Glucose estimation

Glucose estimation

1. Estimation of glucose Dr. Azad Alam Siddiqui Assistant Professor Department of Vocational studies (MMDT) KMGGPGC, Badalpur, GB Nagar, UP

2. Objectives T o k n ow the di f fer e nt met h o d s for estim a tion of blo o d glu c ose T o k n ow the pr e c a utions n e e d ed to get ac c ur a te res u lts and better inter p retation of gly c emic status in relati o n to dise a se co n dition.

3. Introduction Glucose is a simple sugar which is a permanent and immediate primary source of energy to all of the cells in our body. Body tries to maintain a constant supply of glucose for your cells by maintaining a constant blood glucose concentration. The concentration of glucose in blood, expressed in mg/dl , is defined by the term glycaemia . Blood sugar levels are regulated by the hormones insulin and glucagon which act antagonistically. These two hormones are secreted by the islet cells of the pancreas. Essentially blood glucose levels determine the time of secretion of these hormones.  The blood glucose level is easily changed under the influence of some external and internal factors such as body composition, age, physical activity and sex. Diabetes is a disease related by the abnormal metabolism of blood sugar and defective insulin production. So blood sugar levels are an important parameter for the study of diabetes. The level of glucose circulating in blood at a given time is called as blood glucose level . The blood glucose level varies at different time on various part of the day .

4. Introduction cont …. Hypoglycaemia is a possible side effect of diabetes medications in which blood glucose level drops below 70mg/dl . Hypoglycaemia is less frequently observed, but is found in conditions such as insulinoma, hypopituitarism, neoplasms, or insulin-induced hypoglycaemia. In people with diabetes, the body doesn't produce enough insulin or respond to insulin properly. The result is that sugar builds up in the blood stream, damaging the body's organs, blood vessels and nerves. This condition in which too much sugar in the blood stream is called hyperglycaemia . The most frequent cause of hyperglycaemia is diabetes mellitus. Some other factors that contribute to elevated blood glucose are pancreatitis, pituitary or thyroid dysfunction, renal failure, and liver disease. Arterial blood has more glucose than venous blood (because the tissues have typically used some glucose).

5. Entry of glucose into the cell Two specific transport system are used : 1. Insulin –independent transport system: • Carrier mediated uptake of glucose • Not dependent on insulin. • Present in hepatocytes, erythrocytes & brain. 2. Insulin dependent transport system : • Present in Skeletal muscle.

6. For glucose estimation from any material, blood is collect in fluoride containing vial. Fluoride inhibit glycolysis by inhibiting enolase enzyme. In CSF, bacteria & other cells are also present so analysed immediately. For glucose estimation from urine, add 5ml glacial acetic acid as preservative to inhibit bacterial growth.

7. The glucose can be measured in whole blood or separated blood (plasma/serum). Whole blood has a lower glucose level because the red cells don't have much glucose in them. If the serum / plasma is not separated from the cells immediately, the blood cells (which stay alive for many hours in vitro) will use up some of the glucose and usually generate lactate. The other ways to stop the glucose falling in a plasma or serum sample are putting the sample on ice or adding a glycolysis inhibitor such as fluoride or citric acid, or both. The term blood sugar often includes, besides glucose, other hexoses as well as other reducing substances present in the blood. There are two methods to estimate blood glucose: Glucose oxidase method Methods depends on reducing property of glucose

8. Function E N E R G Y SOU R CE G l u c o s e i s a ubiq u i t ou s fuel i n b i o lo g y , p r o v i d i ng a pp r o xi m a t e l y 3 . 7 5 k iloca lories ( 16 k ilojo ules) o f f oo d ene r g y p er g r a m . B r ea k d o wn of ca r bo h y d r a t es (e . g . s t a r ch ) yi elds m o n o - an d d i s a c charide s , mos t o f w hich i s g l u c o s e. G l u c o s e i s a prima r y s ou rc e o f ene r g y fo r the b r ai n , an d h e n c e i t s a v aila b ilit y in f l u en c es ps y chologica l p r o c e s s e s . When g l u c o s e i s l o w , ps y chologica l p r o c e s s es r eq u irin g me n t al e f f o r t (e . g ., s el f- c o n t r ol, e f f o rtf ul deci s io n- m a k in g ) a r e i m p ai r e d . N O RMA L V A L UES Ca t e g o r y of a person F asti n g V a l u e P o s t P r andial Mini m um V alue M ax i mu m V alue V alue 2 hours a f t er c o ns u m i ng g l u c o s e N or m al 70 100 L e s s than 140 E a rl y D iabe t es 101 1 40 140 t o 200 E s tab l ished D iabe t es M o r e than 1 40 - M o r e than 200

9. 1. Glucose Hexokinase method The enzyme hexokinase (HK) catalyses the reaction between glucose and adenosine triphosphate (ATP) to form glucose-6-phosphate (G-6-P) and adenosine diphosphate (ADP). In the presence of nicotinamide adenine dinucleotide (NAD), G-6-P is oxidized by the enzyme glucose-6-phosphate dehydrogenase (G-6- PD) to 6-phosphogluconate and reduced nicotinamide adenine dinucleotide (NADH). The increase in NADH concentration is directly proportional to the glucose concentration and can be measured spectrophotometrically at 340 nm . Glu c ose + A TP + H K  ADP + G 6 P G6P + NAD + G6PD  6 P -glu c onolacto n e + NAD H +H

10. 2. Alkaline copper reduction method ( Folin and Wu method) This method employed for the estimation of blood glucose because of the ability of glucose to reduce alkaline copper solutions. Reagents containing phosphomolybdic acid are frequently used to form a blue complex by combination with the reduced copper. When glucose or other reducing agents are treated with alkaline copper solution they reduce the copper with the result insoluble cuprous oxide is formed. The reaction depends on temperature, duration of heating, degree of alkalinity. The cuprous oxide form is allowed to react with phosphomolybdate to form molybdenum blue coloured complex which can be read calorimetrically using red filter on at 680nm .

