Urinalysis for detection of abnormal constituents

Dr. Rohini C Sane
Urineanalysis(Urinalysis): Abnormal constituents of Urine with
their clinical applications
Malignant
melanoma
Mucopolysaccharidoses
Porphyria

Routinely performed Biochemical Kidney Function Tests
❖Biochemical Kidney Function Tests include :
1. Measurement of NPN in blood (serum creatinine , uric acid and blood urea). The
major route of excretion of all these compounds is urine.
2. Clearance tests
3. Renal plasma flow
4. Concentration & dilution tests
5. Urinary & plasma osmolality
6. Tests to assess renal acidification
7. Excretion of extraneous compounds like PSP
8. Routine complete urine analysis
9. Serum protein , albumin ,globulin ,A/G ratio
10. Serum Cholesterol
11. Measurement of serum and urine electrolytes(Tests to assess renal handling of
sodium ions)

Standard Routine urine analysis
❖The Standard Routine urine analysis include :
1. Physical examination
2. Chemical examination*
3. Microscopic examination of urine

Abnormal constituents of urine

1. Proteins : normal daily excretion is < 100mg (cannot be detected by the usual
chemical test) .Proteins are secreted by the tubular epithelial cells.
Proteinuria: Increased excretion of Proteins in urine.
Proteins in excreted in urine include plasma Proteins , hemoglobin ,myoglobin ,
Bence Jones proteins and mucus.
2. Amino acids :
Amino aciduria: Increased excretion of amino acids in urine.
3. Reducing sugars : such as glucose ,fructose ,galactose , pentoses and homogentisic
acid
4. Ketone bodies: normal daily excretion is 3-15 mg . Ketonuria is increased excretion
of Ketone bodies in urine and it seen in starvation and Diabetes mellitus .
5. Bile salts and bile pigments : seen in jaundice.
6. Blood : presence of blood in urine (hematuria) is seen in nephritis and in urinary
tract lesions.
7. Porphyrins
8. Mucopolysaccharides
9. Melanin

Proteins as the Abnormal constituents of urine and
Proteinuria

Sources of urinary protein
Urinary protein
Hemoglobin Plasma globulin
Bence- Jones
protein
Mucus from
urinary tract
lesions
Semen and
vaginal
secretion
Protein from
pus
Related
products from
red cells
Plasma Albumin
Contaminationofurinesample:
vaginaldischargeorfaecesshouldbe
avoidedusinga mid-stream
specimenor catherization.

Tests to assess severity of renal disease
• In the case of a renal disease , to assess the severity of the disease , the
following additional tests may be performed.
1. Serum total proteins ,albumin ,globulin and A/G ratio
2. Electrophoresis fractionation of serum proteins
3. Serum cholesterol

Proteinuria
❖Protein in urine is an indicator of leaky glomeruli and is the first sign of
Glomerular injury before a decrease in GFR.
❖The glomerular membrane doesn’t usually allow passage of albumin and
large proteins(e.g. gamma globulins). Most common type of proteinuria is
due to albumin.
❖Amountofproteinfoundinurineunderphysiologicalconditions =<30mg/24hr.
❖Proteinuria: Increased excretion of proteins occurs due to:
1. Increased plasma protein concentration
2. Increased glomerular permeability
3. Reduced tubular reabsorption
4. Increased tubular secretion
❖ Excretion of albumin more than 300mg/24hr is indicative of severe /
significant damage to glomerular membrane.

Mechanism of excretion of urinary protein
Glomerular membrane permeability : to
substances with molecular weight < 69,000
therefore plasma proteins (albumin molecular
weight 69,000 , gamma globulin molecular
weight 189,000) absent in a normal urine .
After glomerular filtration , the most of the
proteins is reabsorbed in tubules .
Renal tubules
secrete a
mucoprotein
called as
Tamma- Horsfall
protein.

Tamma-Horsfall protein
• Renal tubules secrete a mucoprotein called as Tamma-Horsfall protein. This
protein is normally excreted in urine.

Functions of Tamma-Horsfall protein

Tamma-Horsfall protein and Hyaline cast
❖Tamma-Horsfall protein:
▪ A glycoprotein secreted by thick part
of loop of Henle and proximal part of
distal convoluted tubule.
▪ Constitute 1/3rd part total urinary
protein.
▪ Forms the matrix of all casts.
▪ The proteins forms a meshwork of
fibrils that can trap any elements
present in tubular filtrate including
cells ,cell fragments or granular
material.

