Diagnosing Diabetes Mellitus

Diagnosing Diabetes MellitusIntroductionIn this practical, enzymatic set abouts were apply to determine the glucose concentration of patients in plasma sample distributions. The use of enzymes as a marker for analytical analysis of plasma samples be very reliable due to the high specificity, reproducibility and sensitivity of enzyme commodevasss (glucose HK as assert kit).The first assay used is a glucose oxidase assay, this assay measures the glucose levels in plasma by utilise enzymes such as glucose oxidase to catalyse the oxidation of D-glucose which is present in plasma into D-Gluconic acid. The reaction is coupled to another chemic reaction where a reduced reverberate of o-Diansidine is oxidised to an oxidised form of o-diansidine. This occurs in combination with a formation of hydrogen peroxide. The hydrogen peroxide is consequently broken down and reacts with H2SO4 to form a coloured compound that can be spectrophotometric bothy detected at a wavelength of 540nm.Ano ther popular glucose measuring assay is the hexokinase assay. This assay catalyses the phosphorylation of glucose present in the sample in presence of adenosine triphosphate (ATP) by using hexokinase to form glucose-6-phosphate (G6P). G6P is then oxidised in the presence of nicotinamide adenine dinucleotide (NAD) to form 6-phospho-gluconate, this reaction is catalysed by the presence of glucose-6-phosphate dehydrogenase (G6PDH). While this reaction is undergoing, the NAD is reduced to NADH. This creates a colorimetric signal that can be detected at a wavelength of 340nm by a spectrophotometer.These assays be two of many assays used to measure glucose in patients. They ar commonly used to determine if the patient is diabetic or for routine uses to monitor patients who are already diagnosed with diabetes.Glucose Oxidase assayMaterialsPipette and tipsEppendorf tubesPotassium phosphateGlucose oxidase (1U/l)Peroxidase (0.01U/l)O-Diansidine hydrochloride moved(p) role samples x6 (2 for each patient)Stop solution (Sulphuric acid H2SO4)96 rise platePlate readerMethodFirstly, 3ml of glucose oxidase reagent was prepared by adding 60l of glucose oxidase, 30l of peroxidase and 60l of o-diansidine hydrochloride in 2,85 ml of 0.1 potassium phosphate to create a solution at a pH of 7.5.CalculationsGlucose Oxidase (1U/l) = (1000 U/ml)/(20 U/ml) =50 (3ml)/50=0.06ml =60lPeroxidase (0.01 U/l) = (10 U/ml)/(0.1 U/ml) = 100 (3ml)/100 =0.03ml =30lO-Diansidine hydrochloride = 150 dilution needed 3ml=3000l 3000/50=60lThen, a set of 6 models were prepared by diluting a 0.1M stock glucose solution in peeing as followed hedge 1. Preparation of standards volumesFinal concentration (mM)0.1 stock glucose solution (l)Purified water (l)00200510190102018015301702040 matchless hundred sixty2550150Then, 50l of the samples (provided) and standards were added in duplicate on a 96 intumesce plate as shown under (See Table 2.).Table 2. 96 intimately plate distribution1234567891011A25QC4Pa tient 1T=1Patient 2T=1Patient 3T=125QC4Patient 1T=1Patient 2T=1Patient 3T=1B20QC7Patient1T=2Patient 2T=2Patient 3T=220QC7Patient 1T=2Patient 2T=2Patient 3T=2C15QC1115QC11D1010E55F00Then 100l of the assay reagent prepared earlier was added to each well.After 30 proceeding of incubation at 37C, the reaction was stopped by adding 100l of sulphuric acid to each well. The plate was then read at 540nm on a plate reader.ResultsAfter reading the plate at 540nm on the plate reader, the following results were obtainedTable 3. Sample Absorbance 1234567891011A0.6400.1260.1600.0990.0870.6860.1290.0820.0900.093B0.5450.1930.1410.1390.0870.5670.2040.1760.1130.041C0.4420.2660.4100.3030.104D0.2610.283E0.1930.144F0.0490.048Results highlighted in red were omitted from further calculations due to preparation errors. 