11. 3. Oxidase Peroxidase method Glucose oxidase is an enzyme extracted from the growth medium of Aspergillus niger . Glucose oxidase catalyse the oxidation of β D- glucose present in the plasma to D glucono -1,5 - lactone with the formation of hydrogen peroxide; the lactone is then slowly hydrolysed to D-gluconic acid. (Glucose oxidase catalyses the aerobic dehydrogenation of β -D-glucose, thus forming gluconic acid and hydrogen peroxide.) The hydrogen peroxide produced is then broken down to oxygen and water by a peroxidase enzyme . Oxygen then react with an oxygen acceptor such as o-toluidine or 4-aminoantipyrine which itself converted to a coloured compound, the amount of which can be measured colorimetrically . Glucose Oxidase Glucose + O 2 +H 2 O Gluconic acid +H 2 O 2 Peroxidase H 2 O 2 + Phenol + 4-aminoantipyrine Quinoneimine +H 2 O

12. Re q ui r ement s : *Samples: B l o o d sampl e s - - W hole blood - - Serum - - Plas m a ( w i t h C a . o xala t es / Na F ), w h i c h is the p r eferred sa m ple Ins t rumen t at i on: P h oto m e t er ad j ust e d on wa v e l en g th 5 05 nm Cu v e t te (light path) 1 cm W a t er bath at 37 ºC A u to m a t ic pipe t t e s, dis p osable t e st tubes , racks and dis p osa b le t i ps f o r the dis p enser s .

13. Protocol Method The test tubes were marked T1 to T6, for those that had to be inoculated with glucose. They were arranged in order of concentration on the rack. Inoculation commenced by transferring glucose into different test tubes T1 being the least concentrated. 0.5mM of Glucose was transferred using a pipette. 0.2ml was inoculated into T2, 0.4 ml into T3, 0.6 ml into T4, 0.8ml into T5 and 1.0 ml into T6. Distilled water was then inoculated into the test tubes using a different pipette to avoid cross contamination. 1ml was inoculated into T1, 0.8 ml into T2, 0.6 ml into T3, 0.4 ml into T4 and 0.2 ml into T5. There was no water inoculated into the last tube T6.

14. Protocol cont … Phenol (0.5ml) was then inoculated into all the test tubes. It was transferred using a different pipette to avoid cross contamination. 1.5ml of GOD-POD reagent was then inoculated into all the test tubes using an automatic pipette and a long pipette tip. The test tubes were then agitated on the rack and incubated in the water bath for (15 -30) minutes at 37 C. The temperature was constantly checked during incubation. After (15-30) minutes, the solutions changed colour from colourless to light pink according to the concentration. These different solutions were then read on a spectrophotometer in a cuvette. The spectrophotometer was zeroed at first then absorbencies of glucose was read and recorded. A cuvette was wiped on the soft side to minimize absorbencies caused by contamination. These different absorbencies were recorded on a table.

15. Procedure Mix and incubate for 15 minutes at 37 C. Mix and read absorbance of standard(S) and test against blank(B) at 505nm. The final colour is stable for 10 hr at R.T. Reagent For 1ml procedure B S T Glucose working Reagent 1.O ml 1.O ml 1.O ml Glucose Standard (conc.100mg/dl) ----------------------- 10µl ------------------------ Sample ----------------------- ------------------------- 10µl

16. Calculations Glucose Conc. (mg/dl) = Abs. of T / Abs. of S × Conc. of standard Glucose

17. GLUCOSE ESTIMATION IN CSF CSF is a fluid that flows through and protects the subarachnoid space of the brain and spinal cord. It is obtained by lumbar puncture, L 3-L 4. In CSF, Glucose is estimated by GOD – POD method. CSF Contain- 15– 45 mg% Glucose

18. GLUCOSE ESTIMATION IN CSF Clinical interpretation: An increased CSF glucose level is seen in hyperglycaemia. Decreased CSF glucose in Bacterial Infection Hypoglycaemia

19. CLINICAL SIGNIFICANCE OF GLUCOSE ESTIMATION Increased glucose : (hyperglycaemia) • Diabetes mellitus, • Hyperthyroidism, • Hyperpituitarism, • Adrenocortical hyper activity, Decreased glucose: (hypoglycaemia) • Hypothyroidism, • Hypopituitarism, • Hypoadrenalism,

20. Conclusion The Folin Wu method though sensitive is nonlinear. The fading of colour during reading of absorbance adds to this imprecision and inaccuracy. Folin Wu method require protein precipitation, centrifugation and boiling which is time consuming. Folin Wu method requires special tubes ( Folin Wu tubes) and accurate boiling time (6 minutes). The absorbance should be read immediately as colour fades rapidly or at fixed interval (such as 5 minutes) which is technically not feasible. The GOD-POD method is linear (up to 500 mg/dl), sensitive (detection limit 0.3 mg/dl), simple (requires 10 microlitre of sample to be incubated for 15 minutes with single reagent at 37 C) and requires simple instrumentation (the absorbance to be read between 505 nm to 550 nm). When the results obtained by this method using Indian kit, is compared with that of Hexokinase method using a kit from Sigma Chemical Co. USA, the average deviation was found to be -0.97 per cent only. Thus the GOD-POD method which is comparatively cheap, the kit for which is readily available and can be adopted using colorimeter, is best method for glucose estimation.