Abnormal constituents of Urine

Amit Jha
Lecturer
UCMS, Bhairahawa
• Urine

– Water (95%) & Solids (5%)]

– Urinary out put: 1-1.5 L per day.

– Almost all substances found in urine are also find in

blood.

– may also contain cells, casts, crystals, mucus &

bacteria.
• Urine:

– Provides information about functioning &

abnormalities of kidneys & urinary tract

– Help in diagnosis of various systemic diseases [+nce

or –nce of several substances in urine]
Normal constituents of urine

Creatinine 0.8 – 1.8 gm/L

Uric Acid 0.5 gm/L
Urea 25-30 gm/L
Ca++ 0.2 gM/L
PO43- 1.7gm/L
K+ 1.7 gm/L
Na+ 3.5 gm/L
Cl- 6-16 gm/L
• Preservation

– For routine analysis, urine is best examined fresh.

– Bacterial growth will ruin a specimen if analysis is

delayed for >3 hrs.

– Refrigeration: best way to preserve if analysis is

delayed.

• Refrigeration for >24hrs isn’t recommended.

• Chemicals used
– Toluene
– Thymol [for sugar estimation]
– Formalin
– Boric acid
– Camphor
– Toluol [for acetone estimation]
– Chloroform
 Changes occur in non preserved specimen

Urea → NH3 ↑es pH

↓ Glucose d/t bacterial utilization
↓ KB d/t volatilization
↓ bilirubin d/t exposure to light
↑ bacterial number
↑ turbidity bacteria & amorphous
Disintegration of RBCs casts
↑ nitrite d/t bacterial reduction of nitrate
Changes in color d/t oxidation or reduction of metabolite
URINE ANALYSIS

• Physical

• Chemical

• Microscopic
MACROSCOPIC EXAMINATION OF URINE
 Color
 Clarity
 Odor
 Volume
 Specific gravity
 pH
Color:

• Normal urine is of amber color due to +nce of urochrome

(urobillin & urobilinogen) in urine.

• Colourless: Dilute urine

• Dark: Concentrated urine.

Colorless Dark
High fluid intake Low fluid intake
Use of diuretic Excessive sweating
DM Dehydration (burns, fever)
DI
Alcohol
 Abnormal colour of urine

Cloudy Excess PO4, Urates, Pus cells, Bacterial contamination

Red Frank Hematuria, hemoglobinuria, Myoglobinuria,

Intake of Pyridium, Phenolphthalein
Ingestion of Beet root, Black berries

Deep yellow Obstructive Jaundice, Ingestion of Vitamin B complex

Greenish Obst. Jaundice [excess Billirubin or billiverdin]
Phenol poisoning

Blue Methylene Blue poisoning

Brown black Hemorrhage in bleeding, Acidic urine, Porphyria

Black Alkaptonuria
Milky +nce of Chyle
Cola Nephritic syndrome
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 Clarity (Transparency)
• Normal urine clear or transparent
• Any turbidity will indicate +nce of either of the following:
• WBCs (pus).
• RBCs
• Epithelial cells
• Bacteria
• Casts
• Crystals
• Lymph
• Semen
• Phosphate
 ODOR

Normal fresh urine Faint aromatic odor d/t +nce of volatile acids
Standing for long time Ammoniac odor
Bacterial action of pus (UTI) Offensive odor
Ketoacidosis Fruity odor
Phenylketonurea Mousy odor
VOLUME

Adult 600 – 2500 ml /24hr 0.5-1ml /kg/hr ~ 1.5L/24hr

Children 200–400ml/24hr 4ml/kg/ hr

Oligouria ↓ in urine flow [< 400 ml]

Polyuria ↑ in urine flow [> 2500 ml]
Anuria <100ml/day
Nocturia ↑ urination during night
• Causes of anuria:
• Severe Renal Defect
• Loss of urine formation mechanism.
• Due to +nce of stone or tumor.
• Post transfusion hemolytic reaction.
• Incompatibility between donor`s & receiver's blood
→ hemolysis → excess Hb causes blockage of
renal tubules → acute renal failure.
Causes of polyuria: Causes of Oliguria:

↑ed fluid intake Water deprivation

↑ed salt & protein intake Dehydration
Addison’s disease Prolonged vomiting
Intravenous saline or glucose Diarrohea
Chronic glomerulonephritis Excessive sweating
Diuretics intake Acute renal failure
Psychogenic polydipsia Hypotension
DM Renal Ischemia
DI Obstruction
[Calculi,Tumor, Prostatic hypertrophy]
• pH
– One of imp. functions of kidney is pH regulation.
– Blood pH: 7.4 & urine pH: ~ 6.0 (4.6 – 8.0)
[due to secretion of H+ & reabsorption of HCO3-]
– Urine pH ≥ 9, indicate that urine is stand for a long
time & must be rejected.

