GLUCOSE AASSAY METHODS By Dr Samson Ojedokun.pptx

Glucose is the most important carbohydrate fuel in the body.

The majority of circulating glucose comes from the diet following meal
However, in the fasting state, gluconeogenesis and glycogenolysis maintain glucose concentrations.

It is a monosaccharide, an hexose and an aldose sugar.

1. METHODOLOGIES OF GLUCOSE ASSAYS Dr S.A Ojedokun Department of Chemical Pathology LTH Ogbomoso, Nigeria

2. Outlines Introduction Type of specimen Specimen collection Assay methods Assay Procedure Factors determining choice of method Advances in Glucose measurement Conclusion References

3. Introduction Glucose is the most important carbohydrate fuel in the body. The majority of circulating glucose comes from the diet following meal However, in the fasting state, gluconeogenesis and glycogenolysis maintain glucose concentrations. It is a monosaccharide, an hexose and an aldose sugar.

4. Glucose monitoring: a key element in diabetes care plan Help assess impacts of : diet, medications, illness, stress & exercise Monitor treatment safety treatment requires constant blood glucose control and monitoring There are so many digital health innovations for diabetes patients

5. Type of Specimens Blood Whole blood Serum Plasma Capillary Cerebrospinal Fluid Urine

6. Timed sample Fasting specimen Post prandial Glucose Tolerance test Random sample

7. Specimen Collection, Storage and Variations Specimen bottles and principle: fluoride oxalate, EDTA, Citrate, Heparin Fasting whole blood sample 10-12% lower than plasma glucose Fasting capillary is 0.1-0.27mmol/L higher than venous blood glucose Post prandial capillary is 1.1-3.8mmol/L (20-25%) higher than venous glucose CSF; analyse immediately or centrifuged and stored at 4 C. 24hrs Urine; preserve with 5mls glacial acetic acid to maintain pH 4-5, then store at 4 C

8. Assay Methods In the past few decades, the majority of the quantitative tests for glucose determination depend on the oxidation of glucose by hot alkaline copper solution o potassium ferricyanide These were replaced by o-toluidine test and then later by enzyme method. Assay methods could be broadly classified into Qualitative, semi-quantitative and quantitative methods.

9. Brief evolution of methods

10. Assay Methods Cont’d Enzymatic methods Reduction Methods Ferriccyanide (Hoffman’s) method Copper sulfate methods Smogi -Nelson method Condensation method: Aromatic Amine Other methods (glucometers)

11. Enzymatic Method 1. Hexokinase Method Glucose + ATP hexokinase ADP + G6P G6P + NAD G6PDHase 6 - Phosphogluconate + NADH + H + Abs of NADPH is measured in the UV region (340nm) This is the reference method and specific for D-glucose because fructose and mannose can react in the first reaction but G6PD is specific exclusively for G6P thus, phosphorylated fructose and mannose do not react.

12. Hexokinase Method cont’d Advantages Disadvantages Interferences Linearity Coefficient of Variation Sensitive, accurate, rapid, covers a wide range of conc. Time consuming, Expensive, Easily affected by interferences Positive interferences from: - Hb >5mg/dL - Bilirubin >85Umol/L - TAG > 55mmol/L - Lipemic sample 27.7mmol/L (600mg/dL) - within run CV = 1.3% - btw run CV = 1.1% - Sensitivity =0.02mmol/L

13. 2 . Glucose Oxidase Glucose + H 2 O + O 2 glucose oxidase Gluconic acid + 2H 2 O 2 H 2 O 2 + O-dianisidine peroxidase H 2 O + Coloured Compound Measured at 505nm Trinder’s method: Spectrophometer or refractometer (film or strip – dry chemistry) e.g POCT, clinistix . In Tinder’s method, hydrogen peroxide react with 4aminophenazone + phenol to give quinonimine and water Polarigraphic method: using O 2 electrode to O 2 utilization

14. Glucose oxidase cont’d Advantages Disadvantages Interferences Limit of linearity Coefficient of Variation Highly specific for Glucose, Suitable for CSF and Urinary glucose - Requires production of different reagents - difficult standardization Negative interferences by inhibition of Peroxidase from: - Bilirubin - Hb - Amino acid - Uric acid - Gluthathione - Ascorbic acid = 27.7mmol/L   - within run CV = 4.8% - btw run = 3.3%

15. 3. Glucose Dehydrogenase Method Glucose + NAD GDH Gluconolactone + NADH+H This is based on the action of glucose dehydrogenase with NAD, the D-glucose and NAD are converted to D-gluconolactone and NADH. The amount of NADH formed is proportional to the concentration of glucose present. The change in NADH absorbance is measured at 340nm.

16. Advantage: Highly specific for glucose; results are close to Hexokinase method; no matrix or anti-coagulants interference. Disadvantage: Time consuming. Not widely use

17. Reduction methods 1. Ferriccyanide (Hoffman’s) method : Utilizes the reductive property of glucose ferricyanide is reduced by glucose. Fe 3+ Fe 2+ Color change from yellow to colorless solution

18. 2. Copper sulfate methods Benedict/ Clinitest : The reagent contains Na-citrate & Na carbonate with CuSO 4 . It gives colour according glucose conc (blue---green----yellow----brown----red). Fehling: using KOH & Na/K tartrate with CuSO 4 Folin-Wu: Alkaline CuSO 4 + Phosphomolybdic acid to molybdenum blue by reducing Cu 2 O CuO 2

19. 3. Somogyi- Nelson Heating of Arseno-molybdate in alkaline medium

20. Reducing method cont’d Demerit: Non-specific Qualitative and Semi-quantitative Merit: Fast. Interferences from other reducing agents

21. Condensation Method Glucose can under go condensation reaction with aromatic compound to give coloured product. Heating Glucose and O- toludine in acidic medium yields a green coloured product, Glucosamine, measured at 630nm The colour is then measured spectrophotometrically to estimate the glucose concentration.

