Metabolism of human insulin after subcutaneous administration: A possible means to uncover insulin misuse

The misuse of insulin for performance enhancement in sport or as toxic agent has frequently been reported in the past. In contrast to synthetic insulin analogues, the administration of recombinant human insulin is hardly recognized by mass spectrometry. The present study was designed to uncover the misuse of recombinant human insulin for doping control purposes as well as for forensic applications. It is hypothesized that an altered metabolite profile of circulating insulin prevails after subcutaneous administration due to exposure of insulin to epidermal proteases.     

The influence of non-specific binding on scatchard analysis used in insulin/receptor binding studies with erythrocytes

Conclusions By equilibrium binding studies it could be demonstrated that erythrocytes have been saturated with specifically bound insulin in the order of 0.10–0.20 nmol/l with 4×10¹² erythrocytes/l. From this an insulin receptor number of 15–30 receptors/normal erythrocyte could be calculated.By the current results we got evidence that in the insulin/erythrocyte receptor system the terminal part of the Scatchard plot was artefactual and might not be used for the calculation procedures because it is strongly influenced by non-specific effects. Because of the fluid change from the specific to the non-specific character of binding we propose to use total ligand concentrations only up to 5 nmol/l with 4×10¹² erythrocytes/l for the investigation of specific binding on erythrocyte insulin receptors.By focussing on the initial part of the Scatchard plot curvilinearity disappeared and the character of this hormone/receptor system was reduced to one binding site with one characteristic affinity constant and a receptor concentration of one characteristic order.

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Der Einfluß der unspezifischen Bindung bei Anwendung des Scatchard Plots zur Untersuchung der Insulin/Receptor-Wechselwirkungen an Erythrocyten

     

Antihyperglycemic effect of insulin from self-dissolving micropiles in dogs

As a percutaneous delivery device, self-dissolving micropiles (SDMPs) composed of chondroitin sulfate and insulin were prepared under room temperature from highly concentrated solution, glue. The mean weight of SDMP was 1.03+/-0.04 mg. One insulin SDMP was percutaneously administered to the shaved abdominal skin of four beagle dogs at insulin dose level of 1.0 and 2.0 IU/dog. After administration, blood samples were collected for 6 h and plasma glucose levels were measured. The time when minimum plasma glucose level appeared, T(min), was 1.38+/-0.2 h for 1.0 IU study and 1.38+/-0.1 h for 2.0 IU study and clear dose-dependent hypoglycemic effect of insulin was observed in the dose range. By comparing the area above the plasma glucose level vs. time curve (AAC) between insulin SDMP and subcutaneous (s.c.) injection solution, the relative pharmacological availabilities were 99% (1.0 IU) and 90% (2.0 IU), respectively. To ascertain the usefulness of insulin SDMP, oral glucose tolerance test (OGTT) was performed. When dogs were treated with insulin SDMPs, 2.0 IU, followed by an OGTT 30 min, glycemia did not appear for 5 h. On the other hand, when OGTT was performed at 1 h after insulin SDMP administration, hypoglycemia appeared as in the case of s.c. injection of insulin solution, 2.0 IU. Insulin SDMP improved the oral glucose challenge for 3 h, with a maximum effect at 30 min before the administration of glucose. Those results suggest the usefulness of a SDMP for the percutaneous delivery of peptide/protein drugs like insulin.     

Insulin binding and degradation by human erythrocytes and erythrocyte membranes

Conclusions By the current results there is evidence that the insulin degrading enzyme activity of the erythrocytes is not located on the inner or outer surface of the plasma membrane but is exclusively cytosolic. On the other hand, specific insulin binding to erythrocytes and erythrocyte membranes has been demonstrated and it could be supposed that the insulin binding region of the plasma membrane is not associated with an insulin degrading activity.In comparison to erythrocytes unsealed and sealed ghosts bound an equal amount of ¹²⁵I-insulin. Since there is binding but not degradation of insulin the membranes of erythrocytes might be a useful tool for the investigation of insulin internalization.

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Insulin-Bindung und -Abbau durch menschliche Erythrocyten und Erythrocyten-Membranen

     

Role of thiol-disulfide exchange in insulin binding to its receptor.

The effects of reduction by DTT, oxidation by DTNB and treatment with NEM on the thiol contents and insulin binding to its receptor in mice liver membranes were studied. Reduction with DTT leads to a parallel increase in the thiol content and the specific binding of insulin to the membrane. Scatchard analysis of the results shows little change in the number of binding sites but a twofold increase of the binding constant. Washing the membrane with bound insulin by a DTT containing buffer results in a more marked increase in the release of bound insulin than washing with buffer alone, suggesting that part of the insulin is bound to its receptor by covalent disulfide linkages through a thiol-disulfide exchange reaction and reduction with DTT leads to a marked increase in this "disulfide-linked" insulin. Treatment with DTNB or NEM of the DTT-reduced membrane seems to reverse the effect of DTT reduction, although the reaction of the untreated membrane with DTNB or NEM had little or no effect on the specific binding of insulin. It is suggested that initially, part of the thiols responsible for the exchange reaction may not be available for reaction with DTNB and reduction with DTT generates further thiols leading to increased specific binding in general and increased insulin binding to the receptor through covalent disulfide linkages in particular.     

