---

Screening of Urine for Inborn Errors of Metabolism & Paper Chromatography

MBBS 1st Year | Biochemistry Lecture Notes

---

PART 1: INBORN ERRORS OF METABOLISM (IEM)

---

1.1 Introduction

• Accumulation of the substrate (before the block)
• Deficiency of the product (after the block)
• Accumulation of abnormal metabolites (via alternate pathways)

---

1.2 Why Screen Urine?

• Many IEMs cause accumulation of abnormal metabolites that spill into urine
• Urine collection is non-invasive - ideal for neonates and infants
• Early diagnosis allows dietary or therapeutic intervention before irreversible damage (especially brain damage) occurs
• Most IEMs causing aminoacidurias present in the neonatal or early infant period

---

1.3 Common IEMs Detected by Urine Screening

---

A. Phenylketonuria (PKU)

Phenylalanine --[PAH blocked]--> Tyrosine (cannot proceed)
       ↓ (alternate pathway)
Phenylpyruvic acid → Phenylacetic acid → Phenyllactic acid

• Elevated phenylpyruvic acid (highest)
• Elevated phenylacetic acid (gives characteristic mousy/musty odor)
• Elevated phenylalanine
• Decreased 5-hydroxyindoleacetic acid
• Elevated urinary indoles (from altered tryptophan metabolism)

• Mental retardation (if untreated)
• Fair skin and hair (reduced tyrosine → reduced melanin)
• Eczema
• Musty body odor

---

B. Alkaptonuria

Phenylalanine → Tyrosine → p-Hydroxyphenylpyruvate → Homogentisic acid
                                                            ↓ [BLOCKED]
                                                    (accumulates in blood & urine)

• Large amounts of homogentisic acid in urine
• Urine turns dark brown-black on standing or at alkaline pH (due to oxidation of homogentisate)

• Dark urine (may be the only early sign)
• Ochronosis - dark-blue to black pigmentation in cartilage and connective tissue (occurs later)
• Arthritis (late complication)

---

C. Maple Syrup Urine Disease (MSUD)

Leucine/Isoleucine/Valine → α-Keto acids --[BLOCKED]--> Cannot be further decarboxylated
                                ↓ (accumulate)
                     Excreted as keto acids in urine

• Elevated leucine, isoleucine, valine
• Corresponding keto acids elevated in urine
• Urine (and cerumen/ear wax) has characteristic maple syrup / caramelized sugar odor

• Severe neonatal vomiting, seizures, lethargy, stupor
• Irregular respirations
• Hypoglycemia
• Rapidly comatose and fatal if untreated

---

D. Tyrosinemia

• Type I (Tyrosinosis): Deficiency of fumarylacetoacetate hydrolase → succinylacetone accumulates → liver failure, renal dysfunction, rickets
• Type II (Richner-Hanhart syndrome): Deficiency of tyrosine aminotransferase → corneal erosions, palmar/plantar hyperkeratosis, mental retardation
• Neonatal tyrosinemia: Benign, transient; lowered p-hydroxyphenylpyruvate hydroxylase activity; common in premature infants

• Tyrosine, p-hydroxyphenylacetic acid, p-hydroxyphenylpyruvic acid excreted
• Tyrosine crystals: fine, silky, brown-to-black; precipitate at acid pH; soluble in alkali

• Nitrosonaphthol test: Nonspecific screening; tyrosine and tyramine form soluble red complexes
• Confirm by chromatography or quantitative serum tyrosine assay

---

E. Cystinuria

Only cystine crystallizes and forms stones (poorly soluble at normal urine pH)

• Recurrent urinary tract calculi (kidney stones)
• Renal colic, hematuria

• Colorless, hexagonal crystals of cystine in urine (first morning specimen)
• Massive cystinuria
• Cystine stones are radio-opaque (contain sulfur)

---

F. Hartnup Disease

• Aminoaciduria of neutral amino acids without elevated blood levels (renal type)

