A Stable Isotope Dilution Assay for the In Vivo Determination of Insulin Levels in Humans by Mass Spectrometry

Abstract

Insulin levels in humans were measured by a new assay, the isotope dilution assay (IDA), based on stable isotope dilution mass spectrometry. A known amount of a deuterated analog of insulin was used as an internal standard and added to the serum samples before sample processing. After isolation by immunoaffinity chromatography and solid phase extraction, followed by a purification step on reversed-phase microbore high-performance liquid chromatography (HPLC), the insulin-containing fraction was analyzed by mass spectrometry. The relative intensity of the signals due to insulin and its deuterated analog in the mass spectrum was used to determine the concentration of insulin in the sample. Using serum samples of 0.5-2.0 ml, we were able to measure insulin levels in the range of 3-1700 pmol/l in several clinical samples from type II diabetic patients. The basal level of endogenous insulin was also determined in two normal subjects and found to be approximately 20 pmol/l. Insulin secretion was followed after the ingestion of 75 g glucose in one healthy volunteer. Finally, the determination of the insulin level of one hemolyzed post-mortem blood sample, for which immunoassays gave inconsistent results, was performed to help forensic investigations. Our results showed a good correlation with standard immunoassay data, except in six samples where much lower values were obtained by our stable isotope dilution assay, suggesting an overestimation of insulin levels by immunoassay in some cases. As it is not subject to immunological interferences by insulin-related compounds, this new assay has a major clinical advantage in that it avoids confusions related to hyperinsulinemia.

Sensitive Quantitative Analysis of C-Peptide in Human Plasma by 2-Dimensional Liquid Chromatography–Mass Spectrometry Isotope-Dilution Assay

“…IDAs are used to measure metabolites (17 ), small drug molecules in biological fluids (23,24 ), and large complex molecules such as peptide hormones (6,7,(13)(14)(15)(16)18 ). For reported C-peptide assays, the lowest amount measured was 30 fmoles on column, which was obtained via initial immunoprecipitation/SPE followed by off-line RP chromatography and direct MS detection (7 ); the detection limit was also 30 fmoles for ultrafiltration followed by RP LC/tandem MS (6 ).…”

“…Human-sequence isotope-labeled C-peptides with relative molecular masses (M r ) of 3038 [Mϩ18] and 3050 [Mϩ30] were synthesized via incorporation of multiple fluorenylmethyloxycarbonyl-labeled amino acids containing either 13 C or 15 N labels on an ABI 433A Peptide Synthesizer by solid-phase chemistry. Peptides were purified by preparative reversed-phase (RP) HPLC.…”

“…represent the intact molecule (6,7,(13)(14)(15)(16); it thus offers the possibility of absolute specificity. Previously published LC/MS methods for quantifying C-peptide in urine and plasma had low sensitivity and low sample throughput, and required complicated sample preparation and large sample volumes (6,7 ).…”

Proteome Kinetics: Coupling the Administration of Stable Isotopes with Mass Spectrometry-Based Analyses

“…We should note that in cases where one aims to determine the kinetics of a single protein and/or a select group of proteins it is possible to use custom synthesized standards, e.g. this strategy has been used for measuring insulin concentration Stocklin et al 1997). A related approach would be to use an "isomer dilution" strategy (Thevis et al 2005), e.g.…”

Kinetic Study of Neuropeptide Y (NPY) Proteolysis in Blood and Identification of NPY3–35

“…The assays used the internal standard methodology with stable isotopes NPY (IDA) (23,24) or porcine NPY as internal standard.…”