Changes of conformation and aggregation state induced by binding of lanthanide ions to insulin

To clarify the mechanism of lanthanide ions (Ln3+) on the across-membrane transport of insulin and subsequent reducing blood glucose, the interactions of Ln3+with Zn-insulin and Zn-free insulin are investigated by spectroscopic methods. The results reveal that the binding of Ln3+ to insulin can induce its structure changes from secondary to quaternary structure, depending on the Ln3+ concentration. In the lower concentration, it triggers the conformational changes of insulin monomer in the binding region with insulin receptor (B(24-30)). It would affect insulin-insulin receptor interaction. Moreover, Ln3+ binding promotes the assembly of insulin monomer from dimer to polymer. The potency of Ln3+ in inducing insulin's aggregation is stronger than that of Zn2+. Furthermore, the aggregation can be reversed partly by EDTA-treatment, indicating that it is not due to denaturation. Similar to Zn2+ effect, Ln3+ can stabilize insulin hexamer in a certain range of concentration, but is stronger than the former.     

Studies of bicarbonate binding by dinuclear and mononuclear Ni(II) complexes

Bicarbonate ion reacts with the dinuclear nickel(II) complex containing the taec ligand (taec = N,N',N' ',N' '-tetrakis(2-aminoethyl)-1,4,8,11-tetraazacyclotetradecane) in buffered aqueous solution to form the mu-eta(2),eta(2)-carbonate complex with a large effective binding constant for bicarbonate ion, log K(B) = 4.39 at pH = 7.4. In contrast, the dinuclear nickel(II) complex containing the o-xyl-DMC(2) ligand (o-xyl-DMC(2) = alpha,alpha'-bis(5,7-dimethyl-1,4,8,11-tetraazacyclotetradecan-6-yl)-o-xylene) does not react with bicarbonate or carbonate ion in aqueous solution. In propylene carbonate, the reaction of [Ni(2)(o-xyl-DMC(2))](4+) with bicarbonate proceeds rapidly to form the mu-eta(1),eta(1)-carbonate complex. The structure of this carbonate complex has been determined by an X-ray diffraction study that confirms the mu-eta(1),eta(1)-carbonate binding mode. A mononuclear analogue of [Ni(2)(taec)](4+), [Ni(2,3,2-tetraamine)](2+) does not form a detectable mononuclear or dinuclear product with bicarbonate ion in aqueous solution, but [NiDMC](2+) (DMC = 5,7-dimethyl-1,4,8,11-tetraazacyclotetradecane) reacts slowly with carbonate ion in aqueous solution to form a 2:1 complex.     

Chloride coordination by oligoureas: from mononuclear crescents to dinuclear foldamers

A series of acyclic oligourea receptors which closely resemble the scaffolds and coordination behavior of oligopyridines have been synthesized. Assembly of the receptors with chloride ions afforded mononuclear anion complexes or dinuclear foldamers depending on the number of the urea groups.     

Transketolase from human erythrocytes. Purification and properties

Human erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase) was purified 8200-fold by adsorption onto hydroxylapatite, DEAE-cellulose treatment, acetone fractionation, and chromatography on Sephadex G-100. The purified transketolase could not be separated from glyceraldehyde-3-phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde-3-phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G-100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde-3-phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose-7-phosphate from xylulose-5-phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°.     

Determination of hypericin and pseudohypericin from Hypericum perforatum in rat brain after oral administration

The popularity of St. John’s Wort (SJW) extracts for treating mild to moderate depression has increased over the last decades and great effort has been devoted to identify the active principle of SJW extract. Previous investigations suggest the contribution of at least three classes of compounds, the phloroglucinols, the quercetin flavonoids, and the phenanthroperylenequinones, to the clinical efficiency of SJW extracts. Up to now, a plausible molecular mechanism of action has been described only for the phloroglucinols. For the flavonoids and the phenanthroperylenequinones different targets were proposed on the basis of pharmacological studies. The vast majority of these targets are located in the CNS and therefore increasing interest in the question of the CNS availability of these substances arose. Recently, the ability of phloroglucinols and flavonoids to penetrate the blood brain barrier could be demonstrated. For the phenanthroperylenequinones an examination of CNS bioavailability is still missing. The aim of this work is to close this gap by developing and validating a HPLC method with electrochemical detection for the quantification of the phenanthroperylenequinones in brain tissue of rodents after oral application of SJW extract or pure hypericin. In our study, the phenanthroperylenequinone content in the CNS tissue was below the lower limit of detection of the analytical method and was thus lower than 16 pmol/g brain for hypericin and lower than 52 pmol/g brain for pseudohypericin after oral administration of 1600 mg/kg SJW extract or pure hypericin (5 mg/kg). Correspondence: Mario Wurglics, Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University/ZAFES, 60438 Frankfurt am Main, Germany.     

