Glucose methodology in clinical chemistry

Glucose methodology in clinical chemistry, Glucose methodology, glucose oxidase, Gluokinase method, hexokinase method, copper reduction, folin wu method

1. GLUCOSE METHODOLOGY DR. OFONMBUK UMOH RESIDENT DOCTOR, DEPT. OF CHEMICAL PATHOLOGY UNIVERSITY OF UYO TEACHING HOSPITAL

2. OUTLINE INTRODUCTION PRE-ANALYTICAL PROCEDURES OVERVIEW OF METHODS IMPORTANT CONSIDERATIONS REFERENCE INTERVALS CONCLUSION & REFERENCES

3. INTRODUCTION Glucose are aldehyde or ketone derivatives of polyhydroxy alcohols, or compounds that yields this derivatives on hydrolysis. A monosaccharide, it is a simple sugar with the molecular formula C₆H₁₂O₆ that can exist either as a straight chain or in a ring form.

4. Carbohydrate rich foods are partially digested by salivary amylase in the mouth to intermediate dextrins . While in the stomach and small intestine, acids & pancreatic amylase acts on these dextrins , breaking them to glucose, fructose and galactose After the digestion of this carbohydrate rich food, it is then transported from the intestinal mucosa via the portal vein to the liver. …introduction

5. …introduction Glucose is the body’s main energy substrate. It is readily available for utilization by all tissues of the body, temporarily stored in the liver and muscle as glycogen – or converted to amino acids and fat. Other energy substrates can be converted to glucose and its subsidiaries, via intermediary metabolism. Vital organs of the body such as the brain, uses glucose as their main source of energy.

6. Specimen: Blood, Urine, CSF FOR BLOOD TESTS: Laboratory : Plasma/Serum; Self-Monitoring/POCT: Whole Blood Sample bottle: preferably Fluoride oxalate. EDTA an Plain bottle can also be used. Forms of test: Random Blood/Plasma Glucose, Fasting Blood/Plasma Glucose,One Hour or Two Hours Post-prandial Glucose, Glycosylated Haemoglobin . PRE-ANALYTICAL CONSIDERATIONS

7. BLOOD SAMPLE COLLECTION: Fasting plasma sample, preferably after an Overnight fast: 8 – 12 hours fast. Patient should be well rested, to avoid the effect of stress – induced counter-regulatory hormones on the result. Whole blood sample collected into Fluoride – Oxalate bottle and separated as soon as possible after collection. This separated sample can last as long as 3 days at room temperature, or up to 21 days when frozen. …pre-analytical considerations

8. Urine sample: freshly voided urine, collected into a plain bottle, midstream. Cerebrospinal fluid (CSF) sample can also be analyzed for glucose concentration. CSF samples for use are freshly collected via Lumbar puncture from the L4/L5 position of the vertebral column, and analyzed as soon as possible for glucose. …pre-analytical considerations

9. PLEASE NOTE: Whole blood glucose is about 5-10% lower than serum glucose due to high permeability of the erythrocyte plasma membrane for glucose and also because the red blood cell has a lower water content than plasma. Fasting capillary blood glucose is 2-5mg/dl higher than in venous blood and this increases to 20-70mg/dl following a carbohydrate load, due to increased glucose utilization by peripheral tissues. …pre-analytical considerations

10. INDICATIONS FOR GLUCOSE ESTIMATIONS Diagnosis & management of Diabetes Mellitus. Management of Hypoglycemia (as in Pancreatic disease with insulin therapy Monitoring of patients on long term therapy with diabetogenic drugs e.g. Corticosteroids, Thiazide diuretics etc Post – gastrectomy “Dumping syndrome” Screening for inborn errors of metabolism (IBEM). Of all the clinical contexts in which glucose is measured, diagnosis & management of diabetes constitute about 95% of the indication for measuring body glucose concentration. …pre-analytical considerations

11. OVERVIEW OF METHODS CHEMICAL METHODS A) OXIDATION-REDUCTION REACTIONS ALKALINE COPPER REDUCTION: FOLIN-WU, BENEDICT RXN, NELSON-SOMOGYI, NEOCUPROINE, SHAEFFER-HARTMANN-SOMOGYI ALKALINE FERRICYANIDE REDUCTION: HAGEDORN – JENSEN B) CONDENSATION METHOD: ORTHO-TOLUIDINE METHOD, ANTHRONE (PHENOL) METHOD ENZYMATIC METHODS HEXOKINASE METHOD GLUCOSE – OXIDASE METHOD GLUCOSE – DEHYDROGENASE METHOD

12. CHEMICAL METHODS Basically obsolete methods, no longer in use as routine and more convenient methods have emerged. A). OXIDATION – REDUCTION REACTION 1. ALKALINE COPPER REDUCTION METHODS Folin Wu: Glucose + Alkaline Copper Tartrate ===  Cuprous oxide (Cu²⁺) Cu²⁺ + Phosphomolybdic acid → Phosphomolybednum oxide (blue end product ). Proteins are removed using tungstic acid. Benedict's method : Modification of Folin -Wu method for qualitative urine glucose.