Glomerular proteinuria
❖Normal levelofUrinaryProtein:100mg/day
Glomerular membrane permeability:tosubstanceswithmolecularweight<69,000thereforeplasma
proteins(albuminmolecularweight 69,000,gamma globulinmolecularweight189,000)absentinnormal
urine.
❖Glomerularproteinuria:
1. Increaseinfilteredloadduetodamagedordiseasedglomeruli /vascularpermeability→–plasma
proteinsappearinurine(glomerulardamagefirstmechanismofproteinuria)
2. AlbuminpredominatesinGlomerularproteinuria.
3. PhysiologicalAlbuminuria:observedinstrenuousexercises,lasttrimesterofpregnancy,adolescents,
severestress,postural(inuprightposition)duetoincreasedhydrostaticpressureinrenalveins.
4. PathologicalAlbuminuriaisclassifiedas
a) PrerenalAlbuminuria:impaired renalcirculationasindehydration,congestiveheartdisease,fever.
b) RenalAlbuminuria:observedinrenaldiseasessuchasnephroticsyndromeand /glomerularnephritis,
urinarytractinfection.
c) PostrenalAlbuminuria:thatoccursinlesionsrenal/urinarytract,bladder,prostrateurethra.
❖Micro-albuminuria:excretionof smallquantityofalbumin(50-300mg/day).It isobservedin Diabetes
Milletus, hypertensionandrenalfailure .

Mechanism of proteinuria
1. Glomerular damage: the capillary walls become more permeable , large
molecular weight proteins mainly albumin is passed in urine.
2. Defect in the reabsorption process of the tubules: diminished tubular
reabsorption low molecular weight proteins are not completely reabsorbed
and hence they are present in urine.
3. The presence of protein may be the first sign of a serious problem.
4. Classification of clinical conditions associated with proteinuria :
a. Prerenal Albuminuria: (which produce proteinuria because of secondary
effects on kidney. Proteinuria disappears when the primary disease is cured).
b. Renal Albuminuria(when a lesion is intrinsic to kidney)
c. Postrenal Albuminuria: proteins may be added in urine as it passes along
urinary tract.

Clinical conditions associated with proteinuria
Glomerular damage proteinuria Defect in the reabsorption process of the
tubules
Glomerular nephritis Pyelonephritis
Hypertension Renal tubular acidosis
Lipoid nephrosis Cystinosis
Interstitial nephritis
Rejection of kidney allografts
Semen and vaginal secretions can also give rise to proteinuria.
Contamination of urine samples ,for vaginal discharge or faeces must be
avoided. To prevent this ,catherization may be essential in female patient ,mid-
stream specimen from the male is satisfactory.

Types of Albuminuria
❖ Albuminuria may be :
1. Physiological Albuminuria: observed in strenuous exercises, high protein diet ,
last trimester of pregnancy ,adolescence, severe stress ,postural (in upright
position)due to increased hydrostatic pressure in renal veins.
2. Pathological Albuminuria is classified as
a) Prerenal Albuminuria(factors operating before blood reaches kidney) : infection,
fever due to toxic effects on kidney, impaired renal circulation as in dehydration,
congestive cardiac failure (CCF), intraabdominal pressure due to tumors ,ascites
etc .
b) Renal Albuminuria( when a lesion is intrinsic to kidney) :observed in renal
diseases such as nephrotic syndrome and /glomerulonephritis.
c) Postrenal Albuminuria: that occurs due to obstruction in lower urinary tract.
❖Micro-albuminuria: small quantity of albumin(50-300mg /day)observed in Diabetes
Mellitus ,hypertension ,renal failure.

Clinical conditions associated with proteinuria
Physiological
Albuminuria
PrerenalAlbuminuria Renal Albuminuria Postrenal
Albuminuria
Strenuous exercises Infection Glomerulonephritis Due to obstruction in
lower urinary tract
High protein diet Fever due to toxic
effects on kidney
in renal diseases such
as nephrotic
syndrome
Lesions of renal
pelvis, bladder and
urethra
Severe stress Fever Lesions of prostate
Last trimester of
pregnancy
Impaired renalcirculation
asindehydration
Adolescence Congestivecardiacfailure
(CCF)/myocardialinfarction
Postural(inupright
position)dueto
increasedhydrostatic
pressureinrenalveins
Intraabdominal pressure
on ascites due to
tumors

Classification of Proteinuria
❖Overflow proteinuria - increased concentration of low molecular weight proteins
leading to their excretion.
❖Hemoglobinuria due to intravascular hemolysis as in : hemolytic conditions such
as sickle cell anemia , thalassemia ,hemolytic anemia ,paroxysmal (induced by
cold and stress).
❖Myoglobinuria : occurs due to break down of muscles as in crush injuries , burns,
electric shock ,malignant hyperthermia, paroxysmal after exercise, following
infraction due to release from myocardium.
❖Presence of Bence Jones proteins in urine : seen in Multi Myeloma patients.
❖Tubular proteinuria : decrease in resorptive capacity due to tubular damage.
❖Nephron loss proteinuria : number of nephrons decreased ,GFR decreased→
remaining nephrons overworking.
❖Urogenic proteinuria – inflammation of lower Urinary Tract
❖Proteins secreted into tract

Overflow proteinuria
• Overflow proteinuria : when small molecular weight proteins are
increased in blood ,they overflow in urine e. g. hemoglobin(molecular
weight 69000) can pass through normal glomeruli .
• If hemoglobin exist in free form , it appears in urine(hemoglobinuria)
e.g. hemolytic conditions .