Acidic urine Alkaline urine

Acidosis Alkalosis
DKA UTI [Proteus]
Starvation RTA
Dehydration Vegetarian diet
Diarrhea
E. coli infection
Muscular fatigue
Clinical significance of pH
1. Determine existence of acid base disorder.
2. Precipitation of crystals to from stone requires specific
pH for each type.
• Hence, pH control may inhibit formation of these
stones.

Crystals in acidic urine Crystals in alkaline urine

Ca oxalate Ca carbonate
Uric acid Ca phosphate
Mg Phosphate
 Specific gravity
• Normal: 1.015-1.025.
• Theoretical extremes: 1.003 to 1.032.
• Contamination during collection & storage gives false value.

Sp. gravity is ↑ed in

•DM [Glycosuria]
•Nephrosis [Albuminuria]
•All cases of oliguria
•Hematuria
•Hemoglobinuria
•Execessive sweating
Sp. gravity is ↓ed in
•Excessive water intake
•DI
•Chronic glomerulonephritis
•All cases of polyuria [except
DM]
• Low fixed specific gravity
– Due to loss of concentrating ability by damaged tubule,
sp. gravity of urine is fixed at 1.010.
– Found in:
• Chronic glomerulonephritis-end stage kidney
• ADH def.
• Polycystic kidney
• Chronic pyelonephritis
• Chemical examination of Urine

Sugar Blood Porphyrin

Protein Mucin 5-HIAA
KB Bile salt
Hb Bile pigment
• Urine examination for +nce of Sugar
– Glycosuria is defined as presence of sugar in urine in
a amount that can be detected by chemical methods.
– Reducing subst. found in urine:

Sugar Non-sugar
Glucose [DM, Endocrine disorder] CHCl3, Formaldehyde [preservative]
Lactose [Pregnancy, Lactation] Homogentistic acid
Fructose Ascorbic acid
 Hyperglycemic glycosuria
• Blood glucose > Renal threshold for glucose →
glycosuria
• Occurs in Endocrinal disorder
» DM
» Cushing’s syndrome
» Hyperpituitarism
» Hyperadrenalism
• Alimentary Glycosuria
High glucose intake at once for > 1 week
↓
↓ed tolerance of body for glucose
↓
Glycosuria
 Renal glycosuria
Defect in renal tubule
↓
Subsequent lowered renal threshold for glucose
↓
Glycosuria
• Occurs in:
– RTA
– Heavy metal poisoning
– Fanconi’s Syndrome
 Benedict’s Test
• General test for Reducing sugars
• Reagent’s composition:

CuSO4 17.3gm Provide Cu++

Na2CO3 100gm Provide alkaline medium
Na-Citrate 173gm Cu++ chelating agent [slowly releases Cu++ ]
Dist. water 1000ml

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 Benedict’s Test
• Copper reduction test in alkaline medium
• Principle:
– Reducing sugars under alkaline medium, tautomerise
to form enediols (powerful reducing agent), which
reduces Cu++ to Cu+.
CuSO4 → Cu++ + SO4--
Cu++ + Na-citrate → Cu-Na-citrate complex
Reducing sugar → Enediol
Enediol + Cu++ → Cu+ + sugar acids
Cu+ + OH- → CuOH
2 CuOH → Cu2O (↓)

27
• Procedure
– 5mL of Benedict’s reagent was taken in a test tube.
– 8 drops of urine was added.
– Mixed well.
– Boiled for 2 min
– Cooled & color was observed.

Observation Inference
Sample A
Sample B
 Benedict’s Test

Blue color -ve

Green colour Trace < 0.5gm%
Green PPT + 0.5-1.0 gm%
Green to yellow PPT ++ 1.0 – 1.5 gm%
Yellow to red PPT +++ 1.5-2.0 gm%
Brick Red PPT ++++ >2.0 gm%

• Final color formed is dependent on amount of reducing

sugars +nt in given sample, thus benedict’s test is known
as Semi-quantitative test.

