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We present a clinical case highlighting the diagnostic challenges in a patient with an insulinoma. A 57-year-old lady initially presented with sweating on waking. Investigation documented a fasting blood glucose of 2 mmol/L (3.9-5.4) and the presence of anti-endomysial antibodies. On the basis of a diagnosis of Coeliac disease, the patient was commenced on a gluten free diet with apparent resolution of symptoms for 9 years. Subsequently, she re-presented with recurrent episodes of confusion which resolved with a ‘sugary’ drink. Consequently, she underwent a prolonged supervised fast: glucose 1.9 mmol/L (neuroglycopaenic symptoms), insulin 76 pmol/L (90% of insulinomas and this case underlines the utility of GLP-1R PET/CT in insulinoma localisation.

We investigated biological determinants that would associate with the response to a diet and weight loss programme in impaired glucose regulation (IGR) people using sequential window acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry (MS), a data acquisition method which complement traditional mass spectrometry-based proteomics techniques. Ten women and 10 men with IGR underwent anthropometric measurements and fasting blood tests. SWATH MS was carried out with subsequent immunoassay of specific peptide levels. After a six-month intervention, 40% of participants lost 3% or more in weight, 45% of patients remained within 3% of their starting weight and 15% increased their weight by 3% or more. Hemoglobin A1c (HbA1C) level was reduced with weight loss with improvements in insulin sensitivity. SWATH MS on pre-intervention samples and subsequent principal component analysis identified a cluster of proteins associated with future weight loss, including insulin-like growth factor-II (IGF-II) and Vitamin D binding protein. Individuals who lost 3% in weight had significantly higher baseline IGF-II levels than those who did not lose weight. SWATH MS successfully discriminated between individuals who were more likely to lose weight and potentially improve their sensitivity to insulin. A higher IGF-II baseline was predictive of success with weight reduction, suggesting that biological determinants are important in response to weight loss and exercise regimes. This may permit better targeting of interventions to prevent diabetes in the future.

Insuline, of beter gezegd hypoglycemie, kan op velerlei wijze aanleiding zijn tot juridische actie. Meestal staat dan de rol van insuline niet werkelijk ter discussie, maar in een enkel geval wel. Dat geldt vooral bij het vermoeden dat insuline verkeerd of met misdadige intenties is gebruikt. De grootste verschillen in onderzoek naar hypoglycemie in een klinische versus een forensische setting zijn dat in de forensische setting de voorgeschiedenis meestal niet betrouwbaar is, er geen goede monsters zijn afgenomen voor het onderzoek of dat deze niet correct zijn bewaard of gelabeld, en dat de resultaten van de analyse hoogstwaarschijnlijk zullen worden aangevochten om redenen van specificiteit, accuratesse en vanwege interpretatie van de gegevens. Een immunometrische bepaling (immunoassay) van insuline zal de standaard blijven bij de medische beoordeling van hypoglycemie, maar in de forensische situatie zal waarschijnlijk massaspectrometrie hiervoor in de plaats komen. Met name nu synthetische insulineanaloga de plaats van humane insuline hebben ingenomen bij de behandeling van diabetes.

Insulin or, more appropriately, hypoglycaemia gives rise to a wide variety of interactions with the law. In most cases its role is not seriously open to question occasionally however, it is. This is especially true of situations in which insulin is suspected of having been used inappropriately or maliciously. The major differences between investigation of hypoglycaemia in clinical and forensic situation are that in the latter the history is often unreliable, appropriate samples for analysis were not collected, preserved or labelled correctly and analytical results are likely to be challenged on grounds of specificity, accuracy and interpretation. Immunoassay remains the mainstay of clinical investigation of hypoglycaemia but likely to become displaced by mass-spectrometry in the forensic situation especially now that human insulin is being replaced by synthetic insulin analogues for the treatment of diabetes.

