EXPERIMENT #10 SALIVARY DIGESTION

Alimentary Digestion

• Ingested food materials are made assimilable

  • Assimilable— can be transported through intestinal mucosa and absorbed into the portal venous system.

• Series of enzymatic hydrolyses:

  • Colloidal food molecules → Simple, non-colloidal solutes

Hydrolytic enzymes

• Involved in digestion

• naturally occuring foodstuffs → assimilable forms

• Catalyse hydrolysis of:

  • Proteins → amino acids

  • Starches → monosaccharides

  • Fats → acyl glycerol

Saliva

• Watery secretion

• Produced by:

  • 3 pairs of large salivary glands

  • & many minute glands (buccal glands)

    • in mucusal lining of the mouth

• speeds up chemical changes in carbohydrates

• no chemical effects on:

  • proteins

  • fats

Components of Saliva

• H20

  • ~99% of salova

• Salivary amylase

  • digestive enzyme which hydrolyses starch to maltose

• mineral salts

  • eg. sodium carbonate

  • Purpose: maintain pH~ 6.5-7.5

    • Optimal for salivary amylase

• mucin

  • sticky material (glycoprotein)

  • Purpose:

    • Binding of food particles

    • lubrication to assist swallowing

pH of Saliva

• pH paper ~ 8.0

• Actual pH: 6.5-7.5

  • Salivary amylase functions best

  • slightly alkaline but neutral due to sodium bicarbonate

Enzyme action

• Mouth and elsewhere: enzyme action dependent on pH surroundings

  • Eg. pH optimum, salivary amylase: 6.5-7.5

• Stomach: very acidic (pH 1.0-3.5)

  • Salivary amylase inactivated

    • no carb digestion in stomach

Qualitative Tests for SALIVA

Test for Mucin

• Mucin

  • sticky

  • Highly glycosylated, extended, and hydrated protein

  • Extensive in salivary secretions

  • functions:

    • Increase viscosity of fluids in saliva

    • Binds and lubricates food particles

  • (+) result: ppt (mucin)

Test for proteins

• Alias: Biuret test

• (+) results: violet solution / purple product

  • Coordination complex

  • intensity of color is dependent on the number of peptide bonds

    

Test for Inorganic Matter

Test for Chlorides

• (+) result: White ppt

• 

Test for Phosphates

• (+) results: yellow ppt

  • (ammonium phosphomolybdate)

Test for Sulfates

• (+) result: white ppt

• 

Test for Calcium

• (+) result: white ppt

• Importance:

  • Maintenance or stabilization of compact formation of a-amylase

  • Maintenance of enzymatically active conformation.

Test for Thiocyanate (FeCl3 Test)

• (+) results: Pale yellow solution with red ppt

• Confirmation: HgCl2

  

CNS

• Metabolite of CN

• End product of detoxification of CN- containing cpds

  • enzyme: rhodanase

• CNS IN SALIVA:

  • Reasearch: present in saliva

  • Sources:

    • smoking

    • food containing cyanide

Digestion of starch paste

• Starch→ (digestion by saliva)→ (smaller saccharide units)

• Salivary digestion complete: Violet-colored product not formed

Achromatic point

• Digestion of Starch will reach Achromatic point when:

  • ↑in duration

  • ↑ lightening of the color of violet-colored complex

• Reaction mixture: no more color change with I2 solution

• Complete breakdown of strach

  • Starch→ Dextrin →disaccharides→monosaccharides

    

Salivary Amylase

• Cleaves a-1-4 linkages starch

• gives different colors in I2:

  • Amylodextrin: purple

  • Erythrodextrin: red

  • Achrodextrin: colorless

  • Maltose: colorless

  Starch→ amylodextrin→Erythrodextrin→Achrodextrin→Maltose

  

Fehling’s Test

• For reducing sugars

• (+) Results: Blue Solution with Brick red ppt

Benedicts Test

• For reducing sugars

• (+) result: green, red, or yellow ppt

  • All sugars except sucrose

• Principle:

  • Aldehydes, ketone -[O] → OA : alkaline copper solution

Phenylhydrazine Reaction

• Salivary digestion of starch→ monosaccharides

• Monosaccharides+ phenylhydrazine reagent → Osazone Crystals

Effect/ influence of free acid

• Free acid: Hinders digestion of starch by salivary amylase

• Same trend with dilute base.