200l instead of 100 l of assay reagent was added to well A9, this diluted the signal creating an inaccurate result. The well was re-prepared in well C9 but the time interval between the subs trate and stop solution was unequal to the duplicate in well A3, therefore, the result of well C9 was omitted from further calculations. Finally, the result of well B11 was omitted from further calculations since the assay reagent was not added to the well, therefore there was no chemical reaction (same value as blank).Table 4. normal Absorbance tireds (mM)0.0005.00010.00015.00020.00025.000AbsorbanceDate 25/01/2017 0.0490.1440.2910.4420.5450.640Date 25/01/20170.0480.1930.2830.4100.5670.686Mean0.0490.1690.2870.4260.5560.663Standard deviation (n=2)0.0010.0350.0060.0230.0160.033CV % (n=2)1.45820.5631.9715.3122.7984.906Table 5. Standards mean absorbance recapitulativeStandardsConcentration (mM)Abs100.049250.1693100.2874150.4265200.5566250.663A standard curve can be plotted using the results generated by the wells containing the standards.Figure 1. Standard curve of the Absorbance versus concentrationThe precision of the standard curve can be persistent by calculating the coefficient o f revolution (CV% where CV=standard deviation (SD)/mean) for each standard as shown down the stairsFigure 2. Comparison of the coefficients of variations percentages of the standard duplicates Results are usually rejected if the difference in values is greater than 20% for more than 75% of the calibration standards. In this case, the precision of the curve is pleasing since all the CVs of the points (except from 1) are below 20 % (EMEA or Desilva).The accuracy and the precision can be further analysed by the character controls (QCs) which were ran in duplicate on the plate.Table 6. Quality control resultsAbsorbanceResultsQC (4 mM)QC (7 mM)QC (11 mM)0.1260.1930.2660.1290.2040.303Mean0.1280.1990.285Standard deviation (n=2)0.0020.0080.026CV % (n=2)1.6643.9189.196As shown above, the CVs of the QCs are below 20% importation that the precision of the duplicates is acceptable.By extrapolating the mean absorbance from the QCs, the corresponding concentrations can be determined. QC pe rformances are determined by calculating the precision and accuracy of these. The precision is determined as a CV% (CV%, where CV = SD/mean) and the accuracy as absolute bias (% RE, where congener error RE = measured value actual value/ actual value). If these are between 20% (25% for low QC and high QC) and the target total error (sum of these two values) is below 30% (40% for low QC and high QC), then the results generated from the standard curve are acceptable (De silva EMEA).Table 7. Quality control precision and accuracyQC (4 mg/ml)QC (7 mg/ml)QC (11 mg/ml)Absorbance0.1280.1990.285Concentration3.296.139.57CV % (n=2)2.5955.09210.958Bias %17.858354712.4860813.03319986Total error20.45317.57823.992Following the results shown above, it can be determined that the precision and accuracy of the results are acceptable for each control.Once this has been determined, the results generated from the samples can be analysed reliably.Finally, by extrapolating the absorbance generated from t he samples on the standard curve, a corresponding concentration can be determined as shown belowTable 8. Sample resultsPatient1(t=1)Patient 1 (t=2)Patient 2 (t=1)Patient 2 (t=2)Patient 3 (t=1)Patient 3 (t=2)absorbance0.1300.1590.0950.1260.090.087Concentration (nM)3.374.531.973.211.771.65Hexokinase assay payable to time constraints, the hexokinase assay was not performed. Despite this, some results were provided to determine the glucose concentrations of the 3 patients.ResultsTable 9. Standards, quality controls and samples results providedStandardsGlucose concentration mMAbsorbance00.04850.154100.366150.496200.725250.854Samples and quality controlsSampleAbsorbanceIQC10.162IQC20.260IQC30.393Patient 1 sample 10.269Patient 1 sample 20.411Patient 2 sample 10.196Patient 2 sample 20.303Patient 3 sample 10.206Patient 3 sample 20.236A standard curve can be plotted using the standard results providedFigure 3. Standard curve of absorbance over concentrationBy extrapolating the absorbance from the quality controls and samples, the corresponding concentrations can be determined as shown belowTable 10. Quality control resultsQC (4)QC (7)QC (11)Absorbance0.1620.260.393Concentration (nM)4.277.1611.10Bias %6.684305052.3473040.871558008The bias calculated is within the 20% range meaning that the assay is accurate and the results generated are validated.Finally, by extrapolating the provided absorbance of the samples on the standard curve, a corresponding concentration can be determined.Table 11. Sample resultsPatient1 (t=1)Patient 1 (t=2)Patient 2 (t=1)Patient 2 (t=2)Patient 3 (t=1)Patient 3 (t=2)absorbance0.2690.4110.1960.3030.2060.236Concentration (nM)7.4311.635.278.445.576.45DiscussionThe results obtained by the glucose oxidase assay are very diametrical from the hexokinase assay, even though the accuracy and precision of both assays are acceptable. Meaning that there must have been an error in preparation of the samples, standards or controls of one of these assays. It ha s been predetermined that a preanalytical error was made during the storage of the patient samples prior to their use in the glucose oxidase assay. The samples were thawed and not kept on ice which immobile the samples, this explains the low values for the patient concentrations. Due to the abnormal results obtained in the glucose oxidase assay (t=1 and t=2 decrease in glucose concentrations for patient 3), the results were omitted from further clinical exposition. The results provided in the hexokinase assay seem to be more correct (increase in glucose concentrations from t=1 to t=2). Taking this into account, the results obtained from the hexokinase assay were used for the clinical interpretation of the patients.The paired samples which were ran on the assays were samples collected during a glucose tolerance test. A glucose tolerance test (commonly called GTT or OGTT) is performed on a patient to help diagnose diabetes. It is used to help diagnose disorders such as pre-diabetes, insulin resistance, gestational diabetes (for pregnant women) and reactive hypoglycaemia for example. The test involves the administration of a glucose dose (usually 75g in liquid form) to a fasting patient. Blood is taken before the ingestion of the dose and 2 hours later. The results from the blood glucose levels help determine glucose disorders(ref).GTT is a widely-used tool for diagnosing gestational diabetes. It is offered to women who are 24-28 weeks pregnant and present a risk factors such as an elevated BMI, family history of diabetes or a previous pregnancy of a large baby. Patient 1 is a 36-year- gray-headed female who is 28 weeks pregnant. She was offered a routine GTT following the last pregnancy since the baby was large. Her fasting glucose was 7.43 mmol/L and her blood glucose levels after 2 hours was 11.6mmol/L. In the UK, according to the National Institute for Health and Care Excellence (NICE) guidelines, a fasting glucose greater than 5.6 mmol/L and/or a 2-hour bl ood glucose level greater than 7.8mmol/L for pregnant women is considered diagnostic for gestational diabetes (NICE guidleines). Therefore, the patient in question is diagnosed with gestational diabetes.Patient 2 is a 67 year old female, she was referred for a GTT following the results of her fasting plasma glucose to be 5.6mmol/L at a routine GP check-up. According to the results from the hexokinase assay, her fasting glucose was 5.27mmol/L and her 2-hour plasma glucose levels were 8.44mmol/L. These results sharpen that the patient has an impaired glucose tolerance (fasting glucose less than 7mmol/L and 2-hour plasma level between 7.8 and 11.1mmol/L). This diagnosis is given when the results do not indicate diabetes but are still abnormal. Further check-ups need to be planned to routinely check for diabetes.Patient 3 is a 24 year old male. He is affected by cystic fibrosis which requires annual pancreatic function tests such as GTTs. His fasting blood glucose levels were 5.57mmol/ L and the 2-hour blood glucose levels were 6.45mmol/L following these results it is safe to say that this patient is healthy (fasting plasma levels less than 5.5mmol/L and plasma glucose levels at 2-hours less than 7.8mmol/L).ConclusionBy using an enzyme assay, blood glucose levels from patients can be monitored. These assays are reliable due to the specificity, reproducibility and sensitivity of the enzymes. By measuring the glucose levels of samples before and after the administration of an oral dose of glucose, the results can help diagnose some(prenominal) types of diabetes such as gestational diabetes in pregnant women. There are a variety of available assays that can measure glucose from serum samples, but they all function on the same principle of catalysing a reaction to produce a detectable signal that is correlated to a concentration. The use of internal quality controls is an indispensable way of testing the reliability of the results.