The insulin-receptor affinity of five human insulin analogues with one to four amino acid substitutions was measured with human hepatoma cells (HepG2). The binding affinities ranged from 0.05% for AspB25 insulin, 18% for AspB9, GluB27 insulin, 80% for AspB28 insulin, and 327% for AspB10 insulin to 687% for HisA8, HisB4, GluB10, HisB27 insulin relative to human insulin. Binding constants obtained by competition experiments at steady state with [125I]TyrA14-labeled insulin and unlabeled analogues and by kinetic studies with [125I]TyrA14-labeled analogues and insulin gave essentially the same values. The kinetic studies showed that differences in affinity between analogues were due to differences in both dissociation and association rate constants. The affinity for insulinlike growth factor I receptor was low, ranging from less than 0.005% for AspB25 insulin to 0.6% for HisA8, HisB4, GluB10, HisB27 insulin. The potencies of insulin analogues in activation of the tyrosine kinase of solubilized and partially purified insulin receptors from HepG2 cells, measured with the exogenous substrate poly(Glu80-Tyr20), ranked in the same order as the binding affinities, the actual values being somewhat elevated for the high-affinity analogues, however. We conclude that these human insulin analogues are active in insulin-receptor binding and tyrosine kinase stimulation but show wide variation in affinity.

SUMMARYThe experimental animals consisted of 71 heifers or cows of the Black and White Danish breed (SDM), 61 heifers or cows of the Red Danish breed (RDM), and 8 heifers or cows of the Jersey breed. Highly significant differences between breeds were found in yield of colostrum at first milking after calving and in the concentration of immunoglobulin (Ig) in colostrum. The RDM animals had the highest colostrum yield but the lowest Ig %. The total yield of Ig was not significantly different between RDM and SDM. The individual variation in colostrum yield, Ig %, and Ig yield was very marked.Heifers had a lower colostrum yield and Ig yield than cows in second and later lactations. For SDM the Ig % was not dependent on the lactation number. In contrast the older RDM cows had a significantly higher Ig % in colostrum than the younger cows. No effect of season of the year was found on colostrum yield, Ig %, and Ig yield. An increase of the interval between calving and the first milking caused a very significant drop in colostrum Ig %. Loss of colostrum from the udder before milking increased the probability of getting colostrum with low Ig %. The repeatability in Ig % at successive calvings was found to be high.

One hundred and forty-one calves, belonging to three different breeds, were each given one single feeding of a variable amount of colostrum from their dams at the age of 2, 6, 10, 14 or 20 hr, respectively. The increase in serum Ig concentration during the first 24 hr after colostrum feeding (A Ig % 24) was a function of the mass of Ig fed to the calf, the age at colostrum feeding, and the birth weight of the calf. Among these three factors the mass of Ig and the age of the calf were the two predominant factors. The absorption coefficient, expressing the absorbed fraction of a given amount of Ig, was primarily determined by the age of the calf at first feeding. Thus the absorption coefficient was reduced linearly to about half by delaying the feeding from 2 to 20 hr. Except for a negative effect of increasing amounts of colostrum in one of the breeds, no other factors were detected as responsible for variation in the efficiency of Ig absorption. Thus, the absorption coefficient appeared to be unaffected by the Ig % in the colostrum and also by the quantity of Ig given to the calf. 627

Twenty "Red Danish" bulls were tested for rate of gain, linear growth, longissimus muscle area and efficiency of feed utilization from the age of l'h to 12 months. These variables were correlated to blood concentrations of testosterone and somatomedin, which were measured at 4 to 5 months, 6 to 7 months and/or 8 to 10 months of age.Average testosterone concentrations of bulls above 6 months of age were found to be correlated negatively (P<.05) with feed utilization (feed units/kg gain). Average somatomedin activities throughout the test period were positively correlated with rate of gain (P<.05) and linear growth (P<.05), and negatively to feed utilization (P<.05). Also, the somatomedin activity was found to increase significantly from 6 to 7 months up to 10 months of age (P<.05). No significant correlation between testosterone and rate of gain, linear growth or longissimus muscle area was found, and, likewise, there was no significant correlation between somatomedin activity and longissimus muscle area.It is concluded that the blood level of somatomedin at an early age may represent a possible indicator of future growth capacity in young bulls. (

After intravenous injection of 125I-labeled human insulin and analogs in normal and nephrectomized rats, we examined their kinetic fate by Q-Sepharose separation into intact ligand, "fragments" (genuine fragments and protein-bound radioactivity), and iodide. Receptor binding association and dissociation constants (kass and kdis, respectively) of the analogs were estimated dynamically in vitro by BIAcore. The very fast disappearance of intact ligand from serum was found to be determined by 1) both kass and kdis of receptor-bearing tissue, thus substantiating our primary hypothesis; 2) elimination by kidneys, and 3) fast extravascularization. The rate of appearance of degradation products from receptor-mediated intracellular processing seems determined by kdis. With the possible exception of a truncated analog, ligand appears protected against degradation while the intracellular receptor-ligand complex remains intact. Non-receptor-mediated processing in kidneys is slow, compared with the receptor-mediated uptake and degradation of ligands with rate constants comparable to those of insulin. We observed binding of insulin and analogs putatively to serum proteins; binding capacity and affinity appeared insignificant for insulin but considerable for some analogs.