Synthesis,crystal structures,DNA binding and cleavage properties and protein binding activities of three mononuclear cobalt(II) complexes

Three new mononuclear cobalt(II) complexes containing ligands with extended planar quinoxaline moieties, {dipyrido[3,2‐a:2′,3′‐c]phenazine (dppz) or dipyrido[3,2‐d:2′,3′‐f]quinoxaline (dpq)}, viz. [Co(dppz)(acac)₂] · CH₃OH ( 1 ), [Co(dpq)(tfnb)₂] ( 2 ) and [Co(dpq)(dbm)₂] ( 3 ), where acac = acetylacetonate, tfnb = benzoyltrifluoroacetone and dbm = dibenzoylmethane, have been synthesized and structurally characterized as octahedral complexes. The binding ability of the complexes with calf thymus (CT)‐DNA has been investigated by spectroscopic and viscosity measurements. Results indicate that all complexes bind to CT‐DNA via intercalative mode, and the DNA binding affinity of dppz complex 1 is apparently stronger than that of dpq complexes 2 and 3 . Furthermore, DNA photocleavage experiments indicate that these complexes are efficient DNA cleaving agents in UV‐A (365 nm) and hydroxyl radical (HO^·), singlet oxygen (¹O₂) and superoxide anion (¹O₂^?) serve as the major cleavage active species. In addition, interaction of the complexes with bovine serum albumin (BSA) was investigated using UV ? visible and fluorescence methods, which indicated that all complexes could quench the intrinsic fluorescence of BSA in a static quenching process. Copyright © 2014 John Wiley & Sons, Ltd.     

On the pH dependence of Cu(II) binding to insulin: ESR and electronic studies

The ESR and electronic absorption spectra have been used to investigate co-ordination of Cu(II) to insulin in aqueous solutions at different pH. Two series of complexes with low- and high copper content were examined and the values of the magnetic tensor components and the shape of the diffuse reflectance transitions suggested that these copper-insulin derivatives have tetragonal symmetry with Cu(II) in a (N_xO_y) ligand field, where oxygen donor groups are predominant at low pH and nitrogenous ligands at high pH. Such a trend was further supported by the presence of superhyperfine structure at pH = 13. Oxygen of the carboxylato groups, nitrogen of α- and ε-amino groups and of imidazoles, all contribute to the coordinations field. At very high pH only, a preferential binding site for Cu(II) is found, which probably involves deprotonated peptide nitrogens.     

Excretion profile of boldenone and its metabolites after oral administration to veal calves

The residue profiles of boldenone (17β-Bol), its epimer (17α-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17α-Bol decreased rapidly after end of treatment; detectable amounts of 17α-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17β-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.     

Anion control of isomerism, crystal packing and binding properties in a mononuclear zinc complex

The coordination chemistry of the tetradentate pyridyl N-donor ligand cis-3,5-bis-[2-pyridinyleneamin]-trans-hydroxycyclohexane (DDOP) has been investigated with zinc(II) nitrate and triflate. The resulting complexes, [Zn(DDOP)(H₂O)(NO₃)](NO₃) (1), and [Zn(DDOP)(H₂O)(OTf)](OTf) (2) differ not only in their counterions, but also the arrangement of the axial ligands and their solid state hydrogen bonded networks. Isothermal titration calorimetry was used to assess the difference in binding properties exhibited by the two zinc complexes at physiological pH in an aqueous environment. A series of coordinating amino acids were found to preferentially bind to the mononuclear zinc triflate (1) complex over the corresponding nitrate (2) assembly, with histidine exhibiting a two centre binding mode.     

