Amylase E-PNPG 7

1. Amylase E-PNPG 7 • An amylase: is an enzyme that catalyses the hydrolysis of starch into sugars. • Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion.

2. Foods that contain large amounts of starch but little sugar, such as rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase degrades some of their starch into sugar. The pancreas and salivary gland make amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy.

3. Plants and some bacteria also produce amylase. As diastase, amylase was the first enzyme to be discovered and isolated (by AnselmePayen in 1833).Specific amylase proteins are designated by different Greek letters. All amylases are glycosides hydrolases and act on α-1,4-glycosidic bonds

4. Principle • E-PNPG 7 Substrate. α-Amylase • Alpha-Amylase Human salivary amylase: calcium ion visible in pale khaki, chloride ion in green. PDB  • article: Alpha-amylase • The α-amylases (EC 3.2.1.1) (alternative names: 1,4-α-D-glucanglucanohydrolase; glycogenase) are calcium metalloenzymes completely unable to function in the absence of calcium. By acting at random locations along the starch CHAIN, α-amylase breaks down long-chain carbohydrates ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin Because it can act anywhere on the substrate α-amylase tends to be faster-acting than β-amylase. In animals it is a major digestive enzyme, and its optimum pH is 6.7–7.0.

5. In human physiology, both the salivary and pancreatic amylases are α-amylases. • The α-amylases form is also found in plants, fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus) •  Reagent for quantitative determination of α-amylase in human serum and urines. •  Reading: kinetic at 405 nm

6. Determination of total protein and Albumin-globulin ratio Serum protein represent a complex mixture containing a number of components which differ in properties and function. Liver is the organ, mainly responsible for formation of plasma albumin and at least 30% serum globulin.

7. The major components of protein in serum include: • Albumin • Globulin • Conjugated protein such as glycoprotein and lipoprotein

8. Laboratory method for serum protein investigation is based on any of the following principles • Nitrogen estimation • Estimation of tyrosine in protein • Biuret method (most commonly used method in clinical practice)

9. Principle One commonly used method for determining the total protein in a sample is the Biuret method. The Biuret method is based on the complexation of Cu2+ to functional groups in the protein’s peptide bonds . 1. The formation of a Cu2+-protein complex requires two peptide bonds and produces a violet-colored chelate product which is measured by absorption spectroscopy at 540 nm

10. Principle Substances which contain two CONH2 groups joined together a single carbon or nitrogen atom and those which contain two or more peptide links,give a blue to purple coloured compound with alkaline copper solution.This method is reliable and reproducible method for quantitative and qualitative determination of proteins.

12. Determination of Albumin-globulin ratio • In the differential estimation of albumins and globulins, the property of globulins to get precipitated by 28% sodium sulphite solution. For this, serum is taken in a tube and treated with sodium sulphite solution, whereupon globulins are precipitated. The contents are centrifuged. The filtrate containing only albumin is treated with biuret reagent.

13. Possible causes of high blood protein include: • Chronic liver disease • Dehydration (which may make blood proteins appear falsely elevated) • HIV/AIDS • Multiple myeloma

14. Lower-than-normal levels may be due to • Bleeding (hemorrhage) • Liver disease • Malnutrition

15. NOTE • Only the peptide part of the protein molecule gives this reaction • The total protein concentration normally ranges from 6.0 to 8.0 g/dl. • Albumin range from 3.5 to 5.5 g/dl • Globulin from 2.5 to 3.5 g/dl. • A:G ratio varies from 1.5:1 to 2.5:1

16. Procedure • Stock solution: 0.1 g/100 ml ( 1000 ppm) of standard protein (or albumin) • Only albumin: pipetted 100 µl of serum into a test tube and add 400 µl sodium sulphite. Mix and allow standing for 5 min. The content is centrifuged. The filtrate containing only albumin is treated with biuret reagent.

17. Standard solutions

18. Working solutions

19. Mix and incubate all the tubes at 37ᵒC for 10 min. after cooling to room temperature read absorbance against reagent blank at 540 nm. • Plot a graph with protein concentration against the absorbance

21. Determination of calcium in serum Calcium is present in plasma and serum in three forms, a protein bound fraction, an ionized part, and one in combination with substances like citrate.

22. The body contains 25 to 35 mol. of calcium. • 98 % is the bones and teeth • The small part is present in serum and other extracellular fluid and very important for muscle contraction. • Very little calcium is present in red blood cells

23. Regulation Three hormones are involved in maintaining a constant ionized calcium concentration. • Parathyroid hormone (PTH) • 1,25-dihydroxy cholecalciferol (DHCC) • Calcitonin Normal serum calcium is 8.5 – 10.3 mg/dl.