22. Demerit Non-specific O-toludine is carcinogenic Qualitative and Semi-quantitative Merit Fast Interferences from reducing substances: Galactose, Fructose

23. Other methods Invasive Glucose Monitoring Glucometers used for Self-monitoring of Blood glucose (SMBG) utilizes disposable electrochemical cell Enzymatic method Reflectance photometry to measure the amount of light reflected from the test pads or electrochemical systems where the enzymatic reaction in the electrode incorporated on the test strip produces a flow of electrons. The current is directly proportional to the sample glucose concentration is converted to a readout device

24. Advantages Disadvantages Quick TAT Portable device POCT Disposable strips No instrument contamination Minimal amount of blood required Automatic calibration or lot-specific code chip or strip and in-built QC Abort testing when sample is insufficient Storage memory Large variabilities among meters in reaction time 15-120sec Range bond 2.2-22.2mmol/L or 0-33.3mmol/L Lack of technique harmonization due to different products or manufacturers and varied performance among meters Error codes require SOP consult Invalid strip leading to wastage User variability Other variables: haematocrit values, hypotension, hypoxia, lipemic samples, increase blood viscosity eg in DKA lowers bld gluc

25. 2 . Minimally invasive Glucose monitoring Ex. is Glucowatch : Application of low-level current induces movement of glucose by electro-osmosis across skin where it is measured by glucose oxidase detector. Glucose conc in transdermal fluid and plasma are highly correlated. Implanted sensors: The widely adopted method is the electrochemical sensor that uses glucose oxidase implanted IV or subcut

26. 3. Non-invasive Glucose Monitoring Uses near-infrared light spectroscopic devices to measure real-time blood glucose levels either the absorption or reflection of light from subcut tissue. Limited by several interferences. The technique is still widely being researched

27. Briefly on: Glycated Haemoglobin Glucose Tolerance test

28. Glucose Measurement Procedure Plasma/CSF (GOD Method) Reagent preparation: R1 Buffer & R2 Enzymes. stable for one month at 4-8 C Instrument and Wavelength: 505 nm (490-550)nm Pipetting into formatted bottles Incubation: Temperature 37 C / 15-25 C Reading of absorbance Calculation of result

29. Reference intervals

30. WHO Guideline Normal: 70-100mg/dL (3.9-5.6 mmol/L) Impaired fasting: 100-125mg/dL (5.6 to 6.9 mmol/L) Diabetes: >126mg/dL (>7mmol/L) or higher on two separate tests ADA Guideline <100mg/dl (<5.6mmol/L) 100-125mg/dl (5.6 to 6.9 mmol/L) >126mg/dl (>7mmol/L)

31. No sex difference Fasting increases by 2mg/dl (0.1mmmol/L) per decade PPBG increases by 4mg/dL (0.2mmol/L) per decade After glucose challenge, it increases by 8-13mg/dL (0.4-0.7)mmol/L per decade

32. Urine Benedict’s test: 5drops of urine + 10drops of Benedict’s rgt + heat Clinitest : 5drops of urine + 10drops of H 2 O + 1tablet Clinistix : dip stick, examined in 10sec use colour chart

33. Factors Determining choice of Method ACCURACY: Many glucose acid methods are highly accurate providing reliable results for clinical and research purposes SENSITIVITY: Some methods can detect glucose at very low concentration making them suitable for application trace amount of glucose need to be measured. SPECIFICITY: Some urine aren’t specific for glucose only, other substance could give false result SPEED: Certain method offer rapid result which can be crucial in emergency medical situation. AUTOMATION: This is possible in many glucose assay methodologies reducing the human error and increasing throughput VERSATILITY: Different method are suitable for various sample types including blood, urine and other biological fluids

34. COST: Some advanced glucose assay methods can be expensive due to the need for specialize equipment and reagents COMPLEXITY: Certain methods require technical expertise and specialize equipment, making them less accessible for smaller clinics or laboratory INTERFERENCE: Some methods may be prone to interference from other substances in the sample leading to inaccurate result INVASIVENESS: Blood-based glucose assay can be invasive which can be uncomfortable for patient

35. Advances in Glucose Measurement 1977 Glucosemeter

36. Current meters

37. Continuous Glucose Monitoring

38. Types Real-time CGM (rt-CGM) Intermittent scanned-CGM (is-CGM ): “Flash” CGM Professional CGM: clinic-owned device and provides either retrospective or real-time glucose data) similar to a 24-hour cardiac Holter monitor that provides information about cardiac rhythms to detect nocturnal hypoglycemia, dawn phenomenon, postprandial hyperglycemia and to assist in management of diabetes therapies  Personal CGM: patient-owned and provides real-time glucose data.

39. Advantages Up to 288 readings per day No pain Accurate, stable and consistent Glucose trends/arrow systems Alerts and alarms with low and high Remote monitoring AGP-based reports to visualize comprehensive glucose data

40. Conclusion Numerous methods that are non-specific are employed for glucose estimation but the specificity has been greatly improved upon with the introduction of enzymatic methods, this should be given preference.

41. References Walker HK, Hall WD, Hurst JW. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990 Henry’s Clinical Diagnosis And Management by Laboratory Methods Twenty-Second Edition, Preanalysis Pg 24-36. Tietz fundamentals of clinical chemistry, 2008 fifth edition chapter 4 pg 63-80. Bracker , J. Diabetes Technol Ther . 2009.11 (suppl. 1) 525-536

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