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Biomedical subjects

J Markussen

Publications and source records attributed to J Markussen.

At least 37 records · Page 2Linked to original sources
PubMed1989

NovoSol Basal: pharmacokinetics of a novel soluble long acting insulin analogue.

OBJECTIVE: To determine the courses of absorption and the interindividual and intraindividual variations in absorption of iodine-125 labelled Ultratard HM and NovoSol Basal injected subcutaneously. DESIGN: Open randomised crossover study. Each patient was tested during two study periods of five days each, during which he or she received a subcutaneous injection of either 125I-NovoSol Basal or 125I-Ultratard HM on four consecutive days. The aim was to detect a reduction in intraindividual standard deviation by a factor of two with a probability 0.95, taking 0.05 as the level of significance. This required 24 degrees of freedom and led to the choice of four courses in each of eight patients. SETTING: Referrals to the diabetes research centre in Hvidøre, Copenhagen. PATIENTS: Eight insulin dependent (type I) diabetics with low or undetectable C peptide concentrations who were receiving a multiple insulin injection regimen. One patient withdrew immediately after recruitment. INTERVENTIONS: After an overnight fast patients received 96 nmol (16 IU insulin) of either 125I-NovoSol Basal or 125I-Ultratard HM injected subcutaneously into the thigh. To ensure that the insulin entered the subcutaneous fat at the same depth, ultrasonography was performed on each patient before the first injection. A different injection site on the thigh was used each day for four days in order to facilitate monitoring of the disappearance of four different depots in each patient. MAIN OUTCOME MEASURE: Residual activity at the injection site was measured roughly every two hours throughout the day. No radioactivity measurements were performed overnight (10 pm till 8 am). The residual radioactivity after the injection on the first day (upper right thigh) was recorded for five days, that after the injection on the second day (upper left thigh) for four days, after the injection on the third day (lower right thigh) for three days, and after the last injection (lower left thigh) for two days. RESULTS: NovoSol Basal was absorbed according to first order kinetics with a mean t50% of 35.3 (SEM 1.4) hours; Ultratard HM was absorbed after a lag phase and the corresponding t50% was 25.5 (2.5) hours. The intraindividual variations in t50% were significantly smaller with NovoSol Basal than with Ultratard HM (18.4% v 44.5%; p less than 0.001). Interindividual variations, however, were not significantly different (25.2% v 36.9%; p = 0.38). The total variation in t50% was substantially smaller with NovoSol Basal than with Ultratard HM (20.3% v 42.8%). CONCLUSIONS: NovoSol Basal seems to be an appreciable advance over Ultratard HM as a soluble insulin preparation for obtaining reproducible 24 hour insulin concentrations in the blood

Absorption↗
PubMed1988

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30.

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.

Absorption↗
PubMed1987

Soluble, prolonged-acting insulin derivatives. I. Degree of protraction and crystallizability of insulins substituted in the termini of the B-chain.

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.

Animals↗
PubMed1987

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30.

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)

Amino Acid Sequence↗
PubMed1987

Isolation and amino acid sequence of insulins and C-peptides of European bison (Bison bonasus) and fox (Alopex lagopus).

Insulins and C-peptides were extracted and purified from bison and fox pancreatic glands. The insulins were reduced and pyridylethylated, and the derived A- and B-chains separated by HPLC. Amino acid sequence determinations of the pyridylethylated A- and B-chains proved bisontine insulin to be identical to bovine insulin and fox insulin to be identical to dog and porcine insulin. Bisontine C-peptide proved to be identical to bovine C-peptide. The isolated fox C-peptide comprises 23 amino acid residues and probably represents a major tryptic fragment of a larger C-peptide. The fox C-peptide fragment is identical to the dog C-peptide (9-31) except for residue 3 (residue 11 in the dog C-peptide), which is aspartic acid as compared with glutamic acid in the dog C-peptide.

Amino Acid Sequence↗
PubMed1985

Structure-function relationships in glucagon. Re-evaluation of glucagon-(1-21).

Glucagon-(1-21) was prepared fully synthetically as well as by carboxypeptidase A digestion of natural porcine glucagon. Neither of the two preparations had glucagon agonistic effects with regard to receptor binding or adenylate cyclase activation in purified rat liver plasma membranes. Nor did these preparations contain lipolytic activity in isolated free fat cells. A preliminary batch of glucagon-(1-21) prepared by carboxypeptidase A digestion did, however, contain 1-2% glucagon bioactivity. This activity was separated from glucagon-(1-21) by high-performance liquid chromatography and quantitatively recovered in four minor hind peaks which eluted close to but not in a position identical to the elution position of native glucagon.

Adenylyl Cyclases↗
PubMed1985

Kinetics of trypsin catalysis in the industrial conversion of porcine insulin to human insulin.

