Two-color electrophoretic immunoassay for simultaneous measurement of insulin and glucagon content in islets of Langerhans

A multianalyte CE competitive immunoassay using two-color detection was developed to measure insulin and glucagon in islets of Langerhans. Insulin was quantified with FITC-insulin (Ins*) and anti-insulin antibodies (Ins Ab) and glucagon was quantified with Cy5-glucagon (Glu*) and anti-glucagon antibodies (Glu Ab). A 3 mW Ar(+) laser at 488 nm and a 25 mW laser diode at 635 nm were used to excite FITC and Cy5, respectively. Fluorescence was split with a half-silvered mirror and passed through a 520 +/- 20 nm bandpass filter or a 663 nm longpass filter for the detection of insulin and glucagon, respectively. The two-color detection format enabled independent quantitation of both analytes even with concentrations of insulin immunoassay reagents 20-fold higher than glucagon reagents. Simultaneous calibration curves were generated and used to determine insulin and glucagon content in islets of Langerhans. Amounts of insulin and glucagon were 56.6 +/- 3.2 and 1.0 +/- 0.5 ng/islet, respectively. LODs were 7 nM insulin and 3 nM glucagon. The assay will be applicable to fast monitoring of multiple peptides secreted from islets of Langerhans and can be applied to other systems for the quantitation of multiple analytes with large differences in concentrations.     

Dynamic monitoring of glucagon secretion from living cells on a microfluidic chip

A rapid microfluidic based capillary electrophoresis immunoassay (CEIA) was developed for on-line monitoring of glucagon secretion from pancreatic islets of Langerhans. In the device, a cell chamber containing living islets was perfused with buffers containing either high or low glucose concentration. Perfusate was continuously sampled by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled glucagon (FITC-glucagon) and monoclonal anti-glucagon antibody. To minimize sample dilution, the on-chip mixing ratio of sampled perfusate to reagents was maximized by allowing reagents to only be added by diffusion. Every 6 s, the reaction mixture was injected onto a 1.5-cm separation channel where free FITC-glucagon and the FITC-glucagon–antibody complex were separated under an electric field of 700 V cm⁻¹. The immunoassay had a detection limit of 1 nM. Groups of islets were quantitatively monitored for changes in glucagon secretion as the glucose concentration was decreased from 15 to 1 mM in the perfusate revealing a pulse of glucagon secretion during a step change. The highly automated system should be enable studies of the regulation of glucagon and its potential role in diabetes and obesity. The method also further demonstrates the potential of rapid CEIA on microfluidic systems for monitoring cellular function.     

Capillary liquid chromatography of multiple peptides with on-line capillary electrophoresis immunoassay detection.

A competitive immunoassay for neuropeptide Y (NPY) based on capillary electrophoresis (CE) with laser-induced fluorescence detection was developed utilizing polyclonal antisera as the immunoreagent and fluorescein-labeled NPY as the tracer. The assay was performed with on-line mixing of reagents, automated injections, and a 3 s separation time. The assay had a detection limit of 850 pM. To detect NPY at lower concentrations, the assay was coupled on-line to reversed-phase capillary liquid chromatography (LC). In this arrangement, 5 microL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol-containing mobile phase. The resulting chromatographic peaks were monitored by the CE immunoassay. With preconcentration, the concentration detection limit was improved to 40 microM and NPY could be measured in push-pull perfusion samples collected from the paraventricular nucleus of freely moving rats. The technique was extended to simultaneous detection of NPY and glucagon secretion from islets of Langerhans.     