13. CHEMICAL METHODS - Oxidation-Reduction reactionS - Alkaline copper reduction contD Nelson- Somogyi method : an improvement that used barium hydroxide and zinc sulfate to precipitate the proteins. This combination also removed most of the non-sugar " saccharoids ", though ascorbic acid and some uric acid remained. Cu² ⁺ + Arsenomolybdic acid→ Arsenomolybednum oxide (blue end product)

14. Neocuproine method : Cu² ⁺ + Neocuproine →Cu² ⁺ Neocuproine complex (Yellow-orange color neocuproine ) Shaeffer -Hartmann- Somogyi : Uses the principle of Iodine reaction with cuprous byproduct and excess I ₂ is then titrated with thiosulphate . CHEMICAL METHODS - Oxidation-Reduction reactionS - Alkaline copper reduction contD

15. 2). ALKALINE FERRICYANIDE REDUCTION METHOD Hagedorn -Jensen: Glucose + Alkaline Ferricyanide → Ferrocyanide ( Colourless end product); other reducing substances interfere with reaction. CHEMICAL METHODS - Oxidation-Reduction reactionS - Alkaline ferricyanide reduction

16. B). CONDENSATION REACTION ORTHO-TOLUIDINE METHOD: 6% o-Toluidine in glacial acetic acid is used to determine glucose in a sample after deprotein-ization with heated trichloroacetic acid 3% (w/v). This forms glycosylamine and a schiff's base which is emerald green in colour . The absorbance is determined at 630 nm. CHEMICAL METHODS - CONDENSATION reactionS -

17. The reaction follows Beer’s Law over a wide range of concentrations. This method has high specificity for glucose, but this chemical can also react with aldoses (glucose, galactose, mannose, also with ascorbic acid) The reagent used is toxic; a big problem is that the acid destroys the tubing on the machines. This procedure is still used, occasionally. CHEMICAL METHODS - CONDENSATION reactionS -….. contd

18. The enzymatic methods These are currently used methods. They are packaged into day –t0 – day laboratory procedures and Point of Care Test kits. These reactions produce an electrical current that is proportional to the initial glucose concentration or a product that when measured spectrophotometrically is proportional to the initial glucose concentration.

19. Three enzyme systems are currently used to measure glucose: glucose oxidase, hexokinase and glucose dehydrogenase. The College of American Pathologists (CAP) control studies determines that all the enzymatic methods exhibit a coefficient of variation (C.V) less than 5% for glucose values on lyophilised serum, with automated methods having CVs less than or equal to 2.6%.

20. Uses two coupled enzyme reactions. The initial reaction is the specific one, while the second is a dye-indicator reaction, and is non-specific. In the initial reaction, the enzyme glucose oxidase, catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide (H2O2). Enzymatic methods THE glucose oxidASE METHOD

21. The hydrogen peroxide from the GOx reaction is consumed by a peroxidase-dye indicator reaction, to be monitored spectrophoto -metrically at 500 nm and is proportional to the glucose concentration in the specimen. The indicator reaction varies in different methods. Enzymatic methods -THE glucose oxidASE METHOD

22. 1). Saifer – Gerstenfeld method: the classical glucose oxidase indicator reaction uses the oxidation of o- Dianisidine by Hyrodgen peroxide. H 2 O 2 + o- Dianisidine → oxidized o- Dianisidine + H 2 O Enzymatic methods -THE glucose oxidASE METHOD

23. 2). Trinder’s Reaction: commercially available reagents uses Phenol and 4 – aminophenazone to react with the hydrogen peroxide, under the catalysis of peroxidase, forming a red – violet quinoneimine dye as indicator. Enzymatic methods -THE glucose oxidASE METHOD

