Reactions of Biochemistry😊😍

Negative Schilling Test

  • Definition
    • Low urinary excretion of radiolabelled Vitamin B12 after oral dose.
    • Indicates abnormal absorption.

Stage-wise Interpretation

  • Stage I
    • Patient given
      • oral radiolabelled Vitamin B12 +
      • IM injection of unlabelled Vitamin B12
    • Correction (normal excretion): > 10%: Dietary deficiency
    • < 10%: Go to Stage II.
  • Stage II (with IF)
    • Correction (normal excretion): Pernicious anemia
    • < 10%: Stage III.
  • Stage III (with antibiotics)
    • Correction: Bacterial overgrowth syndrome.
    • < 10%: Stage IV.
  • Stage IV (with pancreatic enzymes)
    • Correction: Pancreatic insufficiency.
    • < 10% : Ileal disease or ileal resection

D-Xylose Test

  • Stool fat >7 % ⇒ ‘’Greasy stool’’
  • Distinguish
    • mucosal malabsorption vs pancreatic insufficiency
  • Principle
    • D-xylose absorbed in proximal small intestine
    • No pancreatic enzymes required
    • Excreted in urine
  • Procedure
    • Oral dose → measure blood (1–2 hr) + urine (5 hr) levels
  • Results
    • Normal → Normal mucosal absorption
      • Malabsorption due to
        • Pancreatic insufficiency (not mucosal) → ↓↓ Fecal elastase
        • Bile salt deficiency
    • Low:
      • Intestinal mucosal disease (celiac, tropical sprue)
      • Bacterial overgrowth (bacteria use xylose)
        • Corrected with Rifaximin

A patient presents with chronic diarrhea, a normal D-xylose test, and a negative Schilling test. Duodenal biopsies are typical. Which is the most likely diagnosis?

  1. Celiac disease
  1. Ulcerative colitis
  1. Pancreatic insufficiency
  1. Intestinal lymphangiectasia
      2. ANS
      1. Pancreatic insufficiency

NOTES

  • Positive hydrogen breath test : Lactose intolerence → Lactic acid → ↑↑ H+
  • Urea breath test: H Pylori

Urine Tests for Abnormal Constituents

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Test
Constituent
Result
Rothera's test
Ketone bodies

Purple ring
Gerhardt’s test
Ketone bodies
Hard Rot → Ketone bodies
Benzidine test
Heme
Yellow to Dark green/Black

ALKAPTONURIA
Black benz for Human
Sulphosalicylic acid test
Protein
White precipitate
Fouchets test
Bile pigments
Bluish or Green precipitate on filter paper

Mnemonic: Blue green Pigments in a pouch (Fouchets)
Hay's test
Bile salts

Bile salt ↓ surface tension
Sulfur powder sinks to the bottom

Mnemonic: Hey → Salt Sulfur Sinks
  • Bile salts and pigments are elevated in urine during Obstructive jaundice.
    • Due to overflow from biliary obstruction.
    • Enter systemic circulation, then filtration.

Tests for Carbohydrates

General & Reducing Sugar Tests

Benedict’s Test
Benedict’s Test
Test
Purpose
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Molisch Test
General test for all carbohydrates
Positive if ≥ 5 carbon atoms
Benedict’s Test
Detects reducing sugars
Green (0.5 g%) → Yellow (1) → Orange (1.5) → Brick red (2)
(↑ sugar conc.)
Barfoed’s Test
Distinguishes monosaccharides from disaccharides
Positive only for monosaccharides

In Bar → Say 1 or 2 pint (Mono, Di)

Monosaccharides
fructose, glucose, galactose, pentose

Negative + Benedict's positive:
Reducing disaccharides (maltose, lactose).

Barfoed's Test negative indicates a physiological cause (lactose).
Seliwanoff’s Test
Keto vs Aldehyde sugars
Keto sugars (e.g., Fructose)
turn Cherry red
GOD-POD Test
Quantifies glucose (enzymatic)
Glucose → Gluconic acid + H₂O₂ → Color (via POD)
Mucic acid test
Galactose Test
White crystals
Bial's Test
Pentose Test
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Reducing sugars

  • Monosaccharides: All are reducing sugars
    • Glucose, Galactose, Fructose, Mannose
  • Disaccharides: Reducing only if one anomeric carbon is free
    • Reducing: Lactose, Maltose
  • Non-reducing: Sucrose, Trehalose
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  • All Ulose are Ketose, Only fructose is Ctose in ketose

