.05) reduced to 1.2 ± 0.3 ng/ml (n - 17) compared with vehicle-treated rats (2.4 ± 0.5 ng/ml: n - 18). The elevation in plasma i6-kelo-PGI seen 0.5 h (n 17/grottp) after endotoxin infusion was not altered by trans-13-APT. trans-13-APT also significantly (p < 0.05) attenuated the endotoxinindticed fall in platelet count (135 + 27 × 10/mm vs. 350 + 65 × 10/mm and hypoglycemia (73 9 vs. 97 + 7 mg/dl). but not the leukopenia. Since the reticulo-endothelial system may be an important source of iTxB2, and i6-keto-PGF during endotoxemia, in vitro studies were conducted with adherent peritoneal cells. High concentrations of trans-13-APT (50 and 100 μ) significantly reduced (p < 0.05) basal but not endotoxin-induced synthesis of iTxB2 and i6-keto-PGF by isolated adherent rat peritoneal cells. The attenuated rise in plasma i6-keto-PGF in the trans-13-APT-pretreated rats may be the result of lessened shock severity or may represent antagonism of a stimulatory effect of TxA2 on PGI2 synthesis. The results also raise the possibility that thromboxane receptor antagonists may be of benefit during endotoxemia.

Quantitative analysis of 6-keto-prostaglandin F1α using immunoaffinity purification and gas chromatography—mass spectrometry

This paper describes an immunoaffinity purification technique for 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) prior to quantitative analysis by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry (HRGC-NICIMS). Polyclonal antibodies to 6KPGF1 alpha were partially purified using Staphylococcus aureus Protein A immobilized on Sepharose CL-4B. This partially purified fraction was covalently bound to silica gel using N-hydroxysuccinimidyl-functionalized silica. Columns constructed using this gel quantitatively bound 6KPGF1 alpha which could be eluted quantitatively with acetonitrile-water (19:1). Binding capacity was reconstituted by washing with 0.01 M phosphate buffer (pH 7.4). Human urinary and canine plasma 6KPGF1 alpha was sufficiently purified using these columns that HRGC-NICIMS analysis of the methoxime-pentafluorobenzyl-tris-trimethylsilyl derivative was interference-free.

A novel approach for the study of thromboxane A2 and prostaglandin H2 receptors using an 125I-labeled ligand

Previous studies of eicosanoid receptors have utilized 3H-labeled ligands. However, 125I has a higher theoretical specific activity (approximately 2000 Ci/mmole) than 3H (29 Ci/mmole), which provides a potential advantage for 125I ligand binding studies when the receptor density is low. Since eicosanoids do not possess an easily iodinatable structure (e.g. a phenol or imidazole ring), it is not feasible to directly incorporate 125I into the molecule. The thromboxane A2/prostaglandin H2 receptor antagonist, cis-7-(2-p-hydroxyphenylethanolaminocyclopentyl)-heptanoic acid (cis-APO), was synthesized to test the concept that it could be labeled with 125I and used as a ligand for binding studies. cis-APO is a structural analog of 13-azaprostanoic acid, a TXA2/PGH2 antagonist [G. C. Le Breton, D. L. Venton, S. E. Enke and P. V. Halushka, Proc. natn. Acad. Sci. U.S.A. 76, 4097 (1979)], in which the omega aliphatic chain was substituted with 2-p-hydroxyphenylethanol, which contains a phenolic group. [127I]cis-APO was synthesized by insertion of 127I (stable isotope) into the phenolic portion of the molecule under alkaline conditions. [125I]-cis-APO was synthesized via insertion of 125I (unstable isotope) into the molecule in the presence of chloramine T. cis-APO inhibited human platelet aggregation induced by the thromboxane mimetic U46619 [C. Malmsten, Life Sci. 18, 169 (1976)]. The IC50 for cis-APO was 6.4 +/- 0.7 microM and for [127I]-cis-APO was 9.8 +/- 1.3 microM (P less than 0.001). [125I]-cis-APO binding to a human platelet membrane preparation at 4 degrees was time and protein concentration dependent, saturable, and reduced or abolished by trypsin or boiling respectively. The Kd for iodo-cis-APO determined at equilibrium using a Scatchard analysis was 1.48 microM and the maximum binding capacity was 18.7 pmoles/mg protein. The forward rate constant (k+1) was 2.3 X 10(3) M-1 s-1 and the dissociation constant (k-1) was 2.12 X 10(-3) s-1. The Kd determined from k-1/k+1 was 0.92 microM. These observations show that the omega side chain of eicosanoid analogs can be substituted with a phenolic group, iodinated, and retain biological activity. These molecules may then be utilized to study thromboxane A2 or prostaglandin H2 receptors.

Immunoaffinity purification-chromatographic quantitative analysis of arachidonic acid metabolites.

Publisher Summary The chapter presents a study on immunoaffinity purification-chromatographic quantitative analysis of arachidonic acid metabolites. Quantitative analysis of arachidonic acid metabolites, and structurally related compounds of biological and synthetic origin, can be divided into two basic steps. These are extraction-purification procedures and analytical measurement by such techniques as radioimmunoassay (RIA), gas chromatography (GC), GC-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC) with ultraviolet (UV) or fluorescence (FL) detection. The chapter describes methods of immunoaffinity purification using both solid-phase extraction and immunoaffinity precipitation. Quantitative analytical procedures that utilize immunoaffinity purification offer the potential of greatly decreased analysis time. Immunoaffinity purification techniques are, in general, robust, fast, and easy to perform. Immunoaffinity purification is a simple and powerful analytical technique when applied as the initial step in a three-stage analytical process including a high-performance chromatography step followed by a specific detection method. The polyclonal antibody contained in raw serum must be partially purified before immobilization on a solid-phase support.