24. Hypocalcaemia: • Nephrotic syndrome: in which plasma albumin is reduced so the calcium is also low. large amounts of protein in the urine • Hypocalcaemia observed after a massive transfusion of citrated blood which chelates calcium with blood.

25. Commoncausesinclude • hypoparathyroidism • vitamin D deficiency •  kidney failure • pancreatitis Diagnosis should generally be confirmed with a corrected calciumor ionized calcium level

26. Hypercalcemia • Hyperparathyroidism • Malignancy: tumours of the lung and breast has a high association with Hypercalcamia. • Pagetsdisease

27. Principle The principle of estimation is precipitation of calcium as oxalate directly from serum, and after washing, the precipitate by ammonia. The precipitate is dissolved in acid and titrated against standard permanganate solution.

29. Procedure: • To 2 ml of serum is added 2 ml. of DW and 1 ml of 4% ammonium oxalate. This is then centrifuged for 5 min. • The supernatant discarded without disturbing the precipitate. 3ml of 2% ammonia solution is added to the precipitate and mixed by glass rod (NOTE if the precipitate is coloured a few drops of H2O2 andHNO3 may be added and heated to make the precipitate white. The mixture is centrifuged again and supernatant discarded. • To the precipitate 2 ml of H2SO4 is added the mixed well. • The mixture is warmed on a boiling water bath at about 75 C and titrated with 0.01 N KMnO4 solution till a faint pink colour persists for about a minute. (NOTE: at the time of titration the temperature should not be less 70 C) • For blank 2 ml of H2SO4 is titrated with the same permanganate solution.

30. Calculation: Ca/dl serum = Vol. of KMnO4 used for Ca × 10 ( 1 ml of 0.01 N KMNO4 = 0.2 mg of Ca)

31. Determination of Vitamin C by Spectrophotometry in fruit and Vegetable

32. Vitamin C • Vitamin C (Ascorbic acid; Dehydroascorbic acid) is a water-soluble vitamin that is necessary for normal growth and development. • Water-soluble vitamins dissolve in water. Leftover amounts of the vitamin leave the body through the urine.

33. Vitamin C • That means you need a continuous supply of such vitamins in your diet. The body is not able to make vitamin C on its own, and it does not store vitamin C. It is therefore important to include plenty of vitamin C-containing foods in your daily diet.

34. Vitamin C Dehydroascorbic acid Ascorbic acid

35. Function • Vitamin C is needed for the growth and repair of tissues in all parts of your body. It is used to: • Form an important protein used to make skin and blood vessels • Heal wounds and form scar tissue Vitamin C is one of many antioxidants. Antioxidants are nutrients that block some of the damage caused by free radicals

36. vitamin C • All fruits and vegetables contain some amount of vitamin C. • Fruits with the highest sources of vitamin C include:

37. Fruits • Melon • orange • Kiwi fruit • Mango • Pineapple • Strawberries • Watermelon

38. Vegetables • Broccoli • Green and red peppers • Spinach • Sweet and white potatoes • Tomatoes and tomato juice

39. Cooking vitamin C • Cooking vitamin C-rich foods or storing them for a long period of time can reduce the vitamin C content. Microwaving and steaming vitamin C-rich foods may reduce cooking losses. The best food sources of vitamin C are uncooked or raw fruits and vegetables.

40. Side Effects Serious side effects from too much vitamin C are very rare, because the body cannot store the vitamin. However, amounts greater than 2,000 mg/day are not recommended because such high doses can lead to stomach upset and diarrhea.

41. Too little vitamin C can lead to signs and symptoms of deficiency, including: • Anemia • Dry and splitting hair • Bleeding gums • Decreased ability to fight infection • Easy bruising

42. Recommendations • How much of each vitamin you need depends on your age and gender. Other factors, such as pregnancy and illnesses, are also important. • The best way to get the daily requirement of essential vitamins, including vitamin C, is to eat a balanced diet that contains a variety of foods.

43. Determination of Vitamin C by Spectrophotometry in fruit and Vegetable

44. Principle The ascorbic acid was converted to dehydro ascorbic acid by shaking with cupric sulphate solution and then coupled with 2,4- dinitrophenyl hydrazine in the presence of thiourea as a mild reducing agent. Sulphuric acid then converted dinitrophenylhydrazone into a red colored compound, which was assayed calorimetrically. The thiourea is added to prevent oxidation of dinitrophenyl hydrazine reagent by interfering substances.