Cause of Reducing Sugars in Urine

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Barfoed Test

  • 1. Physiological: Disaccharide
    • Pregnancy and lactation:
      • Lactose in urine (→ Galactose + Glucose).
      • Caused by Prolactin excess.
      • Barfoed's Test is negative.
  • 2. Pathological: Monosaccharides
      1. Glucose in urine:
          • Diabetes Mellitus.
          • Renal glycosuria:
            • Damage in proximal convoluted tubules (PCT).
            • PCT fail to reabsorb filtered glucose (e.g., Fanconi syndrome).
          • Alimentary glycosuria:
            • Causes include thyrotoxicosis
              • thyroxine upregulates glucose transporters
            • Occurs after eating.
            • Increased intestinal glucose absorption causes a plasma glucose spike.
            • Glucose tolerance test (GTT) shows a sharp rise and fall.
            • Crosses the renal threshold in urine.
      1. Fructose in urine:
          • Essential fructosuria (fructokinase defect).
          • Hereditary fructose intolerance (aldolase B defect).
      1. Galactose in urine:
          • Galactosemia (galactokinase deficiency).
          • Classical Galactosemia (GalPUT defect)
      1. Pentose in urine:
          • Essential Pentosuria.
          • Xylitol dehydrogenase defect.

Colour Reactions of Amino Acids & Proteins

Test
Target
Color/Result
Ninhydrin Test
α-amino acids
Ninhydrin reagent + alpha-amino group (e.g., lysine) → purple colour
Ninhydrin reagent + imino group (e.g., proline) → yellow colour
If neither group is present → no colour.
Biuret Test
≥ 2 peptide bonds
Purple color → Negative for dipeptides/free AAs
Biuiret → bi peptide bond

Tests for Aromatic Amino Acids

Test
Detects
Result
Mnemonic
Xanthoproteic Test
All aromatic AAs
except phenylalanine???
Yellow color
X → Y → Tryp, Tyr
Hopkin Cole’s / Aldehyde
Tryptophan (indole ring)
Blue color
Try Indian (Indole) Aldi (Aldehyde)
Obermeyer Test
Tryptophan
Blue color
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Millon’s Test
Tyrosine (Phenol)
Red color
Million tyres
Pauly’s Test
Histidine & Tyrosine
Red color
His (Histidine) Imitate (Imidazole) Tired (Tyrosine) Paul ()
  • Phenylalanine (Benzene) on hydroxylation forms Tyrosine (Phenol group)
  • MPS → Shows red color

Tests for Sulfur-Containing Amino Acids

Test
Detects
Result
Sulfur/Lead Sulfide Test
All sulfur AAs except methionine
Black/Brown precipitate
Cyanide Nitroprusside Test
Compounds with –SH / –S–S– group
Positive in Cystinuria, Homocystinuria
Reddish purple/Magenta
Amino Acid(s)
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Test
Colour Reaction
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Arginine
Guanidinium
Sakaguchi test
Red colour
Urge (Arginine) Gone (Guanidinium) when Sakakuchi (old japan guy)
Proline (Iminoacid)
Pyrollidine
Ninhydrin test
Yellow colour
Proline → Nin (Ninhydrin) → Imin (only iminoacid)
All other amino acids
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Ninhydrin test
Purple colour
ㅤ
  • Xanthoproteic → Trp, Tyr
  • MPS → all give a red colour reaction
    • Millon's test
    • Pauly's test
    • Sakaguchi test
  • Phenylalanine, Proline → Yellow
  • Others → Purple

Other Important Biochemical Tests

Test
Detects
Result/Usage
Ferric Chloride Test
phenylpyruvic acid, BCAAs
Positive in PKU

Smell → Phenyl acetate
Guthrie’s Test
Phenylpyruvate,
Phenyl-lactate/acetate
Positive in PKU
DNP Hydrazine (DNPH) Test
α-keto acids in urine
(phenylpyruvic acid in PKU)
due to carbonyl group reaction.
Orange crystals (PKU, HSDU)
VMA Spot Urine Test
Pheochromocytoma detection
Colorless, detected spectrophotometrically
5-HIAA Test
Carcinoid Syndrome (↑ 5-HIAA)
Diagnostic urine test
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Ninhydrin test → Blue-purple (Ruhemann’s purple) with α-amino acids due to oxidative deamination; proline (Imino acid) & hydroxyproline give yellow color
Ninhydrin test → Blue-purple (Ruhemann’s purple) with α-amino acids due to oxidative deamination; proline (Imino acid) & hydroxyproline give yellow color
Sakaguchi: Red color with Arginine due to guanidino group reaction with α-naphthol + oxidant.
Sakaguchi: Red color with Arginine due to guanidino group reaction with α-naphthol + oxidant.
Cyanide test → Cyanide-nitroprusside test → Purple color with cysteine/cystine in urine due to reaction of sulfhydryl groups with cyanide and nitroprusside
Cyanide test → Cyanide-nitroprusside test → Purple color with cysteine/cystine in urine due to reaction of sulfhydryl groups with cyanide and nitroprusside
Rothera test
Rothera test
Biuret test → violet color with proteins due to Cu²⁺ complex with ≥2 peptide bonds.
Biuret test → violet color with proteins due to Cu²⁺ complex with ≥2 peptide bonds.
Xanthoproteic → = nitration of aromatic AAs with HNO₃ → yellow (like picric acid) → orange with alkali
Xanthoproteic →
= nitration of aromatic AAs with HNO₃ → yellow (like picric acid) → orange with alkali
Lead sulfide (Sulfur test): Black precipitate with Cysteine/Cystine due to H₂S + Pb²⁺ → PbS.
Lead sulfide (Sulfur test): Black precipitate with Cysteine/Cystine due to H₂S + Pb²⁺ → PbS.
Dinitrophenyl hydrazine → “DNPH test → Orange-red precipitate with keto acids (phenylpyruvic acid in PKU) due to carbonyl group reaction.”
Dinitrophenyl hydrazine → “DNPH test → Orange-red precipitate with keto acids (phenylpyruvic acid in PKU) due to carbonyl group reaction.”
 