  • Affect optimum pH

  • Inactivate the enzyme

  • ↑ concentration of dil. Na2CO3 → ↑Intensity of Blue color

SUMMARY OF QUALITATIVE TESTS

EXPERIMENT #11 URINE ANALYSIS

Physical properties of Urine

Color

• Color of Normal urine:

  • Yellow to amber

  

  Due to Urochrome:

  • Pigment

  • Urobilin and Peptide

• Set aside undisturbed: will darken

  • Urubilin is released

    

• Abnormal urine:

  • Varies; nearly colorless to black

  • depends on pathological condition

• Milky urine:

  • possible:

    • WBC’S

    • Bacterias

    • Fats

• Reddish-Amber

  • Urobilinogen

    • pigment

    • intestine: Bile or porphyrin-(bacteria)→urobilinogen

    • liver disease, Addison’s disease etc.

      

• Brownish yellow or Green

  • Bile pigments

• Red to smoky brown:

  • Blood and blood pigments

Appearance of abnormal urine

• Cloudy:

  • Possible:

    • phosphates

    • urates

    • WBC

    • Bacteria

    • Epithelial cells

    • fat

Odor or normal and fresh urine

• Slightly aromatic

• set aside undisturbed: Ammonia (NH3) odor

Cause odor change in urine

• drugs

• food

• pathological conditions

Specific gravity

• within 24hours: 1.015-1.025

  • <1.015:

    • excess fluid intake

    • diabetes insipidus

    • chronic renal failure

  • >1.025

    • limited fluid intake/ dehydration

    • fever

    • kidney inflammation

Volume

• V excreted / Day

• 0.5-1.5L

  • ↑ : large amounts lipids

  • ↓ : loss H2O (Perspiration)

pH

• 4.5-8.2

• Factors

  • diet

  • drugs

  • pathological conditions

Organic Compounds

Urea

• end product of protein metabolism (liver in humans)

Uric Acid

• end product of purine metabolism (humans)

• levels high in gout patients

• amount produced in human urine: ~0.5-1.0g/h

  

Creatinine

• excreted by normal healthy adults: 1.2-1.7 g/h

  

• 

  Factors:

  • diet

  • muscular development

    • higher in men tyhan in women and children

• Nitroprusside

  • contraindicated in patients with renal failure

  • 

• Picric acid

  • IUPAC NAME: 2,4,6-trinitrophenol (TNP)

  • Yellow crystalline solid

  • one of the most acidic phenols

Indican

• comes from the putrefactive decomposition of tryptophan in the intestine

• Obermeyer’s Test

  • colorimetric method

  • For: Intestinal putrefaction

    ---

Pathological Components of Urine

• Certain diseases:

  • Dysfunction of liver and kidneys

  • metabolic disorders

Main abnormal constituents

PROTEIN

ALBUMIN

• Proteinuria

  • presence of protein in urine

  • Indicate: Kidney damage or nephritis

• Albuminaria

  • presence of albumin in urine

• Globulins

  • sometimes found in urine with albumin

    • larger molecules

  • indicate more extensive kidney damage

SUGAR

• Glycosuria

  • (+) sugar in urine

  • Glucosoria

    • (+) glucose

  • Possible causes:

    • excessive carb intake

    • diabetes mellitus

KETONE BODIES

• From incomplete oxidation of fats

• Results:

  • accumulation: Acetyl CoA and acetoacetyl CoA

• Indicates: Acidosis

• abnormal amounts excreted

  • depletion of Na+ and K+

  

Conditions

BENCE-JONES PROTEIN

• Monoclonal globulin

• Location: blood or urine

• precipitates at low temperature

• Indicate:

  • multiple myeloma

  • another blood (plasma) disorder

    • Waldenstrom’s macroglobulinaemia (IgM)

    • Chronic lymphocytic leukemia (WBC’s)

    • Amyloidosis (protein amyloid)

EXPERIMENT # 12 INTESTINAL DIGESTION

Small intestine

• most important part: duodenum

  • 3 juices enter the intestine (through duodenum)

    • pancreatic juice

    • bile

    • intestinal juice

Trypsin (Enzyme)