Perfusion and chemical monitoring of living cells on a microfluidic chip

A microfluidic device that incorporates continuous perfusion and an on-line electrophoresis immunoassay was developed, characterized, and applied to monitoring insulin secretion from single islets of Langerhans. In the device, a cell chamber was perfused with cell culture media or a balanced salt solution at 0.6 to 1.5 microL min(-1). The flow was driven by gas pressure applied off-chip. Perfusate was continuously sampled at 2 nL min(-1) by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and monoclonal anti-insulin antibody and allowed to react for 60 s as the mixture traveled down a 4 cm long reaction channel. The cell chamber and reaction channel were maintained at 37 degrees C. The reaction mixture was injected onto a 1.5 cm separation channel as rapidly as every 6 s, and the free FITC-insulin and the FITC-insulin-antibody complex were separated under an electric field of 500 to 600 V cm(-1). The immunoassay had a detection limit of 0.8 nM and a relative standard deviation of 6% during 2 h of continuous operation with standard solutions. Individual islets were monitored for up to 1 h while perfusing with different concentrations of glucose. The immunoassay allowed quantitative monitoring of classical biphasic and oscillatory insulin secretion with 6 s sampling frequency following step changes in glucose from 3 to 11 mM. The 2.5 cm x 7.6 cm microfluidic system allowed for monitoring islets in a highly automated fashion. The technique should be amenable to studies involving other tissues or cells that release chemicals.     

Thermally reversible polymer gels for biohybrid artificial pancreas

As an approach to replacing islets of Langerhans in an implanted biohybrid artificial pancreas, thermally reversible polymers based on N-isopropylacrylamide were synthesized and then evaluated as an extracellular matrix for islets in an immunoprotecting membrane pouch. A high molecular weight poly(N-isopropylacrylamide-co-acrylic acid (2 mole % in feed)) demonstrated gelation at 37°C and became a solution below 30°C. This polymer exhibited minimum syneresis (water separation) upon gelation from a solution state when the temperature was raised from room temperature to 37°C, while poly(N-isopropylacrylamide) exhibited considerable syneresis under the same conditions. These properties influence the efficiency of islet entrapment. The copolymer was able to entrap rat islets almost 100%, but the homopolymer entrapped less than 50%. The static insulin secretion of the islets in the copolymer matrix at high glucose concentration (16.5 mM) was comparable to that of control islets, however, the entrapped islets showed prolonged viability in vitro. These results indicate the potential of developing a rechargeable biohybrid pancreas using thermally reversible polymer gels.     

Highly efficient detection of insulinotropic action of glucagon via GLP-1 receptor in mice pancreatic beta-cell with a novel perfusion microchip

Glucagon exhibits insulinotropic ability by activating cAMP through glucagon or glucagon-like peptide-1(GLP-1) receptors.To investigate the mechanism of endogenous and exogenous glucagon on insulin release,we studied the receptor selectivity on pancreatic islet beta-cells by switching the glucose concentration from 20 mmol/L to 0 mmol/L To measure the exact temporal relationship between glucagon and insulin release,we developed a quick,small volume,multi-channel polydimethylsiloxane(PDMS) microchip.At 0 mmol/L glucose,we observed an insulinotropic effect in both INS-1 cells and islets.Meanwhile,we observed a 63 ± 6.27 s delay of endogenous glucagon-induced insulin release.After treatment with glucagon and GLP-1 receptor antagonists,we found that endogenous glucagon utilized the glucagon receptor,whereas exogenous glucagon primarily utilized the GLP-1 receptor to promote insulin secretion.The microchip can also be used to describe the "glucagonocentric" vision of diabetes pathophysiology.Taken together,the insulinotropic mechanism of different receptors should be taken into account in clinical treatments.     

Altered expression of insulins I and II and their mRNAs in the islets of Langerhans in dexamethasone-induced diabetic rats

Rats have two isomeric insulins (insulins I and II). There have been no reports on the expression of the isomeric insulins in glucocorticoid-induced diabetic rats. To clarify the relation of the expression of each insulin and its mRNAs in dexamethasone-induced diabetic rats, the amounts of the isomeric insulins and mRNAs in the islets of Langerhans were determined in vivo and in vitro. A sensitive and selective HPLC-fluorescence determination method for the isomeric insulins and a newly developed real-time quantitative RT-PCR method for their mRNAs were used. There was a greater reduction of insulin II than insulin I in the islets of Langerhans in dexamethasone-induced diabetic rats. This alteration may be caused by a disproportionate expression of the respective mRNA for the isomeric insulins that resulted from the direct effect of dexamethasone. In addition, continuous hyperglycemia may also suppress the expression of the insulin II mRNA. The overall effects of dexamethasone and hyperglycemia may cause a greater reduction of insulin II than insulin I in the dexamethasone-induced diabetic rat. Conversely, an elevated ratio of insulin I to II in the islets could suggest a diabetic condition.     