---

1.4 Summary Table of IEM Urine Screening Tests

---

1.5 Neonatal Screening Programs

1. Heel-prick capillary blood sample taken ~7 days after birth (when enzyme expression should be full)
2. Blood blotted onto absorbent filter paper (Guthrie card)
3. Disk placed on agar seeded with a phenylalanine-requiring strain of Bacillus subtilis + β-thienylalanine (competitive inhibitor of phenylalanine uptake)
4. At normal phenylalanine concentrations, bacteria are inhibited and do not grow
5. If phenylalanine is elevated (as in PKU), it overcomes the inhibitor and bacteria form visible colonies

---

PART 2: PAPER CHROMATOGRAPHY

---

2.1 Introduction and Definition

• A stationary phase (does not move)
• A mobile phase (moves and carries the sample)

• Stationary phase: A layer of water (or polar solvent) adsorbed onto cellulose paper fibers
• Mobile phase: An organic solvent system that moves through the paper by capillary action
• The mechanism is primarily partition (distribution between two liquid phases)

---

2.2 Principle

• A compound that is more polar stays in the stationary aqueous phase → moves shorter distance
• A compound that is less polar moves with the organic mobile phase → moves longer distance

---

2.3 Rf Value (Retardation Factor)

• Always a value between 0 and 1 (dimensionless)
• Characteristic for each compound under defined conditions (solvent, paper, temperature)
• Used for identification by comparing unknown Rf with known standard run on the same plate
• If Rf values match AND color with detection reagent matches → compound is identified
• If Rf does not match → compounds are different

---

2.4 Procedure for Paper Chromatography of Urine Amino Acids

• Whatman No. 1 filter paper
• Capillary tube or micropipette
• Chromatography tank/chamber
• Solvent system (mobile phase)
• Ninhydrin spray reagent (for detection of amino acids)
• Reference standards

• Ascending: n-Butanol : Acetic acid : Water (4:1:5 upper phase) - most common
• Descending: Phenol : Water (80:20)
• 2D chromatography: Two different solvents run at 90° to each other (for complex mixtures)

• Mark a pencil baseline 2-3 cm from the bottom of the Whatman paper
• Mark spots at regular intervals (2 cm apart) on the baseline

• Apply a small, concentrated spot (~1-2 µL) of urine (or urine concentrate) using a capillary tube
• Apply known amino acid standards in adjacent lanes
• Allow to dry completely (repeat for concentration if needed)
• Keep spots small (2-3 mm diameter) for good resolution

• Pour solvent into the chromatography tank; allow tank to become saturated with solvent vapors (close lid, wait 30 min)
• Dip the bottom edge of the paper into the solvent (the baseline/sample must be above solvent level)
• Allow solvent to rise by capillary action until it reaches ~2 cm from the top
• Remove the paper; mark the solvent front immediately

• Allow the paper to air-dry in a fume hood (organic solvents must evaporate completely)

• Spray uniformly with 0.2% ninhydrin in acetone
• Heat in an oven at 100°C for 5-10 minutes
• Amino acids react with ninhydrin to give purple/violet spots (Ruhemann's purple)
• Exception: Proline and hydroxyproline give a yellow color with ninhydrin

• Measure the distance from baseline to the center of each spot (Ds)
• Measure the distance from baseline to the solvent front (Dt)
• Calculate Rf = Ds / Dt
• Compare with standards

---

2.5 Detection Reagents Used in Paper Chromatography

---

2.6 Application of Paper Chromatography in Urine IEM Screening

1. Aminoaciduria detection: Paper chromatography of urine separates all amino acids present. In PKU, an excess phenylalanine spot and phenylpyruvate spot are visible. In MSUD, leucine, isoleucine, and valine spots are prominent.
2. Confirmatory diagnosis: After a positive chemical screening test (e.g., FeCl3 positive), paper chromatography confirms which exact amino acid or organic acid is elevated.
3. Monitoring treatment: Dietary restriction therapy in PKU or MSUD can be monitored by comparing amino acid patterns over time.
4. 2D paper chromatography: For complex urine samples with multiple aminoacidurias, the paper is developed in one direction, dried, rotated 90°, and developed again in a different solvent - giving a "fingerprint" map of all amino acids.

