Clinical Biochemistry Laboratory – Enzymes, Saliva, Urine, Bile, Blood, Milk & Food Analysis

A comprehensive set of Q&A flashcards covering laboratory tests and biochemical principles for enzymes, saliva, urine, bile, blood, milk, and food nutrient analysis.

Aside from enzyme & substrate concentration, temperature and pH, what three additional factors influence enzymatic activity?

Inorganic salts; presence of activators & inhibitors; concentration of reaction products

What are enzymes?

Complex organic compounds with definite chemical structure secreted by living cells that initiate and hasten chemical reactions without being consumed

What reaction is catalyzed by amylase?

Hydrolysis of starch into simple/reducing sugars

What reaction is catalyzed by catalase?

Decomposition of hydrogen peroxide (H₂O₂) into water and oxygen

What is the optimal temperature range for most body enzymes such as amylase and catalase?

38-40 °C

What happens to most enzymes when the temperature rises above 40 °C?

They denature and lose catalytic activity

Why do certain fruits brown when cut and exposed to air?

Phenolic compounds react with oxygen in the presence of phenolase/oxidase enzymes, forming brown pigments

Which fruit showed the least enzymatic browning in the experiment?

Santol

Why did boiled potato filtrate fail to release bubbles with H₂O₂?

Boiling denatured catalase, preventing decomposition of hydrogen peroxide

How is the presence of oxygen gas confirmed in the liver catalase test?

A glowing splint rekindles when exposed to liberated oxygen

In salivary amylase testing, what color does Fehling’s reagent produce when reducing sugars are present?

Brick-red or orange-red precipitate

What iodine color indicates intact starch?

Blue-black or dark violet

Why was unboiled saliva able to change iodine color from blue-black to yellow-brown?

Active amylase broke starch into smaller dextrins that do not react strongly with iodine

Name the three substances responsible for salivary digestion of carbohydrates.

Amylase, mucin, and water

Why must you rinse your mouth before collecting saliva for analysis?

To remove food particles that could alter test results

What is mucin’s function in saliva?

Makes saliva slippery and viscous for lubrication

What causes mucin to precipitate when acetic acid is added?

Lowered pH decreases mucin solubility, causing coagulation

What positive result is seen in the Biuret test for salivary proteins?

Violet coloration

Which reagent precipitates calcium in saliva?

Potassium oxalate (K₂C₂O₄)

Which reagent pair detects inorganic phosphate in saliva?

Ammonium molybdate and nitric acid

Define erythrodextrin.

Red-colored intermediate dextrin produced during partial starch hydrolysis that gives a red iodine reaction

Define achrodextrin.

Colorless dextrin produced after further starch breakdown; yields no color with iodine

What is the achromatic point in starch digestion?

Stage when starch has been completely converted to achrodextrin and reducing sugars, giving no iodine color

Why does Benedict’s reagent turn red in completely digested starch samples?

Because reducing sugars reduce Cu²⁺ to Cu₂O, forming red precipitate

List the normal inorganic cations of urine.

Calcium (Ca²⁺) and magnesium (Mg²⁺)

List the normal inorganic anions of urine.

Chloride (Cl⁻), sulfate (SO₄²⁻), phosphate (PO₄³⁻)

Name four normal organic constituents of urine.

Urea, uric acid, creatinine, organic sulfates

Which three amino acids participate in creatinine formation?

Glycine, arginine, methionine

Give five abnormal organic constituents that may appear in urine.

Bile pigments, blood, proteins, glucose, indican

How does fresh urine differ in pH from urine left standing for one hour?

Fresh urine is acidic; standing urine becomes alkaline due to bacterial ammonia production

What reagent identifies urinary chloride?

Silver nitrate in nitric acid medium (forms white AgCl precipitate)

Which test specifically identifies uric acid crystals via reddish-violet coloration?

Murexide test

What color change signifies a positive Jaffe’s test for creatinine before acidification?

Red complex with picric acid in alkaline medium

What does the reappearance of white precipitate after boiling filtered urine with BaCl₂ and HCl indicate?

Presence of organic sulfates hydrolyzed to inorganic sulfates

Why must the urine sample not be shaken during the protein coagulation test?

Shaking may mechanically denature proteins, confounding results

What forms the white ring in Heller’s test for albumin?

Protein precipitate at the interface of concentrated HNO₃ and urine

How is the semiquantitative Benedict’s glucose scale interpreted?

Blue = none; green < yellow < orange < red = very high reducing sugar

Which layer shows the blue-green color in the indican test?

Carbon tetrachloride (CCl₄) layer

What major reagent is used in the indican test?

Obermayer’s reagent (ferric chloride in HCl)

What is detected by the benzidine test in urine analysis?

Occult blood

Define bile.

Viscid, alkaline secretion of the liver containing bile acids, pigments, salts, and cholesterol

What is the usual color of bile?

Greenish-brown

Name four important constituents of bile.

Bile acids, bile pigments, inorganic salts, cholesterol

List the inorganic ions present in bile.

Chlorides, sulfates, phosphates

What is another name for Pettenkofer’s test?

Sucrose-sulfuric acid test

What do Pettenkofer’s, Oliver’s, and Hay’s tests detect?

Bile acids or bile salts

What risk is associated with excessive cholesterol in bile?

Formation of cholesterol gallstones

Define emulsification and the role of bile acids in it.

Breakdown of large fat globules into smaller droplets; bile acids/salts act as surfactants to stabilize the emulsion for enzymatic digestion

What positive result indicates bile acids in Pettenkofer’s test?

Red or reddish-violet color

What observation constitutes a positive Hay’s surface-tension test?

Sulfur powder quickly sinks

Which reagent produces a green color with cholesterol in the Lieberman-Burchard test?