Synthesis,characterization and arsenate binding events of new mononuclear copper(II) complexes

Two new mononuclear copper (II) complexes [Cu(L)(H₂O)Cl] (1) and [Cu(L)(H₂O)(SCN)] (2) (HL = 2-[1-(2-dimethylamino-ethylimino)-ethyl]-phenol) have been synthesized and characterized in order to investigate their binding interaction with arsenate ions. Complexes 1 and 2 were synthesized by performing reaction of CuCl₂·2H₂O or CuCl₂·2H₂O/NH₄SCN, respectively, with HL using Et₃N as mild base in MeOH solution at room temperature, and characterized by employing a number of analytical techniques, for example, elemental analysis, molar electrical conductivity, FTIR, UV–Vis and mass spectrometry. Their structures, optimized at DFT/B3LYP/6-311G level, show that the copper atom in 1 and 2 exhibits a distorted square pyramidal geometry. In H₂O/MeOH (3:1; v/v) solution, complexes 1 and 2 were examined for their binding affinity towards arsenate ions. The UV–Vis spectroscopic results specify that the arsenate group binds with 1 and 2 in 1:1 M ratio. The UV–Vis titration data were successfully utilized to calculate the binding constants of arsenate-bound Cu(II) complexes, and the values are found to be 1.723 × 10⁴ M^?1 and 2.161 × 10⁴ M^?1, corresponding to 1/AsO₄^3? and 2/AsO₄^3? assemblies, respectively.     

Binding of sulfonamides to erythrocytes and their components

Binding of zonisamide, a new antiepileptic sulfonamide derivative, was examined to human erythrocytes, their lysate and their carbonic anhydrase by centrifugation for cells or by ultrafiltration for the others. Scatchard plots revealed that the binding to intact and lysed cells was composed of high- and low-affinity components and that to carbonic anhydrase, of the high-affinity component alone. Parameters for high-affinity binding were similar in all three preparations and those for low-affinity binding were similar in the former two preparations. Dissociation constants for these bindings to erythrocytes were smaller than the dissociation constant for serum albumin. These results may explain the concentration of sulfonamides in red cells, and suggest the participation of cellular protein component(s) in addition to previously known carbonic anhydrase in the binding. Acetazolamide, sulthiame, zonisamide, hydrochlorothiazide and sulfanilamide inhibited carbonic anhydrase in a non-competitive manner to different extents. The Ki values of these sulfonamides were of the order of 0.1--0.2 of their respective Kd values determined by ultrafiltration, suggesting that under the present conditions, physicochemical interactions between sulfonamides and carbonic anhydrase primarily occur at common sites that affect the activity of the enzyme.     

Pharmacokinetic properties of N‐nitrosofenfluramine after its administration to rats

N‐Nitrosofenfluramine (N‐Fen), a synthetic adulterant in Chinese herbal diet products, is believed to cause hepatotoxicity in people who use these products. N‐Fen is a relatively new compound, and thus pharmacological and toxicological studies are insufficient. The aim of this work was to (1) define N‐Fen's plasma pharmacokinetics and tissue distribution after single intraperitoneal (i.p.) administration of 25 mg/kg to rats; (2) define its bioavailability; and (3) identify fenfluramine (Fen) and norfenfluramine (Norf) as N‐Fen metabolites. N‐Fen rapidly appeared in the circulation and was distributed to all tissues. Norf was found to be the primary metabolite and not Fen. Plasma and tissue levels of N‐Fen and Norf were low with bioavailability of N‐Fen after i.p. administration was <3%. The AUC_0−t of N‐Fen in the liver and kidney were 6.6 and 12.1 times, respectively, greater than the brain, and 17.8 and 32.6 times, respectively, greater than the plasma. In conclusion, N‐Fen did not show local accumulation in the liver, the site of toxicity, with concentrations represented as percentage of the total dose ranginng from 0.008 to 0.122%; hence the cause of hepatotoxicity could be related to the mechanisms other than toxicity consequences accumulation. Copyright © 2010 John Wiley & Sons, Ltd.     

Crystal structures, cyclic voltammetry and DNA binding of two mononuclear nickel(II) complexes

Two unsymmetrical complexes, [NiL¹]ClO₄ (1) and [NiL²]ClO₄ (2) have been synthesized and characterized by IR, UV, ES-MS and single crystal X-ray diffraction, where HL¹ and HL² are, respectively, the [1+1] condensation products of 2,6-diformyl-4-X-phenol (X = F or CH₃) with N ¹-(2-aminoethyl)-N ²-(4-nitrobenzyl) ethane-1,2-diamine. The coordination geometry of the metal in both complexes can be approximately described as square planar with a mean plane deviation of 0.032 Å in complex 1 and 0.027 Å in complex 2, respectively. The binding activities of the complexes toward calf-thymus DNA have been analyzed by spectroscopy and viscosity methods. The binding constants of 1 and 2 obtained from UV spectroscopic studies are 5.43 × 10⁵ and 1.83 × 10⁵ M^?1, respectively, while the linear Stern–Volmer quenching constants obtained from fluorescence spectroscopic studies are 0.83 × 10³ and 0.71 × 10³ M^?1, respectively. The cyclic voltammograms of the complexes show a pseudo-reversible electrochemical process.