From Tietz Textbook: "Another application [of planar chromatography] was in the screening of urine for drugs or metabolites such as amino acids that accumulate during hereditary disorders."

From Harper's Illustrated Biochemistry: "In most centers, the bacterial inhibition test has been superseded by chromatographic techniques that permit the detection of a variety of abnormal metabolites, and hence the detection of a variety of different inborn errors of metabolism."

---

2.7 Types of Chromatography: Comparison (for MBBS context)

---

2.8 Advantages and Limitations of Paper Chromatography

• Simple, inexpensive, no sophisticated equipment needed
• Multiple samples can be run simultaneously on the same paper
• Non-destructive (spot can be cut out for further analysis)
• Good for qualitative identification in resource-limited settings

• Primarily qualitative; not precise for quantitative measurement
• Slow (several hours for development)
• Requires careful technique (spot size, tank saturation)
• Low sensitivity compared to HPLC or MS/MS
• Currently being replaced in clinical labs by HPLC and tandem mass spectrometry

---

PART 3: INTEGRATION - CLINICAL APPROACH TO A SUSPECTED IEM

---

3.1 Clinical Approach

• Observe color (dark urine → alkaptonuria)
• Smell (mousy → PKU; maple syrup → MSUD; sweaty feet → isovaleric acidemia)

• FeCl3 test
• DNPH test
• Cyanide-nitroprusside test
• Ninhydrin test

• Paper chromatography or TLC of urine for amino acid pattern
• Ninhydrin staining for identification and Rf comparison

• Quantitative plasma amino acids by HPLC
• Tandem mass spectrometry (MS/MS)
• Enzyme assay (gold standard)
• Molecular genetics / DNA analysis

---

PART 4: KEY EXAMINATION POINTS (MBBS 1st Year)

---

Must-Know Facts

1. Garrod coined the term "inborn errors of metabolism" - alkaptonuria was his model disease
2. PKU = phenylalanine hydroxylase deficiency; mousy odor; FeCl3 → blue-green; treat with low-Phe diet
3. Alkaptonuria = homogentisic acid oxidase deficiency; urine turns dark on standing; ochronosis
4. MSUD = branched-chain keto acid dehydrogenase deficiency; maple syrup odor; DNPH test positive
5. Cystinuria = renal transport defect (not enzyme deficiency); hexagonal crystals; cyanide-nitroprusside test
6. Hartnup disease = neutral amino acid transport defect; pellagra-like (tryptophan deficiency)
7. Rf value = distance by compound / distance by solvent front; always 0-1
8. Ninhydrin = detection reagent for amino acids → purple color (proline = yellow)
9. Overflow aminoaciduria (high blood + high urine) vs Renal aminoaciduria (normal blood + high urine)
10. Guthrie test = B. subtilis + β-thienylalanine inhibitor; heel-prick blood at day 7; detects PKU

---

Likely Exam Questions

• Describe the principle and procedure of paper chromatography for separation of amino acids
• What is the Rf value? How is it calculated?
• Name the urine screening tests for PKU and MSUD with their principle
• Differentiate overflow vs renal type aminoaciduria

• Describe the biochemical basis, urine findings, and screening tests for PKU, MSUD, and alkaptonuria
• Describe paper chromatography with its applications in detection of inborn errors of metabolism

---

References

• Henry's Clinical Diagnosis and Management by Laboratory Methods, 23rd Ed. - Urinary Screening for Inherited Metabolic Diseases, Chapter on Aminoacidurias
• Tietz Textbook of Laboratory Medicine, 7th Edition - Planar Chromatography (Chapter 19)
• Harper's Illustrated Biochemistry, 32nd Ed. - Screening Neonates for Inborn Errors of Metabolism; Phenylalanine and PKU

---

---

Clinical Images of IEM Presentations

---

1. PHENYLKETONURIA (PKU)

Fig. 26.32 - PKU patient showing characteristic fair skin, light/blonde hair, and eczematous dermatitis over the neck and upper back. Note the scattered red papules - the dermatitis resembles atopic dermatitis and has a predilection for flexures. The patient is described as blond and blue-eyed due to reduced melanin synthesis (tyrosine deficiency → reduced tyrosinase activity).

