Simultaneous monitoring of multiple hormones from human islets of Langerhans using solid-phase extraction-mass spectrometry.

Islets of Langerhans release peptide hormones in controlled amounts and patterns to ensure proper maintenance of blood glucose levels. The overall release of the hormones is shaped by external factors and by autocrine and paracrine interactions occurring within the islets. To better understand what controls the secretion of islet-secreted peptides, and how these processes go awry in diabetes, methods to monitor the release of multiple hormones simultaneously are needed. While antibody-based assays are typically used, they are most often applied to quantification of a single hormone. Mass spectrometry (MS), on the other hand, is well suited for quantifying multiple hormones simultaneously but typically requires time-consuming separation steps with biological samples. In this report, response surface methodology was used to identify a set of optimal solid-phase extraction (SPE) conditions for the islet-secreted peptides, insulin, C-peptide, glucagon, and somatostatin. The optimized SPE method was used with multiple reaction monitoring and isotopically labeled standards to quantify secretion levels. Calibrations were linear from 0.5 to 50 nM with < 15% RSD peak area ratios. A microfluidic system was used to perfuse 30 human islets with different glucose conditions, and fractions were collected every 2 min for SPE-MS analysis. Results showed the release dynamics of the individual peptides, as well as patterns, such as positively and negatively correlated release and oscillations. This rapid SPE-MS method is expected to be useful for examining other peptide and small-molecule secretions from islets and could be applied to a number of other biological systems for investigating cellular communication.

Small molecules released from islets of Langerhans determined by liquid chromatography - mass spectrometry.

Islets of Langerhans are the endocrine tissue within the pancreas that secrete hormones for maintenance of blood glucose homeostasis. A variety of small molecules including classical neurotransmitters are also released from islets. While the roles of most of these small molecules are unknown, some have been hypothesized to play a critical role in islet physiology. To better understand their role on islet function, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify 39 small molecules released from islets. Benzoyl chloride derivatization of analyte molecules was used to impart retention and facilitate electrospray ionization efficiency. Separation was achieved on a 2.1 × 150 mm column packed with 2.7 µm core-shell C18 particles. Calibration curves showed excellent linearity between the concentration and analyte response, with relative standard deviations of the analyte responses below 15% and limits of detection from 0.01-40 nM. The method was applied to examine small molecules released from murine and human islets of Langerhans after static incubation and perfusion with glucose. Results showed a decrease in secretion rates with increasing glucose concentration for most of the analytes. Secretion rates were found to be higher in human islets compared to their murine counterpart. This method will be useful in understanding the roles of small molecules in biological systems.

A microfluidic system for monitoring glucagon secretion from human pancreatic islets of Langerhans.

Glucagon is a 29-amino acid peptide released from α-cells within pancreatic islets of Langerhans to help raise blood glucose levels. While a plethora of methodologies have been developed for quantitative measurement of insulin released from islets, such methods are not well developed for glucagon despite its importance in blood sugar regulation. In this work, a simple yet robust microfluidic device was developed for holding human pancreatic islets and perfuse them with glucose. The perfusate was collected into 2 min fractions and glucagon quantified using a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay. Simulation of fluid flow within the microfluidic device indicated the device produced low amounts of shear stress on islets, and characterization of the flow with standard glucagon solutions revealed response times within 2 fractions (<4 min). Results with human islets from multiple donors demonstrated either a "burst" of glucagon or a "sustained" glucagon release across the entire period of stimulation. The simplicity, yet robustness, of the device and method is expected to appeal to a number of researchers examining pancreatic islet physiology.

Rapid liquid chromatography-mass spectrometry quantitation of glucose-regulating hormones from human islets of Langerhans.

Glucose homeostasis is maintained through the secretion of peptide hormones, such as insulin, somatostatin, and glucagon, from islets of Langerhans, clusters of endocrine cells found in the pancreas. This report describes an LC-MS method using multiple reaction monitoring for quantitation of insulin, C-peptide, glucagon, and somatostatin secretion from human islet populations. For rapid analysis, a 5 min separation was achieved using a 2.1 × 30 mm (i.d. x length) C18 column with 2.7 µm diameter core shell particles. A sacrificial protein hydrolysate was used with the sample and found to improve signal magnitude, repeatability, and to reduce carryover between runs. At optimized gradient conditions, the gradient run time was 4.55 min producing an average peak width of 0.3 min, a minimum resolution of 1.2, and a peak capacity of 20. As a proof of concept, the method was used to measure secretions from static incubations of human islets from 2 donors. Insulin and C-peptide were quantified and matched well with literature values of these hormones. We expect that this antibody-free quantitation of multiple hormones secreted from islets will provide insights into the temporal relationships of these peptides in the future.