24. Notably, glucose oxidase is highly specific for beta-D-glucose. But glucose in solution exists 36% in the alpha-D-forms and 64% in the beta-D-forms. Hence, commercial preparations of glucose oxidase reagents contain an enzyme – mutarotase , that accelerates the conversion of alpha -D, to beta-D-glucose. Otherwise, extended incubation allows spontaneous conversion. Enzymatic methods -THE glucose oxidASE METHOD

25. The second step reaction, involving peroxidase, is much less specific than the glucose oxidase reaction. Notable interferences to this reaction include: uric acid, ascorbic acid, bilirubin, haemoglobin , tetracycline, glutathione inhibits the reaction by competing with the chromogen for H2O2, thereby producing lesser values. Incorporation of potassium ferrocyanide or Somogyi filtrate, eliminates this 2nd reaction interferences. Enzymatic methods -THE glucose oxidASE METHOD

26. In our facility: The RANDOX GLUC-PAP Reagent For Manual procedure Enzymatic methods -THE glucose oxidASE METHOD R1a – Buffer: Phosphate buffer - 0.1 mol /L, pH 7.0 Phenol - - 11 mmol /L R1b – GOD-PAP Reagent 4-aminophenazone - 0.77 mmol /L Glucose oxidase - - 1.5 kU /L Peroxidase - - 1.5 kU /L CAL - Standard - - - 5.55 mmol /L

27. Enzymatic methods -THE glucose oxidASE METHOD Glucose oxidase technique in our facility:

28. Calculations Result = Abs (Sample) x Standard Concentration Abs (Standard) Limit of Linearity : 28mmmol/L (500mg/dl). Above this value dilute the specimen with saline solution and re-assay taking into account dilution factor. Enzymatic methods -THE glucose oxidASE METHOD

29. Enzymatic methods THE HEXOKINASE METHOD In the presence of adenosine triphosphate (ATP), glucose is phosphorylated by hexokinase to glucose 6 phosphate. This is then oxidized to 6-phosphogluconate by a second enzyme glucose-6-phosphate dehydrogenase.

30. Enzymatic methods -THE HEXOKINASE METHOD cont. The enzyme G6PD converts glucose-6-phosphate into 6-phosphogluconate, with nicotinamide adenine dinucleotide (NAD + ) concurrently reduced to NADH. Every micromole of glucose consumed produces one micromole of NADH. Absorbing light at 340 nm, NADH can be identified via spectrophotometric detection of increased absorbance.

31. A reference method based on this principle has been developed, where serum or plasma is deproteinated by adding solutions of barium hydroxide [Ba(OH2)] and zinc sulphate (ZnSO4). The clear supernatant is mixed with a reagent containing ATP, NAD, Hexokinase and G6PD, incubated at 25 degrees celcius until the reaction is complete, and NADH is measured. Using a specimen blank to correct for interfering substances, the absorbance is read at 340nm Enzymatic methods -THE HEXOKINASE METHOD cont.

32. Enzymatic methods THE GLUCOSE DEHYDROGENASE METHOD Glucose dehydrogenase enables the oxidation of glucose to glucono -lactone during which NAD is reduced NADH. The NADH formed reacts with tetrazolium salt MTT, to form a blue colored compound formazan.

33. The second step reaction is catalyzed by a second enzyme, diaphorase . The production of formazan, which is proportional to the glucose concentration of the sample, is monitored spectrophotometrically at wavelengths 660 and 840 nm. Glucose measuring systems based on glucose dehydrogenase are fewer in number than those based on glucose oxidase or hexokinase Enzymatic methods -glucose dehydrogenASE METHOD

34. The enzyme GLUCOSE DEHYDROGENASE used for this assay is usually isolated from Bacillus cereus. The reaction appears to be highly specific to beta – D – glucose. Hence, mutarotase is usually added to shorten the time necessary to reach equilibrium. The amount of NADH generated is proportional to the glucose concentration. The reaction provides results in close agreement with the hexokinase procedures, and shows no interference from common anticoagulants and substances normally found in serum or plasma. Enzymatic methods -glucose dehydrogenASE METHOD

35. MEASUREMENT OF GLUCOSE IN URINE AND CEREBROSPINAL FLUID ( csf ) Examination of Urine for glucose is rapid, inexpensive, non-invasive and can be used to screen large numbers of samples. Freshly voided urine sample is needed. CSF analysis requires freshly collected sample at the L4/5 below the cauda equina of the spinal cord. It is worthy of note that this tests detects sugars and other substances that reduce Copper, producing a colour , such as: Glucose, Fructose, Lactose, Galactose, Maltose, KB, Arabinose, Xylose, Ribose, Glucuronic acid, Salicylates, Uric acid and Ascorbic acid…and as such, are non-specific.