Background: Insulin-like growth factor binding protein-1 (IGFBP-1) modulates the activity of IGF-I. It exists in serum as phosphorylated and less phosphorylated forms. We wished to measure serum levels of both these forms of IGFBP-1, using a novel assay, in subjects with, or without ischaemic heart disease (IHD). Methods: We measured serum concentrations of the phosphorylated and less phosphorylated forms of IGFBP-1 in 75 subjects (36 with and 39 without IHD). Two immunoassays were used, one which detects non-, and less-phosphorylated forms (LpIGFBP-1), and another which specifically detects the serine phosphorylated form of IGFBP-1 (pIGFBP-1). Results: LpIGFBP-1 concentrations were significantly higher in subjects without IHD than in those with IHD (5.3 +/- 0.5 mu g/L vs. 2.7 +/- 0.4 mu g/L, p < 0.001). pIGFBP-1 levels were also significantly higher in subjects without IHD compared to those with IHD (33.3 +/- 2.0 mu g/L vs. 25.3 +/- 2.2 mu g/L, p < 0.01). The correlation between LpIGFBP-1 and pIGFBP-1 for all subjects was (r = 0.71, p < 0.001). This association was stronger in subjects without IHD (r = 0.76, p < 0.001) than for those with IHD (r = 0.60, p < 0.001). A significant negative association was observed between IGF-I and the ratio between the two forms (r = -0.45, p < 0.0001). Receiver-Operating Characteristic (ROC) curve showed the highest area under the curve for LpIGFBP-1 (0.75) [95% CI: 0.63-0.86] and optimum cut-off value of 2.83 mu g/L with 75% sensitivity and 74% specificity. Conclusions: We propose that low serum concentrations of IGFBP-1 forms could be a marker of coronary risk, and the LpIGFBP-1: pIGFBP-1 ratio may be an index of biologically active IGF-I

We report the case of an 84-year-old man who presented with collapse secondary to hypoglycaemia and was found to have a hepatic Solitary Fibrous Tumour (SFT) secreting Insulin-like Growth Factor II (IGF-II). Solitary fibrous tumours of the liver are very rare tumours and have occasionally been described in association with hypoglycaemia. SFT's of other organs have been associated with production of IGF-II but to our knowledge however there are no reports of a SFT of the liver with confirmed IGF-II hypersecretion, and we suggest that this mechanism underlies the hypoglycaemia seen in this case.

Background: Insulin-like growth factors (IGFs) are anabolic proteins that are essential regulators of cell division, differentiation and growth. We describe the longitudinal changes in IGF-I, IGF-II and the binding proteins IGFBP-1, -2 and -3 before and during normal pregnancy. Method: Serum samples were taken before conception and then at 12, 24 and 36 weeks of gestation in 41 healthy women with uncomplicated pregnancies. We measured IGF-I using an automated chemiluminescent method, IGF-II and IGFBP-2 using in-house radioimmunoassays (RIAs), and IGFBP-1 and IGFBP-3 using commercial enzyme-linked immunosorbent assay (ELISA) and RIA kits, respectively. Because of the potential haemodilution effects during pregnancy, albumin was also measured in all samples. Results: There was a significant fall in IGF-I during the first (36%) and second trimesters (21%) followed by an increase of 25% at 36 weeks. During pregnancy, the mean IGF-II concentrations fell by 12% at 12 weeks, 8% at 24 and 8% at 36 weeks compared with pre-conception values. When IGF-II results were adjusted for the haemodilution of pregnancy, its concentrations increased. During pregnancy, there was a rapid increase in mean IGFBP-1 levels by 17-fold (12 weeks), 24-fold (24 weeks) and 25-fold (36 weeks). IGFBP-2 concentrations fell after conception but started to increase towards term. This increase was more significant when adjusted for haemodilution. In contrast, IGFBP-3 concentrations increased significantly throughout pregnancy. Conclusion: Our data on the physiological changes of IGFs and their binding proteins add further evidence of the vital roles of these hormones throughout normal pregnancy.

Highly enriched, bipotent, hematopoietic granulocyte macrophage colony-forming cells (GM-CFC) require cytokines for their survival, proliferation, and development. GM-CFC will form neutrophils in the presence of the cytokines stem cell factor and granulocyte colony-stimulating factor, whereas macrophage colony-stimulating factor leads to macrophage formation. Previously, we have shown that the commitment to the macrophage lineage is associated with lipid hydrolysis and translocation of protein kinase C alpha (PKCalpha) to the nucleus. Here we have transfected freshly prepared GM-CFC with a constitutively activated form of PKCalpha, namely PKAC, in which the regulatory domain has been truncated. Greater than 95% of the transfected cells showed over a twofold increase in PKCalpha expression with the protein being located primarily within the nucleus. The expression of PKAC caused macrophage development even in the presence of stimuli that normally promote only neutrophilic development. Thus, M-CSF-stimulated translocation of PKCalpha to the nucleus is a signal associated with macrophage development in primary mammalian hematopoietic progenitor cells, and this signal can be mimicked by ectopic PKAC, which is also expressed in the nucleus.