Analysis of protamine peptides in insulin pharmaceutical formulations by capillary electrophoresis

Protamines are a group of highly basic peptides that are sometimes added to insulin formulations to prolong the pharmacological action. In this study, different methods were investigated to identify protamine in insulin formulations. Capillary electrophoresis in aqueous and non‐aqueous media was tested to separate these peptides with very close amino acid sequences. Different buffers (phosphate or formate, both acidified) and various additives (principally negatively charged and neutral surfactants) were investigated to optimize peptide separation. Finally, a micellar electrokinetic capillary chromatography method using a capillary of 120 cm effective length and an aqueous background electrolyte made up of 100 mM phosphate buffer (pH 2) and 50 mM Thesit® gave the best results, providing the separation of the four major protamine peptides within 25 min.     

Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for bile acids in serum

A simple and rapid technique for the simultaneous isolation and analysis of fifteen kinds of bile acid was developed using reversed-phase high-performance liquid chromatography with an automatic dual-precolumn switching system. The serum samples were directly injected onto a first precolumn (hydroxyapatite), which was flushed with 1 mM phosphate buffer. Serum proteins were strongly retained on the hydroxyapatite column, but bile acids were unretained. The bile acids were absorbed on a second precolumn (Serumout-25) and eluted onto the analytical column with solvent B (acetonitrile-methanol-30 mM ammonium acetate, 20:20:60, v/v/v). For the separation of each bile acid, the gradient elution technique was used (solvent A was acetonitrile-methanol-30 mM ammonium acetate, 30:30:40). After separation of the bile acids, NADH was produced by use of immobilized 3 alpha-hydroxysteroid dehydrogenase column and then determined fluorimetrically (gamma em = 460 nm, gamma ex = 350 nm). The recoveries of bile acids in serum generally approached 100%.     

Monitoring oligomer formation from self-aggregating amylin peptides using ESI-IMS-MS

Amyloid diseases are a serious cause for concern world-wide. To understand the mechanism of formation of the fibrillar structures associated with such disorders, it is necessary to study the progression from soluble protein or peptide monomer through an array of oligomers to the final, insoluble, fibrils. The protein IAPP is found in vivo in the form of insoluble amyloid deposits in the pancreatic islets of diabetes type II sufferers. Here, we have studied the in vitro self-aggregation of three fibril-forming peptides from the amyloidogenic core of IAPP. Using electrospray ionization—mass spectrometry coupled with ion mobility spectrometry, the mass and cross-sectional area of each oligomer present in the heterogeneous assembly mixtures can be determined individually in a single, rapid experiment over time. For the three peptides studied, oligomers ≤20-mer were characterized. Conversely, no oligomers higher than a dimer were detected for a non-assembling peptide control. The rate in which the cross-sectional area of the oligomers increases with increasing number of peptide sub-units indicates that assembly for the amyloid-forming peptides proceeds in a linear fashion until an oligomer of a certain size is attained. After this, a step increase in cross-sectional area occurs for the next higher-order oligomer. This behaviour can be explained by molecular modelling of singly, doubly, triply and quadruply stacked β-stranded structures. Using one peptide as an example, the cross-sectional areas of the lower order oligomers (dimer to pentamer) were found to be consistent with a single β-sheet model, whereas the higher order oligomers were consistent with double-stranded (hexamer to decamer oligomers), triply-stranded (11-mers to 15-mers) and quadruply-stranded (16-mers to 20-mers) β-sheet models.     

Simultaneous determination of celecoxib, meloxicam, and rofecoxib using capillary electrophoresis with surfactant and application in drug formulations

A simple and selective CE using surfactant with UV detection is described for the simultaneous determination of selective cyclooxygenase-2 inhibitors, celecoxib, meloxicam, and rofecoxib. The simultaneous analysis of celecoxib, meloxicam, and rofecoxib was performed in Tris buffer (10 mM; pH 11) with 60 mM sodium octane-sulfonate and 20% ACN as an anionic surfactant and organic modifier, respectively. Under this condition, good separation with high efficiency and the required short analysis time is achieved. The linear ranges of the method for the determination of celecoxib, meloxicam, and rofecoxib were over 5-100 microg/mL; the detection limits at 200 nm (S/N = 3; injection 3.45 kPa, 5 s) were 2, 1, and 1 microg/mL, respectively. The small amount of sample required and the expeditiousness of the procedure allow content uniformity to be determined in individual pharmaceutical products.     