Source: Andrews' Diseases of the Skin - Clinical Dermatology

---

2. ALKAPTONURIA / OCHRONOSIS

Image A: Osler Sign - Scleral Ochronosis (Conjunctival pigmentation)

Fig. 26.33 - Osler Sign. Grey-blue/whitish ochronotic pigmentation deposited in the sclera and conjunctiva of both eyes. This is one of the earliest visible signs of alkaptonuria, appearing in the 3rd decade of life as homogentisic acid (HGA) deposits in connective tissues. The pigmentation is caused by oxidation of HGA to benzoquinone acetate which polymerizes and binds collagen.

Source: Andrews' Diseases of the Skin - Clinical Dermatology

---

Image B: Ear Cartilage Ochronosis

Fig. 26.34 - Alkaptonuria (Ear Ochronosis). Dark blue-grey pigmentation of the auricular (ear) cartilage - one of the earliest and most characteristic signs of ochronosis. The cartilage is rich in collagen, which avidly binds the ochronotic pigment. The ear cartilage and the sclera are the two earliest sites of visible pigmentation.

Source: Andrews' Diseases of the Skin - Clinical Dermatology

---

Image C: Scleral Ochronosis (Ophthalmology view - rectus tendon pigmentation)

Fig. 9.16 (Kanski's Clinical Ophthalmology) - Alkaptonuria showing heavy dark ochronotic pigmentation of the sclera and the horizontal rectus tendons of the eye. The dark irregular deposits follow the course of the tendons, and the scleral pigmentation is patchy and deep-black. This is a more advanced presentation than the Osler sign above.

Source: Kanski's Clinical Ophthalmology, 10th Edition

---

3. CYSTINURIA

Fig. 50.8 - Cystinuria (Comprehensive Clinical Nephrology)

• Panel A: Rough and smooth cystine calculi (kidney stones) - orange-brown, granular texture, multiple sizes. They are radio-opaque due to sulfur content.
• Panel B: Plain abdominal radiograph showing a cystine calculus in the right renal pelvis and multiple parenchymal calculi.
• Panel C: Urine microscopy - characteristic flat hexagonal crystals of cystine. Their presence is pathognomonic of cystinuria. They are insoluble at urine pH up to 7.4.

Source: Comprehensive Clinical Nephrology, 7th Edition

---

4. URINE CRYSTALS - MICROSCOPY PANEL (for comparison)

Fig. 4.4 (G-K) - Urinary Crystals - Phase contrast microscopy (Comprehensive Clinical Nephrology)

• Panel G (top left): Cystine crystals - classic hexagonal plates stacked/heaped on one another, with irregular edges and faint rainbow birefringence. These are pathognomonic of cystinuria when found in urine.
• Panels H-K show drug crystals for comparison (2,8-dihydroxyadenine, amoxicillin, ciprofloxacin, indinavir).

Source: Comprehensive Clinical Nephrology, 7th Edition

---

Quick Visual Summary for Exam

---

Clinical Images of IEM Presentations

---

1. PKU - Fair Skin, Light Hair + Eczematous Dermatitis

---

2. Alkaptonuria - Osler Sign (Scleral Ochronosis)

---

3. Alkaptonuria - Ear Cartilage Ochronosis

---

4. Alkaptonuria - Ocular Ochronosis + Rectus Tendon Pigmentation (Ophthalmology view)

---

5. Cystinuria - Stones + X-Ray + Urine Microscopy (3 panels)

• A - Rough, orange-brown cystine kidney stones (radio-opaque due to sulfur)
• B - Plain abdominal X-ray showing cystine calculus in right renal pelvis + parenchymal calculi
• C - Urine microscopy: flat hexagonal crystals - pathognomonic of cystinuria

---

6. Urine Crystal Microscopy - Cystine Crystals (Panel G) vs Others

• G (top left) - Cystine crystals: hexagonal plates stacked on each other, faint rainbow birefringence - pathognomonic when found in urine
• H-K - Drug crystals (2,8-dihydroxyadenine, amoxicillin, ciprofloxacin, indinavir) - for comparison

---

Exam Summary