O'Keefe J H, Sharry L F, Panaretto B A
Division of Animal Production, CSIRO, Blacktown, N.S.W.
Aust J Biol Sci. 1988;41(4):539-52. doi: 10.1071/bi9880539.
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)
通过离子对反相高效液相色谱法纯化质粒衍生的重组小鼠表皮生长因子（rm - EGF）。用包括快原子轰击质谱法、高场质子磁共振法和氨基酸测序法（氨基酸排列和组成）在内的物理化学技术对产物峰（称为rm -α - EGF）进行表征。对rm -α - EGF进行氚标记，通过冻干去除不稳定的氚，然后对产物进行纯化并像对母体化合物那样进行表征，得到除同位素氢取代外与rm -α - EGF相同的化合物。通过对样品尤其是尿液进行冻干来验证标记稳定性。用氚标记的rm - EGF来测定绵羊体内的排泄率和组织分布模式。以120微克/千克活体重的剂量率通过静脉输注给药24小时。从所有绵羊开始给药直至屠宰，定期采集血液、尿液和粪便。在输注开始后24小时（3只绵羊）、48小时（3只绵羊）和192小时（1只绵羊）宰杀绵羊，并采集所有组织和器官的样本。通过液体闪烁计数法对样本进行检测，液体样本直接检测，固体样本燃烧转化为氚水后检测。对于残留研究，所有固体样本在燃烧前冻干至恒重，并从冻干物中测定挥发性氚。尿液排泄广泛且迅速。从输注开始，在24小时时回收了30.1%的给药氚，48小时时为40.4%，192小时时为55.1%。血浆和尿液样本中放射免疫分析（RIA）和氚（³H）的比较表明，表皮生长因子经历了相当程度的代谢。表皮生长因子的粪便排泄也很显著，输注开始后24小时为1.5%，48小时为2.1%，192小时为10.0%。在宰杀时未排泄的表皮生长因子中，41.9%（24小时）、36.8%（48小时）和22.1%（192小时）存在于八个部位：肌肉、肠道、肠内容物、皮肤、血液、肝脏、肾脏和肺。脂肪（网膜、肾周、皮下）中的氚可以忽略不计，在输注开始后192小时采集的羊毛中未检测到³H。未测量通过肺部排泄的挥发性代谢产物（H₂O、CH₄、NH₃）。24小时时总体回收率为97.4%，48小时时为100.5%，192小时时为97.8%，这证实标记处于稳定位置。因此，该结果验证了标记程序和通用标记化合物的使用，并确认了采样程序的有效性。（摘要截短至400字）
