Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter.

The integration of complete analyses systems "on chip" is one of the great potentials of microfabricated devices. In this study we present a new pressure-driven microfabricated fluorescent-activated cell sorter chip with advanced functional integration. Using this sorter, fluorescent latex beads are sorted from chicken red blood cells, achieving substantial enrichments at a sample throughput of 12000 cells s(-1). As a part of the sorter chip, we have developed a monolithically integrated single step coaxial flow compound for hydrodynamic focusing of samples in flow cytometry and cell sorting. The structure is simple, and can easily be microfabricated and integrated with other microfluidic components. We have designed an integrated chamber on the chip for holding and culturing of the sorted cells. By integrating this chamber, the risk of losing cells during cell handling processes is eliminated. Furthermore, we have also developed integrated optics for cell detection. Our new design contributes to the ongoing efforts for building a fully integrated micro cell sorting and analysing system.

Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs.

We have synthesized insulins acylated by fatty acids in the epsilon-amino group of LysB29. Soluble preparations can be made in the usual concentration of 600 nmol/ml (100 IU/ml) at neutral pH. The time for 50% disappearance after subcutaneous injection of the corresponding TyrA14(125I)-labelled insulins in pigs correlated with the affinity for binding to albumin (r = 0.97), suggesting that the mechanism of prolonged disappearance is binding to albumin in subcutis. Most protracted was LysB29-tetradecanoyl des-(B30) insulin. The time for 50% disappearance was 14.3 +/- 2.2 h, significantly longer than that of Neutral Protamine Hagedorn (NPH) insulin, 10.5 +/- 4.3 h (p < 0.001), and with less inter-pig variation (p < 0.001). Intravenous bolus injections of LysB29-tetradecanoyl des-(B30) human insulin showed a protracted blood glucose lowering effect compared to that of human insulin. The relative affinity of LysB29-tetradecanoyl des-(B30) insulin to the insulin receptor is 46%. In a 24-h glucose clamp study in pigs the total glucose consumptions for LysB29-tetradecanoyl des-(B30) insulin and NPH were not significantly different (p = 0.88), whereas the times when 50% of the total glucose had been infused were significantly different, 7.9 +/- 1.0 h and 6.2 +/- 1.3 h, respectively (p < 0.04). The glucose disposal curve caused by LysB29-tetradecanoyl des-(B30) insulin was more steady than that caused by NPH, without the pronounced peak at 3 h. Unlike the crystalline insulins, the soluble LysB29-tetradecanoyl des-(B30) insulin does not elicit invasion of macrophages at the site of injection. Thus, LysB29-tetradecanoyl des-(B30) insulin might be suitable for providing basal insulin in the treatment of diabetes mellitus.

Albumin binding of insulins acylated with fatty acids: characterization of the ligand-protein interaction and correlation between binding affinity and timing of the insulin effect in vivo.

Albumin is a multifunctional transport protein that binds a wide variety of endogenous substances and drugs. Insulins with affinity for albumin were engineered by acylation of the epsilon-amino group of LysB29 with saturated fatty acids containing 10-16 carbon atoms. The association constants for binding of the fatty acid acylated insulins to human albumin are in the order of 10(4)-10(5) M-1. The binding apparently involves both non-polar and ionic interactions with the protein. The acylated insulins bind at the long-chain fatty acid binding sites, but the binding affinity is lower than that of the free fatty acids and depends to a relatively small degree on the number of carbon atoms in the fatty acid chain. Differences in affinity of the acylated insulins for albumin are reflected in the relative timing of the blood-glucose-lowering effect after subcutaneous injection into rabbits. The acylated insulins provide a breakthrough in the search for soluble, prolonged-action insulin preparations for basal delivery of the hormone to the diabetic patient. We conclude that the biochemical concept of albumin binding can be applied to protract the effect of insulin, and suggest that derivatization with albumin-binding ligands could be generally applicable to prolong the action profile of peptide drugs.

Characterization of the three 125I-iodination isomers of human insulin-like growth factor I (IGF1).