Lieberman-Burchard reagent (acetic anhydride + sulfuric acid)

What color sequence signals a positive Lieberman-Burchard reaction for cholesterol?

Purplish/pink ➔ blue ➔ deep green/green-blue

Which forensic test rapidly detects blood by a green or blue color change?

Benzidine test

What is the role of H₂O₂ in the benzidine blood test?

Provides oxygen that the heme catalyzes to oxidize benzidine, forming a colored product

In phosphate testing of blood, what color indicates human blood?

Yellow precipitate

Which reagent reacts with iron in blood to give a faint red color?

Potassium thiocyanate (forming Fe(SCN)₃)

Why is blood aerated in the oxyhemoglobin determination?

Shaking introduces O₂ to convert hemoglobin to oxyhemoglobin

What purpose do Stokes’ reagent and NH₄OH serve in the hemoglobin reduction test?

Act as reducing agents to convert oxyhemoglobin to reduced hemoglobin

Compare colors of oxyhemoglobin and reduced hemoglobin.

Oxyhemoglobin is bright red; reduced hemoglobin is dark bluish-red

What pH reactions were observed with phenolphthalein in milk samples?

Pink in basic, colorless in acidic/neutral samples

Why did sour milk clump instead of forming a film when boiled?

Existing acidity had already denatured proteins; heating intensified denaturation, causing curdling

What does a darker brown color in the hot-alkali milk test signify?

Higher concentration of reducing sugars (Maillard reaction) in milk

What is the objective of precipitating casein from milk?

To separate whey for further analysis of soluble components

What Biuret result confirms protein in whey?

Violet coloration indicates peptide bonds

Which precipitate shows calcium in whey?

White CaC₂O₄ after K₂C₂O₄ addition

Which reagent gives a yellow precipitate for phosphorus in whey?

Ammonium molybdate under acidic conditions

State the principle of the Biuret test.

Copper(II) ions form a violet complex with peptide bonds in alkaline medium, indicating proteins

What two reagents constitute the Carr-Price test for Vitamin A?

Chloroform and antimony trichloride

What color indicates a positive Carr-Price test?

Blue (intense) or blue-green

Which nutrient test result shows a translucent spot when held to light?

Positive test for fats

In starch testing with iodine, which food samples gave positive results?

Green banana, potato, cooked rice (blue coloration)

Which test detects mineral matter in food and what is its positive sign?

Addition of acid & BaCl₂ forming white precipitates

What is the molecular formula for Vitamin A?

C₂₀H₃₀O

Which food item tested positive for glucose with Benedict’s reagent?

Fruit juice (yellow to red spectrum)

Which two food samples showed positive fat tests?

Butter and peanut butter

Which protein-rich foods gave a positive Biuret test?

Examples: cooked egg white, cooked egg yolk, cheese, dilis, etc. (violet color)

Why are reducing sugars important in Benedict’s food test?

They reduce Cu²⁺ to Cu₂O, giving characteristic color indicating carbohydrate type

What is the danger of shaking during the urine coagulation test for protein?

Could falsely enhance turbidity by mechanical denaturation

Why does NH₄OH precipitate Mg²⁺ in urine?

Forms insoluble Mg(OH)₂ in alkaline medium

What two indicators confirmed alkaline pH of bile?

Blue litmus staying blue; phenolphthalein turning pink

Why does sulfur powder sink in Hay’s test when bile salts are present?

Bile salts decrease surface tension, allowing sulfur to wet and sink

What multicolored ring pattern signifies bile pigments in Gmelin’s test?

Sequence of green, blue, violet, red, and yellow rings

What color change occurs in Rosenbach’s modification for bile pigments?

Similar multicolored concentric rings appear on filter paper

How are bile acids precipitated in Oliver’s test?

Acidified peptone forms a white precipitate with bile salts

Which test detects albumin in urine by a fluffy white ring at an acid interface?

Heller’s ring test

What does Obermayer’s reagent contain and what does it detect?

Ferric chloride in dilute HCl; detects indican

Why does Mg²⁺ co-precipitate with phosphate in some urine tests?

Formation of ammonium magnesium phosphate in alkaline solutions

What is the principle behind the sulphate test for urine using BaCl₂?

Ba²⁺ reacts with SO₄²⁻ to form insoluble BaSO₄ white precipitate

Which reagent system distinguishes vitamin A through a blue color in food items like carrots?

Carr-Price (chloroform + SbCl₃)

What is the main visual criterion for a positive benzidine blood test?

Immediate blue or green coloration

In blood phosphate differentiation, what precipitate color indicates pig blood?

White precipitate

Give a reason why the murexide test is clinically significant.

Identifies elevated urinary uric acid (e.g., gout, kidney stones)

Explain why urine becomes alkaline after standing.

Urea splits into ammonia by bacterial urease, raising pH

State the purpose of acetic acid in the Jaffe creatinine test’s second step.

Acidifies mixture, causing red creatinine-picrate complex to revert to yellow picric acid for confirmation

What is formed when calcium reacts with oxalate in saliva tests?

Insoluble calcium oxalate (white precipitate)

Which enzyme is responsible for browning reactions in cut fruits?

Phenolase (polyphenol oxidase)

How does temperature affect enzyme activity below optimum?

Rate decreases because fewer molecules reach activation energy

Why must bile be alkaline for digestive function?

Alkalinity neutralizes gastric acid and optimizes pancreatic enzyme activity

Why are bile salts important for fat absorption?

They form micelles that transport fatty acids and monoglycerides to intestinal mucosa

Describe the color change of Congo red with increasing acidity.

Blue-violet ➔ violet ➔ reddish-violet ➔ red as acidity decreases (becomes more alkaline)