36. SEMI-QUANTITATIVE METHODS: This uses commercially available paper strip impregnated with glucose-specific enzymes in a chromogenic assay. In the measurement of glucose in urine, the strips use are impregnated with glucose oxidase, peroxidase and the dye; o- toludine dye or tetramethylbenzidine (TMB). The test end of the strip is moistened with freshly voided urine and examined after 10seconds. the blue colour develops if glucose is present at a concentration of 100mg/dl or greater. Results are read by comparing the test with a standard colour chart. Semi-quantitative quantitative & Qualitative methods…Urine & CSF Glucose analysis

37. False positives may be produced by the presence of a strong oxidizing agent. Also, exposure of dipsticks to air gives false-positive readings after 7 days. False-negative results may occur with large quantities of reducing substances such as ketones, ascorbic acid, and salicylates. This method lacks sensitivity and specificity and provides no information about blood glucose concentration below the renal threshold (10mmol/L). Semi-quantitative quantitative & Qualitative methods…Urine & CSF Glucose analysis

38. QUANTITATIVE METHODS This uses the Hexokinase, Glucose oxidase and Glucose dehydrogenase methods. It can be applied to the measurement of glucose in urine and CSF samples. QUALITATIVE METHODS Measures Reducing Substances in urine. An example - CLINITEST: a tablet containing Anhydrous Cupric Sulphate +Sodium Hydroxide+ Citric Acid +Sodium bicarbonate. Semi-quantitative quantitative & Qualitative methods…Urine & CSF Glucose analysis

39. 0.25ml urine + 0.5ml of water + one tablet. Allowed to stand for 15 seconds. Mixed and observed for colour . Product: Yellow Cuprous Hydroxide or Red Cuprous oxide. Higher occurrence of false positives. Due to other reducing substances. Semi-quantitative quantitative & Qualitative methods…Urine & CSF Glucose analysis

40. Important considerations Venous plasma - the reference sample Plasma, specifically venous plasma is the sample that affords greatest accuracy and convenience, and the one recommended for diagnosis of diabetes Venous blood is more convenient and safer to collect than arterial blood, and is less prone to sampling error than capillary blood sampling.

41. …Important considerations Because plasma and serum glucose concentration are unaffected by the confounding effect of hematocrit, they are a more accurate measure of circulating glucose than whole blood glucose concentration. Compared with serum glucose, plasma glucose is considered less likely, in the context of routine clinical practice, to be affected by the potential inaccuracy associated with in vitro glycolysis.

42. It is recommended that if whole blood glucose concentration is measured, results should be converted to ‘equivalent plasma values’ using a constant factor 1.11, i.e. Plasma equivalent glucose ( mmol /L or mg/ dL ) = whole blood glucose ( mmol /L or mg/ dL ) x 1.11. This factor is the theoretical ratio of plasma glucose concentration to whole blood glucose concentration derived from blood with normal hematocrit (43 %) and assumes, that water occupies 93 % of plasma volume and 71 % of red cell volume. …Important considerations

43. REFERENCE INTERVALS Adult Fasting Plasma Glucose Random Plasma Glucose Two hours Post-prandial Cerebrospinal fluid Gluc . 24hours Urine Glucose 3.5-5.5mmol/L 3.6-7.8mmol/L <7.8mmol/L (2.5-4.5mmol/L) - 60% of plasma value (0.1-0.8mmol/L Children : Fasting Plasma Glucose 3.5-5.5mmol/L Term Neonates : Fasting Plasma Glucose 1.7-3.3mmol/L Premature Neonates : Fasting plasma glucose1-1-3-3mmol/L

44. conclusion Circulating glucose concentration can be assessed using a range of samples and variable technology. For the most accurate interpretation of individual patient results, due consideration in specimen collection, handling and preservation techniques, should be given, for proper diagnosis, management and monitoring of patients.

45. REFERENCES American Diabetes Association (ADA). Diagnosis and Classification of Diabetes. Diabetes Care 2005; 28: 28: S37-42. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics; fifth Ed., by Burtis et al. Clinical Chemistry; Principles, Techniques and Correlations, 7th Ed., by Bishop et al. HENRY’S Clinical Diagnosis & Management by Lab. Methods 21/e

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