Human insulin-like growth factor I (IGF1) was labeled with 125I and the resulting mixture of iodination isomers was separated by reverse-phase HPLC. Three major radioactive peaks were isolated and identified by sequencing as the expected three monoiodinated species. The ranking of the affinities of the three isomers for the human IGF1 receptor was found to be Tyr24(125I) > Tyr31(125I) >> Tyr60(125I). The Tyr31(125I) isomer was shown to have an affinity similar to that of unlabeled IGF1 and is thus the tracer of choice for IGF1. The tracers were stable upon storage at -20 degrees C for at least 3 months.

Interactions of a hybrid insulin/insulin-like growth factor-I analog with chimeric insulin/type I insulin-like growth factor receptors.

We have examined, by use of a hybrid insulin/insulin-like growth factor-I analog and chimeric insulin/type I insulin-like growth factor receptors, the interplay between ligand and receptor structure in determining the affinity and specificity of hormone-receptor interactions in the insulin and insulin-like growth factor-I systems. Our findings, obtained through the study of radiolabeled peptide binding to detergent-solubilized full-length receptors and to soluble truncated receptors, show that (a) the two-chain hybrid analog exhibits significant cross-reactivity with both receptor systems, (b) the exchange of appropriate domains in chimeric receptors enhances the receptor binding affinity of the analog by 3.5-21-fold, and (c) the affinity of the hybrid analog for the chimeric receptors actually exceeds that of either natural insulin or natural insulin-like growth factor-I. We conclude that the specificity-conferring domains of the insulin and type I insulin-like growth factor receptors reside in different regions of a common binding site, and that the exchange of domains between pairs of related hormones and between pairs of related receptors can yield new ligand-receptor systems with significantly altered affinities and selectivities.

The reactivity of anti-porcine gastric inhibitory polypeptide (GIP) rabbit serum R65 with synthetic human and porcine GIP.

Porcine gastric inhibitory peptide (GIP) has been used extensively as a standard and as [125I]-GIP in radio-immunoassays (RIAs) to determine immunoreactive GIP (IR GIP) in human plasma even though porcine and human GIP have slightly different amino acid sequences. A rabbit anti-porcine GIP serum (R65) which has been widely used in the RIA for IR GIP reacts with a part of the GIP molecule which is identical in human and porcine GIP. In order to validate the use of R65 the IR GIP contents of synthetic human and porcine GIP were measured, using natural porcine GIP as standard, and found to be 40% and 35% on a weight basis respectively. These materials were considered unsuitable for use as RIA standards. The IR GIP in samples of human plasma was then assayed with 125I-labelled synthetic human or porcine GIP using natural porcine GIP as standard. The values measured for IR GIP did not depend on the labelled peptide used (p = 0.84, n = 81) and were linearly correlated (r = 0.98, p less than 0.001, n = 81). It is concluded that the value of IR GIP measured in human plasma using R65 and standards of porcine GIP is linearly proportional to the level of human GIP in plasma.

Receptor binding and tyrosine kinase activation by insulin analogues with extreme affinities studied in human hepatoma HepG2 cells.

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.

Scintigraphic studies in rats. Kinetics of insulin analogues covering wide range of receptor affinities.

Whole-body kinetics of 123I-labeled human insulin and five insulin analogues were investigated by scintigraphic studies in rats. The amino acid substitutions and the relative receptor affinities (RAs), determined by binding to HepG2 cells, were: GluB12, des-B30 insulin, RA 0.15%; AspB9, GluB27 insulin, RA 18%; AspB26 insulin, RA 80%; AspB18 insulin, RA 327%; and HisA8, HisB4, GluB10, HisB27 insulin, RA 687%. All analogues were compared with human insulin (RA 100%). The analogue with RA 0.15% showed a significantly slower disappearance in the heart window, no liver uptake, and the greatest kidney radioactivity, the latter probably caused by high plasma concentrations. The low-affinity analogue (RA 18%) reached a surprisingly high hepatic peak value, although significantly lower than insulin. Kidney radioactivity was higher than for insulin. The analogue with RA 80% showed liver and kidney radioactivities that were not significantly different from those of insulin. The two high-affinity analogues (RAs 327 and 687%) showed peak liver radioactivities not significantly different from insulin. However, liver radioactivity after the peaks declined significantly more slowly. Compared with insulin, the kidney radioactivity curves were not significantly different. We conclude that high-affinity insulin analogues will bind to any available receptor that, because of the large number of receptors in the periphery and the distribution of cardiac output, favors extrahepatic elimination. In contrast, low-affinity analogues bind to receptors several times before they are eliminated. This leads to recirculation and, thus, hepatic elimination due to the high receptor density there. It follows that hepatopreferential binding cannot be expected solely by use of an insulin analogue with a particular receptor affinity.