The kinetics of coupling and transpeptidation reactions catalysed by trypsin were studied in media with low water contents to see whether the usual Michaelis-Menten kinetics applied under the unusual conditions used in peptide bond synthesis, to obtain information about the magnitude of rate constants and activation energies, and to gain insight into the mechanism of catalysis. Porcine insulin, des-(AlaB30)-porcine insulin, human insulin-ThrB30-OMe and human insulin-ThrB30(But)-OBut were used as substrates. Two threonine esters (Thr-OMe and Thr(But)-OBut) were used for transpeptidation and coupling. The reactions progressed according to first-order kinetics until about 35% conversion, and the experimental data were adequately explained by Michaelis-Menten kinetics. The rates of the coupling and transpeptidation reactions in media with low water contents were orders of magnitude below the rates of peptide bond hydrolysis by trypsin in water. It was not possible to approach saturation of the enzyme with substrate so determination of Km was impossible, but for each substrate a value larger than 0.1 M was estimated from the Lineweaver-Burk plot. The rate of release of alanine from porcine insulin depended on the type of threonine ester present; for example, Thr-OMe inhibited the reaction. Coupling occurred faster than transpeptidation. However, in the medium used, the activation energies for the two reactions were similar (about 50 kJ/mol), so the difference in reaction rates is probably due to different transmission coefficients in the activated transition states. Computer simulations enabled us to obtain quantitative descriptions of the reaction progress curves from fitted rate constants.

Alanine↗
PubMed1985

Comparative reduction/oxidation studies with single chain des-(B30) insulin and porcine proinsulin.

The single chain des-(B30) insulin molecule (SCI) has been reduced and reoxidized together with porcine proinsulin (PPI). Yields of correctly folded and reoxidized SCI and PPI were analyzed by HPLC. The concentrations of both proteins were 10(-3) M during reduction and 10(-5) M during oxidation. The pH during reoxidation was varied from 8.6 to 9.2 and the temperature from 4 to 37 degrees. Under all conditions tested, the recovery of SCI was substantially higher than that of PPI. The recoveries peaked after 24-72 h. It is suggested that the "miniproinsulin" SCI folds correctly up more efficiently than porcine proinsulin, resulting in higher yields of reoxidized SCI.

Chromatography, High Pressure Liquid↗
PubMed1985

Single chain des-(B30) insulin. Intramolecular crosslinking of insulin by trypsin catalyzed transpeptidation.

Single chain des-(B30) insulin (SCI) has been synthesized from porcine insulin by trypsin in a medium with a low content of water. Trypsin catalyzes an intramolecular transpeptidation reaction in which the glycineA1 residue substitutes the alanineB30 residue, rendering a LysB29 -GlyA1 peptide link between the A- and B-chains of insulin. The insulin derivative has been purified by column chromatography and appears to be homogeneous in HPLC and disc electrophoresis. The structure was proven to be B(1-29)-A(1-21) insulin by proteolysis with Armilliaria mellea protease followed by a few steps of Edman degradation. The electrophoretic mobility indicates that SCI has a more condensed structure than that of insulin. Perfect rhombohedral crystals were obtained under conditions resembling those under which insulin crystallizes in the same form. SCI was devoid of effect in the blood sugar lowering assay in mice, the estimated potency being less than 0.1% of that of insulin.

Amino Acids↗
PubMed1983

Human monocomponent insulin. Chemistry and characteristics.

The primary structure of different insulins is reviewed and the properties, identification tests, purity, potency and immunogenicity of human insulin are summerized. Novo Research Institute has developed a method, simply using an enzymatic conversion reaction to substitute the B30 alanine of porcine insulin with threonine to manufacture human insulin. This process is basically an extension of the process currently used to manufacture the Novo purified insulins which are commercially available.

Amino Acids↗
PubMed1982

The effect of oxidation of the Met27 residue of [125I]monoiodoglucagon on receptor-binding affinity.

When glucagon is iodinated by the chloramine-T method, the Met27 residue is oxidized. This is not the case when the iodination is performed by the lactoperoxidase method. The two preparations can be purified to the same specific activity using QAE-Sephadex A-25 ion exchange chromatography. Receptor-binding studies in isolated rat adipocytes or hepatocytes revealed that the oxidized from possessed an average-binding affinity which is only about two thirds of that of the non-oxidized form. The reduced affinity of the oxidized tracer cannot be explained by an increased rate of dissociation.

Adipose Tissue↗
PubMed1981

Glucagon: structure-function relationships investigated by sequence deletions.