Efficient identification and quantification of proteins using isotope-coded 1-(6-methylnicotinoyloxy)succinimides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We describe a convenient and useful method for the identification and relative quantification of proteins using light and heavy reagents, 1-(6-methylnicotinoyloxy)succinimides (6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS, respectively). This method is based on the chemical derivatization of amino groups of tryptic peptides with these reagents, i.e., the basic moiety of the reagents thus incorporated into both the N-terminal amino group and the epsilon-amino group of the lysine residue would improve the ionization efficiency of tryptic peptides. An increase in protein sequence coverage is achieved by derivatization with these reagents or by combination of mass values before and after derivatization. Since a combination of 6-CH(3)-Nic-NHS and d(3)-labeled reagent (6-CD(3)-Nic-NHS) generates a 3 Da mass difference per reaction site, the d(3)-labeled reagent shifts the mass values of d(0)-labeled peptides according to the number of reactive amino groups in the peptides. In the case of tryptic peptides, the mass values of C-terminal arginine and lysine peptides are shifted by 3 and 6 Da, respectively. Further, the 3 Da mass difference between 6-CH(3)-Nic-NHS and 6-CD(3)-Nic-NHS offers a means for the relative quantification of protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.     

The role of copper(II) and zinc(II) in the degradation of human and murine IAPP by insulin‐degrading enzyme

Amylin or islet amyloid polypeptide (IAPP) is a 37‐residue peptide hormone secreted from the pancreatic islets into the blood circulation and is cleared by peptidases in the kidney. IAPP aggregates are strongly associated with β‐cell degeneration in type 2 diabetes, as demonstrated by the fact that more than 95% of patients exhibit IAPP amyloid upon autopsy. Recently, it has been reported that metal ions such as copper(II) and zinc(II) are implicated in the aggregation of IAPP as well as able to modulate the proteolytic activity of IAPP degrading enzymes. For this reason, in this work, the role of the latter metal ions in the degradation of IAPP by insulin‐degrading enzyme (IDE) has been investigated by a chromatographic and mass spectrometric combined method. The latter experimental approach allowed not only to assess the overall metal ion inhibition of the human and murine IAPP degradation by IDE but also to have information on copper‐ and zinc‐induced changes in IAPP aggregation. In addition, IDE cleavage site preferences in the presence of metal ions are rationalized as metal ion‐induced changes in substrate accessibility. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous separation of nine metal ions and ammonium with nonaqueous capillary electrophoresis

A simple and fast method for simultaneous separation of nine metal cations Ni2+, Cu2+, Co2+, Zn2+ Cd2+, K+, Na+, Mg2+ and Ca2+, and NH4+ in methanol is reported. The optimization for separation these 10 cations was achieved by using 0.5% acetic acid and 10 mM imidazole as electrolyte. The effects of water and ionic strength in the sample are discussed. The sensitive detection of transition metal ions was accomplished at 191 nm. The optimized method demonstrated high efficiency and good reproducibility, and was applied successfully to the qualitative and quantitative determination of transition metal ions in water samples, chemical reagents, oral zinc gluconate solution and human plasma.     

Mechanistic studies of peptide self-assembly: transient α-helices to stable β-sheets

The pathologic self-assembly of proteins is associated with typically late-onset disorders such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown, but there is increasing evidence supporting the role of transient α-helices in the early events. Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide that self-assembles into aggregates that are toxic to the insulin-producing β cells. To elucidate early events in the self-assembly of IAPP, we used limited proteolysis to identify an exposed and flexible region in IAPP monomer. This region includes position 20 where a serine-to-glycine substitution (S20G) is associated with enhanced formation of amyloid fibrils and early onset type 2 diabetes. To perform detailed biophysical studies of the exposed and flexible region, we synthesized three peptides including IAPP(11-25)WT (wild type), IAPP(11-25)S20G, and IAPP(11-25)S20P. Solution-state NMR shows that all three peptides transiently populate the α-helical conformational space, but the S20P peptide, which does not self-assemble, transiently samples a broken helix. Under similar sample conditions, the WT and S20G peptides populate the α-helical intermediate state and β-sheet end state, respectively, of fibril formation. Our results suggest a mechanism for self-assembly that includes the stabilization of transient α-helices through the formation of NMR-invisible helical intermediates followed by an α-helix to β-sheet conformational rearrangement. Furthermore, our results suggest that reducing intermolecular helix-helix contacts as in the S20P peptide is an attractive strategy for the design of blockers of peptide self-assembly.     

