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 growthfactor, rm-EGF, was purified by ion pair reversed phase high performance liquid chromatography. The product peak (termed rm-a-EGF) was characterized by physicochemical techniques including fast atom bombardment mass spectrometry, highfield proton magnetic resonance and amino acid sequencing (amino acid arrangement and composition). The rm-a-EGF was tritiated, labile tritium removed by lyophilization: and the roduct purified and characterized as for the parent compoundto yield a compound identical to rm-a-EGF except for the isotopic hydrogen substitution. Label stability was validated by lyophilization of samples, especially urine. The tritiated rm-a-EGF was used to determine the excretion rate andtissue distribution pattern in the sheep. It was administered by intravenous infusion for 24 h at a dose rate of 120 p-gkg - 1 live weight. Blood, urine and faeces were collect atfrequent 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 ofall 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 fromthe lyophilisate. Urinary excretion was extensive and rapidFrom the start of the infusion 30.1 % of the administered tritium was recovered at 24 h, 40.4% at 48 hand 55' 1 % at 192 h. Comparison of RIA and tritium eH) in plasma and urine s mples indicated that the EGF had undergone considerable metabolism. Faecal excretion of EGFwas also significant, being 1.5 % at 24 h, 2.1 % at 48 hand 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, ut content, skin, blood, liver, kidney, and lung. Tritium in fat (omental, perinephric, subcutaneous) was negligible, and no 3H was detected in the plucked fleece 192h after the start of the infusion. Volatile metabolic products (H20, 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) confirmthat the label was in stable positions.This result thus validates the labelling procedure and the use ofa generally labelled compound, and confirms the efficacy ofthe sampling procedure.Complete inhibition of wool growth, as shown by wool shedding 192 h after the start of the infusion indicated that this biological activity of the compound was not affected by the labelling procedure. These residue data were used to assess the risk to humans from the consumption of meats and offal from sheep treated with EGF. It was concluded. © 1988 ASEG.