This chapter explains glucose regulation in normal individuals and the clinical disorders of type 1 and type 2 diabetes. Clinical conditions covered include hypoglycemia, hyperglycemia, type I insulin-dependent diabetes mellitus, ketosis, ketoacidosis, type 2 diabetes, nephropathy and albuminuria. Immunoassays relevant to the investigation and diagnosis of growth hormone disorders are reviewed: insulin, proinsulins, C-peptide, glycated hemoglobin, glycated albumin, urine albumin, islet cell cytoplasm autoantibodies, endogenous insulin autoantibodies , tyrosine phosphatase autoantibodies (IA- 2A and IA-2βA), glutamic acid decarboxylase autoantibodies (GAD65A), zinc transporter 8 autoantibodies (ZnT8), adipokines, leptin, adiponectin, tumor necrosis factor alpha, resistin, visfatin, retinol-binding protein. For many of the test analytes the biological function is explained, with the clinical applications of the test and its limitations. Typical assay technology is also described. The type of sample and frequency of use are included, with an example reference interval (for background information only). For some analytes, desirable assay performance characteristics are included and comments about standardization if this is an issue. The oral glucose tolerance test is also mentioned.

The advent of highly sensitive chemiluminescence immunoassays capable of measuring C-peptide in the picomolar range has enabled scientists to prove that the majority of people with long duration Type 1 Diabetes have functioning beta cells. Until now, the received wisdom was that that the autoimmune process destroyed all insulin producing beta cells. However new research shows that around three quarters of patients with the condition possess a small number of beta cells that are not only producing low levels of insulin, but also respond to a meal stimulus (Oram et al 2014). The exciting observation suggests these patients have a few remaining beta cells that are either escaping immune destruction, or they are regenerating. Understanding why some people keep some residual insulin production, whilst others lose it, may help answer key questions about the biology of Type 1 diabetes and help advance research towards  new potential therapeautic targets for the disease.”

Immunoassay has been the predominant method for the measurement of both insulin and C-peptide in biological fluids, though more recently liquid chromatography – mass spectrometric methods have been published.  These methods have been technically demanding and not suited to routine laboratory use.  One such method for C-peptide has been listed as a reference measurement method ( http://www.bipm.org/jctlm, Stoyanov AV et al 2011).

However, Van Houcke and colleagues (Van Houcke et al 2013) investigated the use of the all-procedure trimmed mean (APTM) to achieve harmonisation of immunoassay results for insulin.  They based their proof of concept on the analysis of the insulin standardization study data using 11 immunoassays and their ID-MS  method and principal component analysis.  An excellent correlation was found between the APTM and ID-MS (r2=0.9970).  Given that the APTM is a composite of the calibrations of the immunoassay, the authors indicate that it would be necessary to ensure continuity of calibration of the APTM that there are overlapping measurements in the calibration panels.

Analysis of complex biological fluids for insulin may be required for a number of reasons: for diagnostics, forensic investigation, manufacturing and sporting investigations.  For the detailed investigation of the structure of insulin, its prescursors and degradation products mass spectrometric methods may be the method of choice.  For routine diagnostic use the main limitations have been the need for preliminary isolation of the peptide by immunological means.  Improvements in the analytical sensitivity of mass spectrometers has allowed the development of  MS methods where this step is not required and based on detection of the B chain of insulin after reduction and liberation from the intact molecule (Chen et al 2013).  The authors suggest that this approach will be suitable for the high-throughput Clinical Laboratory.

Insulin sensors that are able to measure insulin ‘in an instant’ are a long held goal for the assessment of islet cell function and as part of insulin pumps for in vivo analysis.  The requirements of sensitivity, ‘instant’ results from whole bloods/interstitial fluid obtained continuously are severe.  Whether antibody based sensors will emerge ahead of other technologies remains to be seen, but such technology has been described (Heyduk et al 2010).

Oram, R.A., Jones, A.G., Besser, R.E., Knight, B.A., Shields, B.M., Brown, R.J., Hattersley, A.T., McDonald, T.J. The majority of patients with long-duration type 1 diabetes are insulin microsecretors and have functioning beta cells. Diabetologia 57 (1),187-91 (2014).

Stoyanov, A.V., Rohlfing, C.L., Connolly, S., Roberts, M.L., Nauser, C.L., Little, R.R.  Use of cation exchange chromoatography for human C-peptide isotope dilution-mass spectrometric assay. J. Chromatog. A, 1218, 9244-9249 (2011).

Van Houcke, S.K., Van Aelst, S.V., Van Uytfanghe, K., Thienpont, L.M.  Harmonization of immunoassays to the all-procedure trimmed mean-proof of concept by use of data from the insulin standardization project.  Clin. Chem. Lab. Med. 51, e103-e105 (2013).

Chen, Z., Caulfield, M.P., McPhaul, M.J., Reitz, R.E., Taylor, S.W., Clarke, N.J.  Quantitative insulin analysis using liquid chromatography-tandem mass spectrometry in a high-throughput clinical laboratory. Clin. Chem. 59, 1-8 (2013).

Heyduk, E.,  Moxley, M.M., Salvatori, A., Corbett, J.A., Heyduk, T. Homogeneous Insulin and C-Peptide Sensors for Rapid Assessment of Insulin and C-Peptide Secretion by the Islets Diabetes 59, 2360–65 (2010).

Dr Penny Clark is Honorary Consultant Clinical Scientist at the Queen Elizabeth Hospital Birmingham (University Hospitals of Birmingham NHS Foundation Trust) and Senior Lecturer at the University of Birmingham. Previously, until 2012, she was head of the Regional Endocrine Laboratory at UHB and Director of the Supra Regional Assay Laboratory with a research interest in immunoassay development, diabetes and thyroidology/thyroid cancer.

Tim McDonald is employed 50% of his time as the sole clinical biochemist in Professor Andrew Hattersley’s diabetes research team at the University of Exeter Medical School. Working with the team, Tim gained a PhD investigating the utility of biomarkers for identifying patients with MODY. Tim was a key member of the research team that investigated the possibility of measuring C-peptide in urine rather than in a blood sample, identifying the clinical utility and optimal storage parameters that enable measurement from a posted urine sample. The measurement of urinary C-peptide/Creatinine ratio (UCPCR) is now provided as a routine NHS pathology test to assess endogenous insulin secretion.
Tim is currently the lead scientist coordinating the central laboratory on the DIRECT study – a €40 million EU Innovative Medicines Initiative grant on stratification of treatment in Type 2 diabetes. In 2012 Tim was awarded the Department of Health’s Young Healthcare Scientist of the year award for translational research and was also awarded an NIHR CSO Fellowship.

Diabetes, diabetes mellitus, hypoglycemia, hyperglycemia, type I insulin-dependent diabetes, ketosis, ketoacidosis, glucose, type 2 diabetes, nephropathy, microalbuminuria, albuminuria, proteinuria, insulin, proinsulins, C-peptide, glycated hemoglobin, glycated albumin, urine albumin, islet cell cytoplasm autoantibodies, endogenous insulin autoantibodies , tyrosine phosphatase autoantibodies (IA- 2A and IA-2βA), glutamic acid decarboxylase autoantibodies (GAD65A), zinc transporter 8 autoantibodies (ZnT8), adipokines, leptin, adiponectin, tumor necrosis factor alpha, resistin, visfatin, retinol-binding protein, oral glucose tolerance test.