Eduard Rogatsky

Insulin enhances glucose-stimulated insulin secretion in healthy humans

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.

Standardization of C-Peptide Measurements

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. METHODS We sent 40 different serum samples to 15 laboratories, which used 9 different routine C-peptide assay methods. We also sent matched plasma samples to another laboratory for C-peptide analysis with a reference mass spectrometry method. Each laboratory analyzed 8 of these samples in duplicate on each of 4 days to evaluate within- and between-day imprecision. The same 8 samples were also used to normalize the results for the remaining samples to the mass spectrometry reference method. RESULTS Within- and between-run CVs ranged from <2% to >10% and from <2% to >18%, respectively. Normalizing the results with serum samples significantly improved the comparability among laboratories and methods. After normalization, the differences among laboratories in mean response were no longer statistically significant (P = 0.24), with least-squares means of 0.93-1.02. CONCLUSIONS C-peptide results generated by different methods and laboratories do not always agree, especially at higher C-peptide concentrations. Within-laboratory imprecision also varied, with some methods giving much more consistent results than others. These data show that calibrating C-peptide measurement to a reference method can increase comparability between laboratories.

Acute elevation of NEFA causes hyperinsulinemia without effect on insulin secretion rate in healthy human subjects.

Abstract: Increased circulating levels of nonesterified free fatty acids (NEFA) have been observed in such hyperinsulinemic states as obesity, impaired glucose tolerance, diabetes, and dyslipidemia where they have been causally linked to the development of insulin resistance and hyperinsulinemia. The concentration of NEFA in plasma is believed to have direct modifying effects on insulin secretion and clearance. It remains controversial whether acute increases in NEFA potentiate insulin secretion in human subjects. We studied the effect of an acute elevation of NEFA during lipid‐heparin infusion compared to a glycerol‐only control on glucose‐stimulated insulin secretion and clearance during a 120‐min hyperglycemic (10 mM) clamp in 7 healthy normoglucose‐tolerant volunteers. The metabolic clearance rate of C‐peptide (MCRCP) was measured in each subject during the study by simultaneous infusion of C‐peptide. Insulin secretion rate (ISR) was calculated from deconvolution of C‐peptide data after correction for the rate of C‐peptide infusion. Clearance rate of insulin (MCRINS) was calculated based upon endogenous ISR. Plasma glucose (mg/dL): basal (90‐115 min) 90.2 ± 2.8 vs. 90.2 ± 2.3; clamp (150‐240 min) 180.5 ± 2.8 vs. 180.9 ± 1.3. Plasma insulin (pmol/L): prebasal (fasting) 29.6 ± 10.0 vs. 29.8 ± 10.6; basal (90‐115 min) 30.1 ± 9.2 vs. 34.5 ± 12.1; second phase clamp (210‐240 min) 127.6 ± 18.2 vs. 182.5 ± 17.3*. Plasma NEFA (mM): prebasal 0.47 ± 0.08 vs. 0.52 ± 0.09; basal 0.35 ± 0.05 vs. 0.98 ± 0.02*; clamp (122‐240 min) 0.06 ± 0.02 vs. 0.77 ± 0.06*. ISR (pmol/min): prebasal 72.7 ± 7.5 vs. 72.0 ± 7.9; second phase clamp (210‐240 min) 268.5 ± 27.2 vs. 200.2 ± 23.7. MCRINS (mL/min): prebasal 3393 ± 488 vs. 3370 ± 511; clamp 2284 ± 505 vs. 1214 ± 153* (*p < 0.05 glycerol vs. intralipid/heparin). This study demonstrates that acute NEFA elevation causes hyperinsulinemia due to a significant decrease in systemic insulin clearance without increasing rates of insulin secretion.

Primary folding dynamics of sperm whale apomyoglobin: core formation.

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.