• hydrolyze peptide linkages involving Arginine and Lysine

• Optimum pH: 7-9

• Active form of trypsinogen

  • activated by enteropeptidase (enterokimase)

• 

BAPNA (Substrate)

• synthetic

• Colorless→ yellow

Effects of Temperature on Trypsin

• T optimum, trypsin: 40 C

• ↑ T: Trypsin denaturation

  • Result: Inactivation

  • other enzymes and proteins also denatures

• ↓ T: trypsin rigid and less active

  • cause decrease in mobility of atoms and molecules

Pancreatin

• commercial mixture of enzymes (amylase,lipase and protease)

• secreted also by normal human pancreas

• extracted from pigs

• For problems digesting fats starches and proteins

LIPASE

• Family of enzymes for hydrolysis of fats

• substrate:

  • Triglycerides

  • Cholesterol

  • Phospolipids

  • Lipid-soluble vitamins

  • etc

• T optimum= 37 C

• Same as trypsin

  • ↑ T: Trypsin denaturation

  • ↓ T: trypsin rigid and less active

Bile Salt

• Emulsifying agents for lipids

• ↑ Surface area lipid

  • improve efficency of hydrolysis

• ↓ T: Activity of lipase

  • (+) : red solution - 37C

  • (-) : blue solution - 0 C

  

Practice Questions

Conceptual Questions (Q1–Q10)

1. What is the primary purpose of salivary amylase in the digestive process?
   a. Hydrolyzing proteins into amino acids
   b. Hydrolyzing starch into maltose
   c. Hydrolyzing fats into glycerol
   d. Lubricating food particles

2. Which pH range is optimal for salivary amylase activity?
   a. 1.0–3.5
   b. 6.5–7.5
   c. 7.5–9.0
   d. 4.0–6.0

3. What is the function of mucin in saliva?
   a. Hydrolyzing proteins
   b. Binding and lubricating food particles
   c. Maintaining salivary pH
   d. Catalyzing starch breakdown

4. Which of the following is a qualitative test for mucin?
   a. Precipitation test (ppt)
   b. Biuret test
   c. Fehling's test
   d. Benedict’s test

5. What is the result of Fehling’s test if reducing sugars are present?
   a. Violet solution
   b. Yellow crystals
   c. Blue solution
   d. Brick-red precipitate

6. What is the achromatic point in starch digestion?
   a. The point where starch turns blue with iodine
   b. The point where no color change occurs with iodine
   c. The point where starch digestion stops
   d. The point where starch forms dextrin

7. Which enzyme activates trypsinogen into its active form, trypsin?
   a. Lipase
   b. Salivary amylase
   c. Enteropeptidase
   d. Pancreatin

8. What is the optimum temperature for trypsin activity?
   a. 25°C
   b. 37°C
   c. 40°C
   d. 50°C

9. What condition is indicated by the presence of Bence-Jones protein in urine?
   a. Diabetes insipidus
   b. Multiple myeloma
   c. Addison’s disease
   d. Gout

10. What does a yellow precipitate indicate in a phosphate test?
    a. Presence of sulfates
    b. Presence of phosphates
    c. Presence of thiocyanates
    d. Presence of chlorides

Analytical Problem Solving Questions (Q11–Q20)

11. You mix saliva with a starch solution and add iodine. Initially, the mixture is violet. Over time, the color fades. What does this indicate?
    a. Starch is not digested
    b. Salivary amylase is inactive
    c. Starch is being digested into simpler sugars
    d. The pH is too low for digestion

12. If urine appears reddish-amber, which condition is most likely the cause?
    a. Gout
    b. Kidney stones
    c. Liver disease
    d. Dehydration

13. A sample shows a violet color in the Biuret test. What does this indicate?
    a. Presence of reducing sugars
    b. Presence of lipids
    c. Presence of proteins
    d. Presence of phosphates

14. Why does salivary amylase become inactive in the stomach?
    a. Lack of sodium bicarbonate
    b. Presence of lipase
    c. Low stomach pH
    d. High stomach temperature

15. Which condition increases the specific gravity of urine above 1.025?
    a. Diabetes insipidus
    b. Dehydration
    c. Excess fluid intake
    d. Chronic renal failure

16. You perform a Fehling’s test on a solution and observe a brick-red precipitate. Which carbohydrate is most likely present?
    a. Glucose
    b. Starch
    c. Sucrose
    d. Cellulose