A series of glucagon analogues, des-(1-4)-glucagon, des-(5-9)-glucagon, des-(10-15)-glucagon, des-(16-21)-glucagon, des-(22-26)-glucagon and des-(27-29)-glucagon, were prepared by condensation of synthetic fragments and characterized biologically and immunologically. Fully synthetic glucagon was also characterized. The potencies with regard to glucagon receptor binding in purified rat liver plasma membranes were, in decreasing order: synthetic glucagon 108%, des-(1-4)-glucagon 5.7%, des-(27-29)-glucagon 0.92%, des-(5-9)-glucagon 0.47%, des-(10-15)-glucagon 0.0028%, des-(16-21)-glucagon 0.0017% and des-(22-26)-glucagon 0.00060% relative to that of natural porcine glucagon. Des-(27-29)-glucagon was the only analogue that activated the adenylate cyclase in rat liver plasma membranes or stimulated the lipolysis in isolated free fat cells from rat epididymal fat pad. The potencies were 0.16% and 0.20% of that of glucagon, respectively. Des-(1-4)-glucagon was a glucagon antagonist in the adenylate cyclase assay. The immunoreactivities of the glucagon analogues were determined with two commonly used anti-glucagon sera, K 5563 and K 4023, directed towards the C-terminus and some segment in the sequence 2-23, respectively. In the K 5563 assay, des-(27-29)-glucagon and des-(22-26)-glucagon had potencies of 0.0009% and less than 0.09% of that of glucagon, respectively. The remaining analogues had potencies varying from 45% to 141% of that of glucagon. In the K 4023 assay, the analogues showed a non-linear dilution effect. The combined results indicate a partition within the glucagon molecule with regard to receptor binding and adenylate cyclase activation. The region 10-26 appears to be the most important for receptor binding, whereas 1-4 is essential for adenylate cyclase activation. The C-terminal segment 27-29 is important for the maintenance of full receptor binding but non-essential for adenylate cyclase activation.

Adenylyl Cyclases↗
PubMed1979

Formation and synthesis of 3'-t-butyltyrosine.

During acidolysis by TFA of the t-butyl protecting group from Z-Tyr(But) or from Ser (But) in the presence of tyrosine, C-t-butylation occurred in the aromatic nucleus in Z-Tyr or tyrosine, respectively, to an extent of 0.5-1.0%. CF3COOBut formed during the acidolysis slowly C-t-butylates tyrosine. Tyr(3'But) is formed. The synthesis of Tyr (3'But) . HCl is described.

Chemical Phenomena↗
PubMed1978

Removal of t-butyl and t-butoxycarbonyl protecting groups with trifluoroacetic acid. Mechanisms, biproduct formation and evaluation of scavengers.

The trifluoroacetic acid-mediated removal of t-butyl groups in protected amino acids leads to the formation of t-butyl trifluoroacetate. This t-butyl ester alkylates in trifluoroacetic acid methionine and tryptophan. The t-butyl trifluoroacetate ester can be destroyed by scavengers commonly employed for t-butyl cations, and the reaction rates of the scavengers with the ester are used in the evaluation of scavengers. Scavengers of sulphide structure react with t-butyl trifluoroacetate to form sulphonium compounds, which possess alkylating properties. In the presence of a scavenger during acidolysis, the trifluoroacetic acid and the scavenger will compete in reacting with the t-butyl cations. Kinetic studies show comparable reaction rates with thiophenol as scavenger. The usefulness of adding scavengers to trifluoroacetic acid in deblocking reactions is due to the removal of t-butyl trifluoroacetate in addition to the removal of t-butyl cations. Isobutene reacts with trifluoroacetic acid and yields t-butyl trifluoroacetate. The reaction reaches an equilibrium displaced in favour of the ester at room temperature. Hence no isobutene can be expected to escape during a deblocking reaction in trifluoroacetic acid.

Alkylation↗
PubMed1978

Kinetics of human connecting peptide in normal and diabetic subjects.

The metabolic clearance rate (MCR) of synthetic human connecting peptide (C-peptide) was measured with a single-dose injection technique in six normal and seven diabetic subjects and with a constant infusion technique in one normal subject. The MCR of C-peptide did not differ in normal subjects (4.4 ml/min per kg; range, 3.7-4.9) and in diabetic subjects (4.7 ml/min per kg; range, 3.7-5.8). Employment of both techniques in one subject gave similar MCR. The average half-life of C-peptide in plasma calculated from the last 1-h period of the single-dose injection studies was longer in the insulin-dependent diabetics (42.5 min; range, 39.4-48.5) than in the normal subjects (33.5 min; range, 24.9-45.3). These results indicate that the beta-cell secretory capacity of normal and insulin-dependent diabetic subjects can be compared by measuring the C-peptide concentration in peripheral venous plasma. The difference in the half-life of C-peptide in plasma between diabetics and normals suggests an altered kinetics of the disappearance of the peptide, while the overall metabolism, as expressed by the MCR, is similar.

Adult↗
PubMed1978

Characterization of seven C-peptide antisera.

The plasma C-peptide immunoreactivity (CPR) in 10 normal subjects varied considerably when measured with different antisera in parallel assays. The CPR level correlated with the blank "CPR" value measured in plasma devoid of C-peptide and to a lesser degree with the sensitivity of the standard curves obtained with the individual antisera. Storage of plasma samples at different temperatures and for different lengths of time before the analyses were carried out resulted in further variation in the CPR results. This was caused by a time- and temperature-dependent fall in CPR, which was more pronounced with some antisera than with others. This sensitivity to storage of plasma did not correlate with the antigenic characteristics of the antisera as determined by their reactivity with 11 specific fragments of the C-peptide molecule. The contribution of human proinsulin to the CPR concentration relative in normal subjects was considered to be negligible even though the relative immunoreactivity of human proinsulin and C-peptide ranged from 11 to 143 per cent among these antisera. These results suggest that differences in C-peptide antisera are a major reason for the variation in the concentration of circulating CPR as measured in different C-peptide immunoassays.

Animals↗