Quantitative Analysis of 25-OH Vitamin D Using Supported LiquidExtraction and Liquid Chromatography - Mass Spectrometry

We report a low sample volume LC/MS method for 25OH Vitamin D analysis. The method requires only 40 μl serum, is fully automatable, fast and sensitive. The method was successfully implemented for clinical analysis of infants for vitamin D deficiency. The 6 min method has a sensitivity range from 2 to 100 ng/ml. A 3 min method was validated from 5 to 100 ng/ml range. Fast analysis, low sample volume and high sensitivity were achieved by combination of supported liquid extraction sample preparation, UHPLC chromatography and fused-core chromatographic column for separation, and highly sensitive mass spectrometry for detection. We emphasize the importance of sample preparation quality for rugged LC/MS analysis. Using SLE-based sample preparation we successfully used only 40 μl of serum while achieving a LLOQ of 2 ng/ml. We found from assayed samples, that 3-12 month old infants were not Vitamin D deficient, compared to adults. The average level of 25(OH)D3 in infants was 57.3 compared to 38.0 ng/ml for adults in a Bronx, NY patient population.

Flow inconsistency: The evil twin of column switching—Hardware aspects

Solvent flow, generated by HPLC pumps is consistent and accurate. This statement, while true for single column (one dimensional) liquid chromatography applications, may not apply to column switching applications. Connection of pumps and/or columns to one flow path may cause substantial pressure changes. Immediate post valve switch pressure differences between pumps can cause backflow where the mobile phase stored at higher pressure will temporary flow into the lower pressure area. A more common side effect of column switching is flow inconsistency during pump pressurization. For the duration of pump pressurization, liquid flow through the column will be smaller than expected since the HPLC column acts like a flow restrictor.

Human C-peptide Quantitation by LC-MS Isotope-Dilution Assay in Serum or Urine Samples

In this communication we report a simple and efficient approach to C-peptide quantitation using isotope dilution mass-spectrometry analysis. The method facilitates quantitation of C-peptide levels at least one order of magnitude lower compared to concentration levels achieved with an IDA method reported previously. The improvement was due to more intensive sample preparation procedure that, in turn, makes it possible to increase the sample load without a corresponding increase in matrix effects. We also show the results of a comparison study with a second laboratory using a similar previously reported method for C-peptide quantitation.

Absolute measurement of internal volume changes inside the pulse damper: discrepancy between delay and dwell volumes.

We have developed a novel technique for the absolute determination of the mobile phase volume stored inside of a variable volume pulse damper at different pressures. Using an Agilent HPLC pulse damper we found a linear volume increase of approximately 1 microL/bar. We found that pump pressurization is a relatively slow process and takes approximately 1 min to reach 90% and takes approximately 2 min to reach 99% equilibration at flow rates below 1 mL/min. During pump pressurization, column flow rate will be less than the pre-set, since part of the mobile phase is retained inside of the pulse damper. During our experiments we observed a discrepancy between data obtained by UV techniques and direct absolute measurements. This difference can be explained by a fundamental difference between the gradient delay volume and dead (dwell) volume.

Isotope dilution assay in peptide quantification: The challenge of microheterogeneity of internal standard

Isotope dilution analysis allows quantitation of elements and different compounds in complex mixtures. The quantitation is based on a known amount of reference material (internal standard, IS) added to a sample that makes the result critically dependent on the value assigned to the standard. In the case of peptides, IS concentration is determined by nitrogen and amino acid analysis while purity is normally assessed by methods such as chromatography or electrophoresis that might not be able to detect many possible amino acid modifications, either naturally occurring or chemically induced. Microheterogeneity of the IS, if it is not accounted for when assigning a reference value to the standard, results in highly overestimated values in target analyte quantitation. In this viewpoint article, we illustrate the problem of internal standard microheterogeneity by analyzing synthetic human C‐peptide labeled analogs.

Reduction in delay time of high-dwell volume pumps in LC-MS applications using short-term low-ratio split flow.

We present a simple hardware design which reduces run time of gradient-based LC/MS applications and improves system equilibration. Our approach does not sacrifice efficiency of chromatographic separation, and does not affect analyte retention time and therefore does not require revalidation. Our technical design is based on a six-port/two-position switching valve and flow splitter installed prior to the LC column. This design minimizes time delays caused by the high-dwell volume of some LC pumps. Implementation of short-term (40-55 s) low-ratio (1:10) flow splitting reduced delay times by over four-fold in our application. This approach allowed hardware-associated time delays to be minimized. Alternative plumbing suggestions are also discussed.