Case report A 2-year-old girl presented to the hospital with symptoms of weight loss, thirst and polyuria over the past 2 weeks. Her blood glucose level was high at 30.7 mmol/L and urine showed evidence of glycosuria of 500 mg/dL and ketonuria of ≥160 mg/dL. Blood tests demonstrated an HbA1c level of 94 mmol/mol (reference range 20-43 mmol/mol). She had a positive IA2 antibody level of 184 IU/mL (reference range 0–10 IU/mL). Her anti-GAD antibody level was 1,1 as opposed to beta cell recovery in a newly diagnosed young infant. Second opinion was sought that corroborated our concern 1 Department of Paediatrics, University Hospitals of Leicester NHS Trust, Leicester, UK 2 Royal Surrey County Hospital NHS Foundation Trust, Guildford, Surrey, UK 3 German Sport University Cologne, Center for Preventive Doping Research/Institute of Biochemistry, Cologne, Germany 4 Department of Biochemistry, University Hospitals of Leicester NHS Trust, Leicester, UK

Objective To develop an alternative method to immunoassay for the quantitative analysis of insulin-like growth factor 1 (IGF-1) using a mass spectrometry (MS)-based approach. Study design and patients A stable isotope dilution Ultra High Performance Liquid Chromatography tandem MS (uHPLC-MS/MS)-based method for the quantification of IGF-1 was developed. The method employed Selected Reaction Monitoring (SRM) of two tryptic peptides derived from IGF-1, and utilised solid phase extraction for enrichment of the peptide fraction containing IGF-1 rather than immunocapture, so was less susceptible to assay interference. Plasma samples from 25 consecutive unselected patients with newly diagnosed acromegaly, collected both before and after 24 weeks of primary medical therapy with Lanreotide Autogel(®), were analysed by a widely used commercial immunoassay (Siemens Immulite 2000(®)) and by uHPLC-MS/MS. Results The uHPLC-MS/MS method showed good correlation with the immunoassay over a wide range of IGF-1 concentrations. The Passing and Bablock regression was: uHPLC-MS/MS (nmol/l) = 1.37 (95% confidence interval: 1.26-1.46) × immunoassay (nmol/l) + 3.14 (95% confidence interval: -2.71 to 10.32). Six patients had discordant growth hormone (GH) and IGF-1 levels following primary medical therapy, and in all six the immunoassay and uHPLC-MS/MS platforms returned comparable results. The method was not affected by concentrations of IGFBP3 up to 12,500 ng/ml. Conclusions uHPLC-MS/MS offers an independent method for determining/validating IGF-1 in subjects with acromegaly. Further studies, including the establishment of age- and sex-matched reference ranges and calibration to the new International IGF-1 standard IS 02/254, are now required to allow its introduction in to routine clinical use.

Insulin assays are utilized in various clinical scenarios, including the assessment of insulin therapy compliance or of suspected insulin overdose. In an interpretative exercise carried out by UK National External Quality Assessment Service (NEQAS), serum sent to the participating laboratories was spiked with 30 pmol/L of the short-acting insulin analogue Human Actrapid. Only two out of 24 participant laboratories had sufficient assay cross-reactivity with Actrapid to interpret the results as suggestive of insulin administration. The development of specific insulin assays has led to deterioration in the ability to detect non-compliance or overdose with recombinant insulin treatment. Clinicians should be aware of this significant limitation, which could lead to misdiagnosis.

Summary objective To compare the outcome of different treatment options used in several cases of non-islet cell tumour hypoglycaemia (NICTH). patients Eight cases of NICTH were referred for diagnosis and monitoring following either surgical or medical treatment. methods Serum samples collected throughout the time-course of each case were analysed for glucose, insulin, C-peptide, IGF-I, total IGF-II, total IGF-II to IGF-I ratio and, in most of the cases, big IGF-II. results Surgical excision was successful in the relief of symptoms and normalization of the biochemical parameters. Therapeutic treatment with glucocorticoids confirmed previous studies showing the suppressive effect on tumour (big) IGF-II production. The present data show that the effect was dose-dependent and reversible if doses were below a critical level. conclusions Within the limits of the cases studied, and the time-scales involved, moderate- to high-dose glucocorticoid therapy had immediate beneficial influence on symptomatic hypoglycaemia and, if tolerated in the long term, was effective in correcting the underlying biochemical dysfunction, unlike other therapeutic regimens. This effectiveness was only achieved when the dose exceeded a threshold level specific to the patient. In addition, reduction of the dose or withdrawal of the drug caused a return of the abnormal biochemical profile. Surgical removal of the malignancy, where this was an option, was successful within the periods studied.

The public already has concerns about 'the new genetics', and it is clear that confidence can only be maintained by scrupulous attention to quality. Standards can be improved by harmonization of methods, discouraging poor practice and using appropriate internal and external quality controls. At present, despite the profound implications of genetic test results, few genetic tests are subject to sufficient scrutiny. The Human Genome Project will lead to the identification of numerous genetic variations contributing to multifactorial diseases, and high-throughput technologies will permit the generation of disease-susceptibility profiles. Clinical laboratories will need to develop the wherewithal to handle these data and present them in a format that is clinically useful.