Somatostatin-like immunoreactivity in man. Measurement in peripheral plasma.

A radioimmunoassay for the determination of somatostatin-like immunoreactivity (SLI) in plasma has been developed using antibodies raised in rabbits against synthetic somatostatin conjugated to hemocyanin and 125I-Tyr1-somatostatin. The detection limit was 2 pg/ml. A combination of EDTA and aprotinin (0.15 M and 80 Kallikrein Inhibitor Units = KIU/ml) inhibited the tracer degradation, but did not eliminate it completely. In addition somatostatin immunoreactivity in unextracted plasma showed a strong dilution effect and the estimates depended on the antiserum used (e.g. a plasma sample (1 : 5) assayed with antiserum R 141 apparently contained 150 pg/ml, whereas antiserum K 5615 yielded a value of 300 pg/ml). When plasma was extracted with acid ethanol, the values obtained with the two different antisera were practically the same, recovery of somatostatin added to plasma was approx. 80% and dilution of specimens gave proportional readings. Consequently, extraction of plasma is regarded as obligatory to ensure valid results. Mean basal level was 26.5 +/- 1.2 pg/ml (mean +/- SEM). When plasma extracts were subjected to chromatography on Sephadex G-25 at neutral pH, approx. 30% of the SLI was recovered in the MW1600 fraction; whereas the major part of the remaining immunoreactivity (37-60%) eluted earlier in one fraction and the residual immunoreactivity in the void volume.

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.

Homogeneous mono-(125)i-insulins. Preparation and characterization of mono-(125)i-(tyr a14)-and mono-(125)i-(tyr a19)-insulin.

Mono-(125)I-(Tyr A19)-insulin (Mono*A19) was prepared by iodinating MC porcine insulin with (125)I in acid medium using iodate (as oxidizing agent), followed by anion-exchange chromatography. Mono-(125)I-(Tyr A14)-insulin (Mono*A14) was prepared by iodinating MC porcine insulin with (125)I, using H(2)O(2)/lactoperoxidase at neutral pH, followed by anion-exchange chromatography. The specific radioactivities were in the ranges of 120-200 and 220-300 mCi/mg for Mono*A19 and Mono*A14, respectively. Analyses of the intramolecular distributions of (125)I demonstrated that the preparations were 97-98% radiochemically pure. In both preparations, 98-99% of the radioactivity was capable of binding to insulin antibodies for up to 6 months of storage of the tracers. The IRI concentration decreased with the duration of storage. The greatest observed fall in IRI concentration was 70%. The ime course could be explained by the assumption that the disintegration of a (125)I-nucleus destroys the immunoreactivity of the insulin molecule in which the decay occurs.

Studies on polypeptides, VI. Synthesis, circular dichroism and immunological studies of tyrosyl C-peptide of human proinsulin.

The synthesis of tyrosyl human C-peptide, a sequence of 32 amino acids, by the fragment condensation of the N-terminal octapeptide and C-terminal tetracosapeptide is described. The t-butyl protecting groups were removed by trifluoroacetic acid to obtain N-benzyloxycarbonyl-tyrosyl C-peptide. The hydrogenolytic debenzyl-oxycarbonylation of this derivative proceeded to an extent of only 80-90%, and tyrosyl C-peptide was purified by preparative electrophoresis. This purified tyrosyl C-peptide led to an improved sensitivity of the radioimmunoassay. The synthetic tyrosyl C-peptide in an immunoassay using anti human b-component serum reacted slightly differently from the synthetic human C-peptide. After labelling tyrosyl C-peptide with 125I and then purifying the radioactive product, we observed that 80% of the radioactivity could be bound when reacted with an excess of the serum. The circular dichroism spectrum of tyrosyl C-peptide is very similar to that of synthetic human C-peptide. An analysis of the spectrum indicates that 3-7 amino acids are in the beta-structure and the rest in random coil conformation.