Islet β-cells express both insulin receptors and insulin-signaling proteins. Recent evidence from rodents in vivo and from islets isolated from rodents or humans suggests that the insulin signaling pathway is physiologically important for glucose sensing. We evaluated whether insulin regulates β-cell function in healthy humans in vivo. Glucose-induced insulin secretion was assessed in healthy humans following 4-h saline (low insulin/sham clamp) or isoglycemic-hyperinsulinemic (high insulin) clamps using B28-Asp insulin that could be immunologically distinguished from endogenous insulin. Insulin and C-peptide clearance were evaluated to understand the impact of hyperinsulinemia on estimates of β-cell function. Preexposure to exogenous insulin increased the endogenous insulin secretory response to glucose by ≈40%. C-peptide response also increased, although not to the level predicted by insulin. Insulin clearance was not saturated at hyperinsulinemia, but metabolic clearance of C-peptide, assessed by infusion of stable isotope-labeled C-peptide, increased modestly during hyperinsulinemic clamp. These studies demonstrate that insulin potentiates glucose-stimulated insulin secretion in vivo in healthy humans. In addition, hyperinsulinemia increases C-peptide clearance, which may lead to modest underestimation of β-cell secretory response when using these methods during prolonged dynamic testing. beta cell | type 2 diabetes mellitus | insulin resistance | insulin clearance |
BACKGROUND: C-peptide is a marker of insulin secretion in diabetic patients. We assessed within-and between-laboratory imprecision of C-peptide assays and determined whether serum calibrators with values assigned by mass spectrometry could be used to harmonize C-peptide results.
From the viewpoint of regulatory guidelines, validation of LC-UV and LC-MS based methods have the same requirements. Matrix effects are not considered for most method validations if they do not influence reproducibility or assay linearity. Since matrix effects can strongly suppress ionizaton efficiency and therefore reduce sensitivity, they must be evaluated (and discussed in the context of method development)-prior to method qualification. The severity of matrix effects is directly dependent upon chromatographic performance. We suggest that evaluation of matrix effects and LC efficiency is essential information for method assessment, optimization and transfer to other mass spectrometers, and should be a mandatory part of routine LC/MS method validation. (J Am Soc Mass Spectrom 2005, 16, 1757-1759 LC/MS analysis of low purity biological samples can cause signal suppression, elevated background, and other negative matrix effects. In this situation, it is only possible to increase S/N while simultaneously maintaining high throughput performance by implementing the SRM mode. This, of course, implies a much higher cost of initial investment in sophisticated triple quadrupole instruments. An alternative approach to increasing S/N is to improve the quality of chromatography purification. This decreases matrix effects and makes possible the use of comparatively less expensive single quadrupole instruments while still achieving high analytical performance. Implementation of assays on single quadrupole mass spectrometers can be a good alternative for non-time intensive or budgeted limited projects. Therefore, enhancement of chromatography by implementation of two-dimensional LC methods or even off-line purification of analyte (especially from complex biological matrices) with resultant reduction of matrix effect can significantly improve the sensitivity of mass spectrometers by increasing ionization efficiency of a purer analyte. From the viewpoint of method development and optimization, information about the strength of matrix effects and chromatographic efficiency is important if the goal is to achieve optimum sensitivity.From a regulatory viewpoint, method validation procedures review the following parameters: precision, recovery, specificity, linearity, limit of detection (LOD) and limit of quantitation (LOQ), ruggedness, and robustness [1]. From the viewpoint of regulatory guidelines, validation of LC-UV and LC-MS based methods have the same requirements. In other words, the mass spectrometer is considered just as another type of passive detector. Matrix effects are not considered for most method validations if they do not influence reproducibility or assay linearity. Not directly addressed are technical issues unique to mass spectrometry based assays such as impact of matrix effects on theoretical maximum sensitivity. As mentioned previously, matrix effects can strongly suppress ionization efficiency and sensitivity, and therefore, must be evaluated (and discussed in the context of method development-before method ...
The development of a sensitive assay for the quantitative analysis of carbohydrates from human plasma using LC/MS/MS is described in this paper. After sample preparation, carbohydrates were cationized by Cs ϩ after their separation by normal phase liquid chromatography on an amino based column. Cesium is capable of forming a quasi-molecular ion [M ϩ Cs] ϩ with neutral carbohydrate molecules in the positive ion mode of electrospray ionization mass spectrometry. The mass spectrometer was operated in multiple reaction monitoring mode, and transitions [M ϩ 133] ¡ 133 were monitored (M, carbohydrate molecular weight). The new method is robust, highly sensitive, rapid, and does not require postcolumn addition or derivatization. It is useful in clinical research for measurement of carbohydrate molecules by isotope dilution assay. (J Am Soc Mass Spectrom 2005, 16, 1805-1811
We describe for the first time the direct quantitative analysis of human C-peptide from urine by isotope dilution assay. Implementation of 2-D reverse phase-reverse phase chromatography (2-D RP-RP) with SIM detection resulted in high assay sensitivity (LOQ= 5 pg on column), accuracy, and method ruggedness. Our experiments demonstrate the strong resolving capability of our proposed 2-D RP-RP platform which significantly reduces strong matrix effects and their resulting quantitation error over a wide range of urine concentrations. In contrast, single column methods (both SIM and multiple reaction monitoring) were found acceptable only for strongly diluted urine samples.
The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH(*) 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of approximately 26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 micro s for a 7 degrees jump to 52 degrees C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 micro s and 56 micro s for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.
We investigated the impact of one dimension (single reverse phase (RP) column) and two dimension (two different RP columns) chromatographic methods on SIM (MS) and multiple reaction monitoring (MRM; MS/MS) performance from human plasma. We find that MRM analysis is clearly preferable for 1-D applications; however, implementation of SIM detection in conjunction with 2-D separation technique resulted in an over 60-fold increase in analyte peak area and improved S/N compared to MRM for our analyte, human C-peptide. Implementation of a 2-D RP-RP technique with SIM detection is capable of eliminating matrix effects and greatly increases signal response and data quality. For two large peptides in complex biological samples, we found that a 2-D approach performed better than high quality sample preparation together with 1-D chromatography and MRM, even on a high-end mass spectrometer.
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