 

Q. 1 mL of ninhydrin reagent was taken in 3 test tubes. Few drops of solutions A, B and C were added to the three tubes and were kept in a boiling water bath. The resultant color is shown. Solutions A, B, and C, respectively, are?

  • (Image showing test tubes A (purple), B (no color), C (yellow)).
    • A. Arginine, glucose, lysine.
    • B. Lysine, glucose, Arginine.
    • C. Lysine, fructose, Proline.
    • D. Hydroxyproline, fructose and Lysine.
        • Explanation:
        • Ninhydrin reagent + alpha-amino group (e.g., lysine) -> purple color.
        • Ninhydrin reagent + imino group (e.g., proline) -> yellow color.
        • If neither group is present -> no color.

Q. A person gives her urine sample for the routine screening test. Benedict's test result is as follows. All of the following can be a cause except.

  • (Image showing Benedict's test result: Control (blue), Urine (red)).
    • A. Multiple Myeloma.
    • B. Pregnancy.
    • C. Fanconi's syndrome.
    • D. Galactosemia.
        • Explanation:
        • Benedict solution is blue.
        • Patient's urine is red, indicating a positive Benedict's test.
        • A positive test means a reducing substance is in the urine.
        • Multiple myeloma: Bence Jones protein is in urine (not a reducing substance).
        • Pregnancy and lactation: Lactose is in urine (a reducing disaccharide).
        • Fanconi's syndrome: Results in renal glycosuria.
          • PCT normally reabsorbs 60-70% of glucose.
        • Galactosemia: Galactose is in urine (a reducing substance).

Q. A person gives her urine sample for a routine screening test. Benedict's test is positive. Below is the result of Barfoed's test. Which of the following is the most probable cause?

  • (Image showing Barfoed's Test as negative).
    • A. Diabetes mellitus.
    • B. Pregnancy.
    • C. Fanconi's syndrome.
    • D. Galactosemia.

NOTE: Different Fanconis

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Fanconi disease/syndrome
• Proximal tubular reabsorption problem → Type 2 RTA
• Glycosuria, aminoaciduria
Fanconi anemia
(Not syndrome)
• Pancytopenia + radial ray
Fanconi Bickel syndrome
• Mutation in GLUT-2  
• Bickel → Bi → 2 (GLUT 2)

Defect in glucose sensing → ↓ insulin release
• Postprandial Hyperglycemia.
• Fasting Hypoglycemia
• Glycogen accumulation disorder
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VACTERAL Holt - Oram (ASD + Radial Ray) TAR (thrombocytopenia + absent radius) Congenital torticollis → Cock robin position
VACTERAL
Holt - Oram (ASD + Radial Ray)
TAR (thrombocytopenia + absent radius)
Congenital torticollis → Cock robin position
Stranger things characters
  • Dustin (Cleido cranial dysplasia)
  • Robin (Cock robin position)
  • Ray (Radial Ray) Hopper (Holt Oram ASD)

Q. A person's urine answers Benedict's test. The biochemist runs a battery of urine tests to detect the reducing sugar present in urine. Based on the images provided below, find out the substance found in urine. Which of the following is the most probable cause?

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  • (Images showing positive Barfoed's and Mucic Acid tests, negative Seliwanoff Test).
    • A. Diabetes Mellitus.
    • B. Fructosuria.
    • C. Fanconi's syndrome.
    • D. Galactosemia.

Q. On prolonged starvation, which of the following tests will be positive?

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  • (Image showing three test results; C is Rothera's test).
  • Ans. C - Rothera's test - Detects ketone bodies.
      • Explanation:
      • Prolonged starvation causes ketosis.
      • C → Rothera's test is positive in urine (purple color ring).
      • A → Xanthoproteic acid test: aromatic amino acids.
      • B → Benedict's test: reducing substances.
      • D → Benzidine test: blood.
      • D → Urine turning dark on standing: Alkaptonuria.