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 KETONURIA
• Usually found ketone bodies in human body & urine are:-

β-Hydroxy butyrate --Acetoacetate------ Acetone

[Primary]
• Normal level of ketone bodies in blood: 70mg/dl
• Renal threshold for ketone bodies: 1mg/dl
• normallly excreted in urine. [<20mg/day]
↑ed KB in urine
Intake of high fat & low carbohydrate diet
Starvation
Uncontrolled DM
Prolonged vomiting
 • Rothera’s test [Nitroprusside test]
– Reagents:

(NH4)2SO4 Crystals Precipitate protein

NH3 solution Provide alkaline medium
Freshly prepared 5% Na-Nitroprusside solution
• Rothera’s test [Nitroprusside test]

– Principle: Saturation of urine with (NH4)2SO4 leads

to settling down of proteins as precipitate.
– KB remains at surface.
– In alkaline medium, KB reacts with sodium
Nitroprusside to give purple/pink ring (at interface).
• Rothera’s test [Nitroprusside test]
– Procedure:
– 2 mL of urine was taken in a test tube.
– 3 drops of nitroprusside solution was added to it.

– 2 mL of NH3 solution was added slowly along the

side wall of tube.
Observation Inference
Sample A
Sample B
 Urinary protein (proteinuria)
• Tamm horse fall protein: protein normally found in urine
(<30 mg/24hr). [undetectable by routine methods]
• Proteinuria: defined as +nce of protein in urine that can be
detected by routine methods.

Pre renal Renal Post renal

Cardiac disease Glomerulonephritis
Fever Nephrotic syndrome
Cancer Nephritic syndrome
Collagen disease Carcinoma of kidney
Intra-abdominal tomors Pyelonephritis
Rejection of kidney allograft
Severe UTI
Lesions of renal pelvis
Lesions of bladder
Lesions of prostate
Lesions of urethra
• Classification:
– Glomerular
– Non glomerular
» Tubular
» overflow
• Glomerular
– Causes:
a. Immune complex deposition

b. AGE deposition [Diabetic Nephropathy]

– ↑ in glomerular permeability due to: ↑ in pore size of

glomerular memb. & loss of -ve charges due to podocyte foot
process retraction & basement memb. damage.

– >3.5gm/24hr: hallmark for diagnosis of Nephrotic

syndrome
• Tubular
– Low mol. Wt. Proteins are normally filtered by
glomerulus & completely reabsorbed in PCT.
[eg: β2-microglobulin, Ig- light chains, & RBP]

Loss of tubular function

↓
↓ reabsorption
↓
Tubular proteinuria
– Causes: Toxic agents [Heavy metal, Drugs]
• Overflow:
– Caused by excess production of Ig light chains.
– Multiple myeloma or monoclonal gammopathy of
uncertain significance (MGUS)
 Microalbuminuria
• Albuminuria: well-known predictor of poor renal function in
patients with type 2 DM & in essential HTN.

• Microalbuminuria: Urinary albumin 30 - 300 mg/24-hr urine.

• Overt Albuminuria (Macroalbuminuria)

– Urinary albumin excretion of ≥300 mg/24hr urine
• Urinary albuminuria comprises 20–70% or urinary total
protein excretion
• Bence Jones Protein
– Abnormal low mol. wt. globin consisting of light chains
of Ig (kappa or lamda).

– Characteristics feature:
• Precipitate at 40-60°C
• Precipitate dissolve at 100°C.
• Precipitate reappear on cooling

– Bence jones proteinuria occurs in Multiple myeloma.

 Detection of proteinuria by Heat & acetic acid test
• Heat & acetic acid test:
– Principle: Native protein (Albumin) when heated at pI,
due to unfolding of quarternary, tertiary & secondary
structure gets denatured & thus precipitated.
– Phosphates in alkaline urine also gives similar results.
– Addition of acetic acid, confirms presence of protein if
white coagulum is formed.
• Procedure
– 2/3rd of test tube was filled with urine.
– Upper part of tube was heated.
– Observed & few drops of acetic acid was added.

Observation Inference
Sample A
Sample B
• Heller’s test
– Principle: Conc. HNO3 form acid meta protein in
reaction with protein to form precipitate at the interface.
– Procedure
• 3 mL of conc. HNO3 was taken in a test tube.
• 2 mL of urine was added along the side wall of tube.