17. Which of the following tests would confirm the presence of chlorides in saliva?
    a. Biuret test
    b. Benedict’s test
    c. Precipitation test (white ppt)
    d. Phenylhydrazine reaction

18. What effect does a high concentration of sodium carbonate have on the digestion of starch?
    a. Stops digestion
    b. Changes starch to glycogen
    c. Increases intensity of the blue color with iodine
    d. Denatures salivary amylase

19. In which qualitative test does ammonium phosphomolybdate form a yellow precipitate?
    a. Chlorides test
    b. Phosphates test
    c. Sulfates test
    d. Thiocyanate test

20. What happens to trypsin activity when the temperature is increased beyond 40°C?
    a. Activity increases indefinitely
    b. Trypsin denatures and becomes inactive
    c. Trypsin remains rigid and functional
    d. The reaction slows down but does not stop

Identification and Chemical Structures (Q21–Q30)

21. Which of the following forms a pale yellow solution with a red precipitate in the presence of FeCl₃?
    a. Thiocyanate
    b. Chloride
    c. Phosphate
    d. Sulfate

22. What is the chemical formula of picric acid, used in some urine analysis tests?
    a. C₆H₆O₃
    b. C₆H₃N₃O₇
    c. C₆H₅NO₂
    d. C₆H₆N₆

23. What color is associated with amylodextrin in iodine tests?
    a. Purple
    b. Red
    c. Yellow
    d. Colorless

24. Which compound is the final product of starch digestion by salivary amylase?
    a. Dextrin
    b. Disaccharides
    c. Maltose
    d. Polysaccharides

25. What is indicated by the formation of osazone crystals in a phenylhydrazine reaction?
    a. Presence of starch
    b. Presence of amino acids
    c. Presence of monosaccharides
    d. Presence of fatty acids

26. Which color change occurs when erythrodextrin reacts with iodine?
    a. Purple
    b. Red
    c. Yellow
    d. Colorless

27. What is the effect of bile salts on lipid digestion?
    a. Emulsifies lipids to increase surface area
    b. Breaks down lipids into amino acids
    c. Converts lipids into sugars
    d. Destroys lipid-soluble vitamins

28. What compound is a metabolite of cyanide and is often detected in saliva?
    a. Thiocyanate
    b. Chloride
    c. Urea
    d. Indican

29. Which enzyme in the intestine hydrolyzes peptide bonds involving arginine and lysine?
    a. Lipase
    b. Pancreatin
    c. Trypsin
    d. Amylase

30. What is the appearance of a positive result in the test for sulfates?
    a. Yellow precipitate
    b. Blue solution
    c. White precipitate
    d. Red precipitate

Conceptual Questions (Q31–Q40)

31. What is the role of sodium carbonate in maintaining salivary pH?
    a. Acts as a digestive enzyme
    b. Neutralizes acids in saliva
    c. Maintains the pH at 6.5–7.5
    d. Activates salivary amylase

32. Which of the following substances contributes to the yellow color of urine?
    a. Urochrome
    b. Indican
    c. Creatinine
    d. Uric acid

33. What does the Biuret test specifically detect in a sample?
    a. Reducing sugars
    b. Salts
    c. Peptide bonds in proteins
    d. Glycoproteins

34. Which of these components is essential for emulsifying fats during digestion?
    a. Pancreatic amylase
    b. Bile salts
    c. Mucin
    d. Sodium bicarbonate

35. What happens to starch when digested by salivary amylase?
    a. It forms peptides
    b. It forms monosaccharides directly
    c. It is broken down into maltose and dextrins
    d. It is converted into cellulose

36. Which physical property of urine is typically measured with a hydrometer?
    a. Color
    b. Odor
    c. Specific gravity
    d. pH

37. What type of bonds does trypsin hydrolyze in proteins?
    a. Disulfide bonds
    b. Peptide bonds involving arginine and lysine
    c. Hydrogen bonds
    d. Glycosidic bonds

38. Which pH range supports optimal lipase activity?
    a. 7.0–9.0
    b. 5.5–6.5
    c. 1.0–3.0
    d. 8.5–10.5

39. Which of the following is a pathological component in urine?
    a. Creatinine
    b. Albumin
    c. Urobilin
    d. Sodium