Analysis of free amino acids in islets of Langerhans by high-performance liquid chromatography using pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

A simple high-performance liquid chromatographic method with pre-column derivatization and fluorescence detection was developed and used for the analysis of free amino acids in islets of Langerhans; 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) served as pre-column derivatization reagent. Islets of Langerhans were separated from the pancreas of normal and obese rats, treated with pre-cooling methanol-water (80:20, v/v), and ultrasonicated to fragmentize the islets and effect deproteination. Several parameters influencing the derivatization reaction and chromatographic separation were optimized. Amino acid derivatives obtained under optimal conditions were separated on a C18 column with acetonitrile-acetate buffer as mobile phase and detected at 470 nm/540 nm (Ex/Em). Matrix effects were investigated and good linearities with correlation coefficients better than 0.9972 were obtained over a wide range of 0.42-42.11 microM for most of the amino acids. The detection limits (S/N = 3) were within the range of 6.1-51 nM. The precision of the method and recoveries were in the ranges of 1.43-10.76% (RSD%) and 85.07-108.82%, respectively. The analytical results showed that the serine content was markedly higher in normal rats than in obese rats, whereas methionine was of relatively lower content in both normal and obese rats.     

Immunoassay by capillary electrophoresis with quantum dots

The application of quantum dots in capillary electrophoresis immunoassay was studied for the first time. Quantum dots were conjugated with antibody and subsequently tested by electrophoretic separation of free antibody and antibody-antigen complex. Antibody was fluorescently labeled by quantum dots via conjugation procedures and its electrophoretic characteristics were effectively modified due to the attachment of quantum dots. The determination of human IgM by direct CE based immunoassay could be easily achieved by simply changing the pH value of separation buffer. Polymer additive influenced the separation too but the effect was not as significant as buffer pH adjustment. Satisfactory separation of complex from free antibody could be achieved with 20mM sodium tetraborate as separation buffer, at pH 9.8. The immunoassay application of quantum dots in CE offers considerable advantages and can be readily applied to other large bio-molecules.     

Micro-plate magnetic chemiluminescence immunoassay and its applications in carcinoembryonic antigen analysis

A micro-plate magnetic chemiluminescence immunoassay was developed for rapid and high throughput detection of carcinoembryonic antigens (CEA) in human sera. This method was based on a sandwich immunoreaction of fluorescein isothiocyanate (FITC)-labeled anti-CEA antibodies, CEA antigens, and horseradish peroxidase (HRP)-conjugated anti-CEA antibodies in mi- cro-plate. The immunomagnetic particles coated with anti-FITC antibodies were used as the solid phase for the immunoassay. The separation procedure was c...     

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.     

Simultaneous determination of cimetidine, famotidine, nizatidine, and ranitidine in tablets by capillary zone electrophoresis.

A simple capillary zone electrophoresis (CZE) method is described for the simultaneous determination of cimetidine (CIM), famotidine (FAM), nizatidine (NIZ), and ranitidine (RAN). The analysis of these drugs was performed in a 100 mM phosphate buffer, pH 3.5. Several parameters were studied, including wavelength for detection, concentration and pH of phosphate buffer, and separation voltage. The quantitative ranges were 100-1,000 microM for each analyte. The intra- and interday relative standard deviations (n = 5) were all less than 4%. The detection limits were found to be about 10 microM for CIM, 20 microM for RAN, 20 microM for NIZ, and 10 microM for FAM (S/N = 3, injection 1 s) at 214 nm. All recoveries were greater than 92%. Applications of the method to the assay of these drugs in tablets proved to be feasible.