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A little refresher: MIMS and MODA Data handling Christopher Cheng, Nikhil Sing & Eamonn GilmoreA few notes • Do NOT panic or worry about these questions • You are NOT expected to understand what is going on at this stage • These questions are taken from an end of year exam paper • We only want to seeow you would approachthese questions and the data presented in them2018 practical section II, q I Explanation of questions and answers Question 1 total 56 marks • A man collapsed suddenly at home. The ambulance crew found him to be shaking, confused and disorientated. A test for blood alcohol was negative. He made a rapid recovery after ingesting a glucose-containing soft drink and eating a chocolate bar. • 1(a) Suggest a likely diagnosis. What initial test should the ambulance crew have carried out to assist with the diagnosis? • 1b) Are there any aspects of his case history that is unexpected? • On follow-up investigation, the man admitted to a history of light-headedness and “feeling queasy” that typically occurred several hours after eating a meal. Tests for insulinoma were negative. • 1(c) Why were tests for insulinoma carried out? Question 1 • carried out to assist with the diagnosis?tial test should the ambulance crew have • There appears to be an excess of insulin for some reason • Maybe he is T1D who has taken too much insulin • Ambulance crew should test his blood glucose level as hypoglycaemia suspected • 3 marks • 1b) Are there any aspects of his history that are unexpected? • had low blood glucose levels. medications and taken a pasta meal so should not have • 3marks • 1(c) Why were tests for insulinoma carried out? • This could explain if there is too much insulin causing the hypoglycaemia • 3 marksData Try to summarise the data in these plots1(d) Suggest a suitable assay that could be used to measure total insulin concentration. Illustrate how the assay is conducted using a diagram. IGNORE! • 1(e) What is insulin C-peptide, and why was it measured? • Ignore! • 1(f) From the data in Figure 1, comment on the patient's blood glucose and insulin levels. Can the data explain the patient’s symptoms?• 1(f) From the data in Figure 1, comment on the patient's blood glucose and insulin levels. Can the data explain the patient’s symptoms? . • Blood glucose rises transiently due to glucose uptake • This stimulates insulin secretion • Total insulin profile declines more slowly for patient; C peptide decline are similar for normal and patient • C peptide may be removed slightly faster maybe a different mechanism • The persistence of insulin does explain the excessive decline in blood glucose and hypoglycaemia • 4 marksGel filtration dataCalibration data • Logarithmic graph • The y axis increases by factors of 10, not by a fixed amount • Suggests an exponential relationship • i.e. for every increase in “fraction number” there is a huge increase in the molecular weight1h using the data estimate the size of insulin in both normal and patient • The fractions are 55 and 225 • This correlates to 5 kDa for the normal • Patient 5kDa and 150 kDa • 3 marks1(j) Comment on these values in comparison to the results obtained with serum taken before breakfast. Data for before breakfast1(i) How do the results from this assay explain the gel filtration experiments? • This shows that there is human anti insulin IgGpresent in patients serum • This can explain gel filtration profile • IgG has a Mr of about 150kDa • 3 marks1(j) Comment on these values in comparison to the results obtained with serum taken before breakfast. • Apparently less insulin present after breakfast compared to before • Incompatible with figure 1.1 • Insulin would be expected to be higher after breakfast • Most likely explanation is that insulin in the serum is now binding serum IgG • This prevents it binding to the insulin on the plate • Hence a reduction in apparent binding ( an artefact of the assay) • 5 marksFigure 1.4 western blot data phosphorylated and total insulin receptor samples 1 no addition; 2 insulin 5 nM; 3 insulin 5 nM + patient serum ;4 insulin (5 nM + healthy individual serum 1(k) What is the significance of insulin receptor phosphorylation in this experiment?1(k) What is the significance of insulin receptor phosphorylation in this experiment? • It indicates insulin activated receptor • Leading to insulin stimulation of glucose uptake • Glycogen synthesis • 2 marksFigure 1.5 Western blot detection of Glut4 in plasma membrane fraction and total cell extract. Samples are as follows: 1 - No addition; 2 - Insulin (1.5 nM); 3 - insulin (1.5 nM) + patient serum; 4 -insulin (1.5 nM) + healthy individual’s serum.• 1(m) Comment on the results in Figures 1.4 and 1.5. Can these results alone explain the differing blood glucose profiles shown in Figure 1.1? • Following insulin treatment as expected the insulin receptor becomes phosphorylated and Glut 4 moves to the plasma membrane • Normal serum has no effect on this • Patient serum has a slight enhancing effect • Anti IgG in the patient serum does not inhibit insulin action even though it binds insulin • The reduction in blood glucose seen in patient post meal is likely to be associated with more persistent insulin levels there may be other factors • Too • 5 marks