title: The fate of tritiated rm-epidermal growth factor in the sheep: validation of the labelling procedure and rate of tissue clearance.
abstract: Plasmid-derived recombinant mouse epidermal growth factor, rm-EGF, was purified by ion pair reversed phase high performance liquid chromatography. The product peak (termed rm-alpha-EGF) was characterized by physicochemical techniques including fast atom bombardment mass spectrometry, high field proton magnetic resonance and amino acid sequencing (amino acid arrangement and composition). The rm-alpha-EGF was tritiated, labile tritium removed by lyophilization, and the product purified and characterized as for the parent compound to yield a compound identical to rm-alpha-EGF except for the isotopic hydrogen substitution. Label stability was validated by lyophilization of samples, especially urine. The tritiated rm-alpha-EGF was used to determine the excretion rate and tissue distribution pattern in the sheep. It was administered by intravenous infusion for 24 h at a dose rate of 120 micrograms kg-1 live weight. Blood, urine and faeces were collected at frequent intervals from all sheep up to slaughter. Sheep were slaughtered at 24 h (3 sheep), 48 h (3 sheep), and 192 h (1 sheep) from the start of infusion and samples of all tissues and organs collected. Samples were assayed by liquid scintillation counting, directly for liquids, and after combustion to tritiated water for solids. For residue studies all solid samples were lyophilized to constant weight before combustion, and volatile tritium determined from the lyophilisate. Urinary excretion was extensive and rapid. From the start of the infusion 30.1% of the administered tritium was recovered at 24 h, 40.4% at 48 h and 55.1% at 192 h. Comparison of RIA and tritium (3H) in plasma and urine samples indicated that the EGF had undergone considerable metabolism. Faecal excretion of EGF was also significant, being 1.5% at 24 h, 2.1% at 48 h and 10.0% at 192 h after the start of the infusion. Of the EGF not excreted at the time of slaughter, 41.9% (24 h), 36.8% (48 h) and 22.1% (192 h) was present in eight locations: muscle, intestine, gut content, skin, blood, liver, kidney, and lung. Tritium in fat (omental, perinephric, subcutaneous) was negligible, and no 3H was detected in the plucked fleece 192 h after the start of the infusion. Volatile metabolic products (H2O, CH4, NH3) excreted via the lung were not measured. The overall recoveries of 97.4% (24 h), 100.5% (48 h), and 97.8% (192 h) confirm that the label was in stable positions. This result thus validates the labelling procedure and the use of a generally labelled compound, and confirms the efficacy of the sampling procedure.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Division of Animal Production, CSIRO, Blacktown, N.S.W.
authors.forename: J H
authors.initials: JH
authors.lastname: O'Keefe
authors.forename: L F
authors.initials: LF
authors.lastname: Sharry
authors.forename: B A
authors.initials: BA
authors.lastname: Panaretto
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004815:Epidermal Growth Factor / Q000097:blood / Q000493:pharmacokinetics* / Q000652:urine; D005243:Feces / Q000032:analysis; D005260:Female; D008460:Meat / Q000032:analysis; D008657:Metabolic Clearance Rate; D009132:Muscles / Q000378:metabolism; D011994:Recombinant Proteins / Q000493:pharmacokinetics; D012756:Sheep / Q000378:metabolism*; D014018:Tissue Distribution; D014316:Tritium
chemical_list: D011994:Recombinant Proteins; D014316:Tritium; D004815:Epidermal Growth Factor