It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.

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It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrophilic insulins, more positively charged than human insulin at neutral pH, have been prepared by substitution with basic amino acids at the termini of the B-chain and by blocking the C-terminal carboxyl group of the B-chain. The isoelectric pH of the insulin is thereby moved from 5.4 towards physiological levels. Slightly acid solutions of derivatives, in which charge has been added in the C-terminus of the B-chain, have a prolonged action in vivo, in particular if the carboxyl group is blocked. It is found that the prolonged-acting hydrophilic insulins crystallize instantly when the pH is adjusted to 7. The prolonged action is ascribed to this readiness to crystallization combined with a low solubility, which may be further decreased by increased concentration of zinc ions. Hydrophobic insulins have a prolonged action independent of the site of substitution even if the derivative is soluble at physiological pH. Some derivatives were prepared from porcine insulin by tryptic transpeptidation. N-terminal B-chain substituted insulins were prepared by alkylation of a biosynthetic single-chain insulin precursor, followed by tryptic transpeptidation rendering the double chain insulin derivative. The observed blood glucose lowering in the rabbits implies that neither N- nor C-terminal B-chain substitution results in substantial deterioration of biological potency. An index for the degree of protraction based on the blood glucose data is used to compare the insulins.

Comparisons were made of the accumulation of selenium, histopathological damage, and reproductive status of redear sunfish (Lepomis microlophus) collected in July 1986 from Martin Lake (a contaminated site) and Lake Tyler (a reference site). Hepatic concentrations of selenium were four times higher in Martin Lake sunfish (7.6 +/- 0.5 ppm) than in fish from the reference lake (2.1 +/- 0.2 ppm). Redears collected from the contaminated lake had lower condition factors than individuals collected from the reference site. Sunfish with elevated levels of hepatic selenium had substantial alterations in the liver including necrosis, cytoplasmic vacuolation, and Kupffer cell proliferation. The ovaries of mature fish collected from Martin Lake frequently had atretic follicles, abnormally shaped follicles, connective tissue hypertrophy, asynchronous oocyte development, and an overall reduction in the number of developing oocytes. These histopathological changes in the ovaries of Martin Lake sunfish were not accompanied by alterations in gonadal steroid titers in the blood. No histopathological lesions could be detected in the testes of Martin Lake fish. Most of the males collected from the contaminated site were immature and had lower circulating levels of sex steroid hormones than reference males. The results show that tissue burdens of selenium have declined by 25% since this sunfish population was sampled last in 1981. Further, the results of this study indicate that the overall health and reproductive status of selenium-contaminated fish collected from Martin Lake is still seriously impaired.