Observation Inference
Sample A
Sample B
• Bile pigments found in urine

In Normal urine [< 0.02mg%] In abnormal urine

•Urochrome [Chemical nature unknown]
•Traces of urobilin [Small amount can’t
be detected]
•Bilirubin [in freshly voided urine]
•Urobilinogen
•Biliverdin [develops on standing urine
from oxidation of bilirubin]
•Urobilin [decomposition product of
bilirubin or urobilinogen due to action of
light or action of bacteria]

• Significance of bilirubinuria
– Only conjugated bilirubin appears in urine.
– It occurs with even minimal degree of jaundice &
may be detected before clinical jaundice is evident.
• Fouchet’s test
– Reagents:
• 10% BaCl2
• Fouchet’s reagent [FeCl3 in TCA]
– Principle:
• BaCl2 react with sulphate radicals in urine to form
BaSO4.
• Bile pigment gets adhered toBaSO4.
• Bilirubin (yellow) is oxidised to biliverdin (green)
with FeCl3 in presence of TCA.
• Fouchet’s test
– Procedure:
• 5 mL of urine was taken in a test tubr.
• 5 mL of BaCl2 & pinch of MgSO4 was added to it.
• Filter & dry the filter paper.
• Few drops of fouchet’s reagent was added to filter
paper & was dried.

Observation Inference
Sample A
Sample B
• Bile salts found in urine
– Na-taurocholate
– Na-glycocholate
• Bile salt appear in urine in obstructive jaundice.
 Hay’s surface tension test [Sulphor test]
• Reagent:
– Sulphor powder

• Principle:
– Presence of bile salts ↓es surface tension of urine
allowing sulphor powder to sink.

False +ve hay’s test False -ve hay’s test

CHCl3 Thymol
Turpentine Excess urobilin in urine
 Hay’s surface tension test [Sulphor test]
• Procedure:
– 4mL of urine was taken in a test tube.
– A pinch of sulphor powder was sprinkled on the
surface of urine.

Observation Inference
Sample A
Sample B
• Determination of bile pigment & bile salt in urine is useful
in differential diagnosis of Jaundice.

Pre Hepatic Hepatic Post Hepatic

Bile salt -nt +nt +nt
Bile pigment -nt Trace to 4+ 2+ to 4+
Urobilinogen 3+ to 4+ 2+ +nt/-nt
 Benzidine test
• Capable of detecting even minute amount of blood.
• Detect free Hb/Mb.
• Glacial acetic acid:
– Ruptures RBC
– Act as solvent for Benzidine powder.
• Reagents‘:
– Saturated solution of benzidine

– 3% H2O2
 Benzidine test
• Principle:
– Heme has peroxidase like property causing H2O2 to
release nascent oxygen that reacts with benzidine
solution to give greenish/ bluish color.

Faint green Trace

Green +
Greenish blue ++
Blue +++
Deep Blue ++++
 Benzidine test
• Since urine is free of Hb & Mb.
– So, a positive test should be followed by exact cause
& origin of abnormal findings.
• Benzidine test is +ve in:
» Hematuria
» Hemoglobinuria
» Myoglobinuria
• Hematuria: > 10 intact RBC /HPF in urine
Renal Post renal General disease Disease of neighbouring
organ affecting kidney
Neoplasm Causes of ureter Malaria Carcinoma of vagina
Neoplasm
Calculus
Ureterocele
TB of kidney Causes in bladder Purpura Carcinoma of uterus
Neoplasm
TB
Calculus
Acute Causes of Prostate Scurvy Carcinoma of rectum
glomerulonephritis Benign hyperplasia
Prostatic neoplasm
Prostatic TB
Nephritic Embolism of kidney Acute appendicitis
syndrome from SABE
Pyelonephritis Hemophilia Acute inflammation of
fallopian tube
Pyonephrosis Malignant HTN of
kidney
• Hemoglobinuria
Blood Hb > Hb binding capacity of haptoglobin
↓
Hb filtered
↓
Hb appears in urine
[Hemoglobinuria]
– Occurs in:

Malaria
Septicemia [hemolytic streptococcal infection]
Sickle cell anemia
Thallasemia
Incompatible blood transfusion
Effect of chemicals on RBC [Sulphonamide, Phenylhydrazine, Arsenic, etc]
• Myoglobinuria
Injury to cardiac/ skeletal muscle
↓
Mb released
↓
Excreted via urine
• Mb: toxic to kidney [high concentration may lead to Acute
renal failure.

MI
Infarction of large skeletal muscle
Muscle damage [Injury, Electric shock, Heat stroke]
Trauma