40. What does a green precipitate in Benedict’s test indicate?
    a. Absence of reducing sugars
    b. Low concentration of reducing sugars
    c. High concentration of reducing sugars
    d. Presence of proteins

Analytical Problem Solving and Identification Questions (Q41–Q50)

41. You add iodine to a reaction mixture and observe a purple color. What is present in the mixture?
    a. Glucose
    b. Maltose
    c. Amylodextrin
    d. Achrodextrin

42. A urine sample is tested and found to contain high levels of uric acid. What condition does this most likely indicate?
    a. Gout
    b. Diabetes insipidus
    c. Chronic renal failure
    d. Liver disease

43. In a phenylhydrazine test, what kind of crystals are formed when monosaccharides are present?
    a. Maltose
    b. Osazone crystals
    c. Glycoside crystals
    d. Polysaccharides

44. What happens when thiocyanate reacts with FeCl₃?
    a. A violet solution forms
    b. A yellow precipitate forms
    c. A pale yellow solution with a red precipitate forms
    d. No visible reaction occurs

45. What is the effect of increasing the temperature above 40°C on lipase?
    a. Lipase denatures and loses activity
    b. Lipase activity remains constant
    c. Lipase becomes rigid and more active
    d. Lipase breaks down into smaller enzymes

46. What does a white precipitate indicate in a test for sulfates?
    a. Presence of sulfate ions
    b. Absence of sulfates
    c. Presence of thiocyanates
    d. Presence of proteins

47. Why is pancreatin extracted from pigs used in commercial enzyme preparations?
    a. It is a source of bile salts
    b. It mimics human amylase
    c. It contains enzymes for digesting fats, proteins, and starches
    d. It activates trypsin

48. What abnormal urine characteristic might result from the presence of WBCs or bacteria?
    a. Milky appearance
    b. Reddish-amber color
    c. Cloudy appearance
    d. Dark brown color

49. What test confirms the presence of indican in urine?
    a. Biuret test
    b. Benedict’s test
    c. Phenylhydrazine reaction
    d. Obermeyer’s test

50. What does a colorless solution indicate in the iodine test for starch digestion?
    a. Starch digestion is complete
    b. Starch digestion has not started
    c. Amylodextrin is present
    d. Erythrodextrin is present

Questions on Indicators (Q51–Q60)

51. What disease is commonly indicated by the presence of albumin in urine (albuminuria)?
    a. Gout
    b. Diabetes insipidus
    c. Kidney damage or nephritis
    d. Liver failure

52. High levels of ketone bodies in urine suggest which condition?
    a. Acidosis
    b. Hypoglycemia
    c. Chronic renal failure
    d. Excess fluid intake

53. The presence of Bence-Jones protein in urine is most often associated with which disease?
    a. Multiple myeloma
    b. Diabetes mellitus
    c. Addison’s disease
    d. Gout

54. What abnormality is indicated by the reddish-amber appearance of urine?
    a. Presence of lipids
    b. Dehydration
    c. Liver disease (e.g., urobilinogen presence)
    d. Chronic renal failure

55. The presence of uric acid in excess quantities in urine is most commonly associated with which condition?
    a. Liver failure
    b. Kidney stones
    c. Hypernatremia
    d. Gout

56. Cloudy urine may indicate which of the following conditions?
    a. Diabetes insipidus
    b. Presence of WBCs, bacteria, or epithelial cells
    c. Liver failure
    d. Dehydration

57. Which condition is suggested by the presence of glycosuria (glucose in urine)?
    a. Diabetes mellitus
    b. Liver disease
    c. Protein deficiency
    d. Hyperlipidemia

58. A positive test for indican in urine can indicate what underlying issue?
    a. Intestinal putrefaction (tryptophan metabolism)
    b. Diabetes mellitus
    c. Kidney inflammation
    d. Hypertension

59. What does a high concentration of creatinine in urine suggest?
    a. Diabetes insipidus
    b. Hypothyroidism
    c. High muscular activity or protein intake
    d. Liver dysfunction

60. A green or brownish-yellow color in urine is often an indicator of what condition?
    a. Kidney damage
    b. Diabetes mellitus
    c. Presence of bile pigments due to liver disease
    d. Dehydration