Tubular proteinuria
❖Tubularproteinuria(specificproteinuria) :
❖Proteinuriaisanindicatorofleakyglomeruli.
❖β2 microglobulinand α1macroglobulin aresmallproteinswhicharefilteredatglomeruliand
reabsorbedbythetubularcellswhenrenaltubularfunctionisnormal.
❖Thepresenceoftheseproteinsinurineisasensitiveindicatorofrenaltubularcelldamage.
Theyaredetectedbyurinaryelectrophoresisandnotnormallyseeninplasmaelectrophoresis.
❖Whenfunctionalnephronsarereduced,GFRdecreasedandremainingnephronsare
overworking.
❖Decreaseintubularreabsorptioncapacityduetotubulardamagesolowmolecularweight
proteinsappearinurine.e.g.retinolbindingprotein(RBP)andAlpha-1-microglobulin.
❖Theseproteinsaresynthesizedinliverandfilteredbytheglomerulus.
❖ Tubulardamageresultsinthereleaseofintracellularcomponentsintheurinarytractandmay
beusedasamarkeroftubulardamagee.g.β-D-glucoaminidase,lysozyme.

Nephron loss proteinuria
❖Nephron loss proteinuria :
In Chronic Kidney Diseases , number of functioning nephrons is
decreased. The compensatory rise in glomerular filtration by other
nephrons .This increases the filtered load of proteins .Even if there is no
glomerular permeability changes ,tubular proteinuria is seen.

Urogenic proteinuria
❖Urogenic proteinuria:
This is due to inflammation of lower urinary tract , when proteins
are secreted into the urinary tract . Accumulation of proteins in
tubular lumen can trigger inflammatory reaction.

Biochemical Tests for Proteinuria
❖Biochemical Tests for Proteinuria : All the methods are based on principle of
precipitation of protein by chemical agents(acids) or coagulation/denaturation by
heat . The Proteinuria is commonly assessed by heat test and Acetic acid test.
❖Overnight first voided urine sample (early morning urine EMU) may be used for
measurement of protein as it rules out the possibility orthostatic albuminuria.
1.Acetic acid test : Urine + 33% Acetic acid dropwise (to achieve isoelectric protein of
albumin)→ heat the upper layer of urine → cloudiness → proteinuria
Calcium & Magnesium phosphate precipitate on heating (precipitate soluble in acidic
medium)
2. Esbach’s test
3. Elisa
4. Radial micro diffusion (detection of microalbumin)
5. Immuno-turbidometry
6. Dipstick replacing the old method

Preparation of a urine sample for Qualitative Tests
for detection of urinary protein
• The urine specimen should be clear.
• If is turbid ,it is necessary to centrifuge it . In that case supernatant is tested
for protein and sediments for microscopic examination.
• Even after centrifugation if the urine is turbid then filter it.
• If the urine is alkaline, add glacial acetic acid dropwise and make it slightly
acidic.

Qualitative Tests for detection of urinary protein
Test Observation Inference
Heat coagulation Test:
Take half test of urine (10ml) heat upper
portion of urine. When turbidity is seen
add 1% of glacial acetic acid) dropwise
till isoelectric pH(PI) is reached and
maximum precipitate observed .
If turbidity disappears after adding
acetic acid ,turbidity is due to presence
of phosphates. (Testsensitivity5mg/dL)
Turbidity due
to coagulum of
protein
No turbidity →
proteins absent.
More reliable
than
Sulfosalicylic
Acid test.
Proteins present in urine
(proteinuria). Proteins are heat
coagulable .
Test is not quantitative but
amount of protein may be roughly
assessed .
(Report protein as trace ,+1,+2 and
+3 depending on amount of
precipitate produced).
SulfosalicylicAcidtest:3mlcentrifuged urine
+20%sulfo-salicylicaciddropwise.
Turbidity after
5 minutes
(proteinuria). Graded as +1 to +4.
Heller’s Test: 3ml concentrated HNO3
+0.5ml of urine from side of test tube to
form upper layer.
White ring at
junction
(proteinuria).

Heat coagulation test and Heller’s Test for detection of
urinary protein
Heat coagulation Test Heller’s Test
Heller’s Test

Esbach’s test to detect Proteinuria
Biochemical test to detect Proteinuria :
Esbach’s reagent (picric acid +citric acid )+ Urine → 24 hr. standing → ppt (gm/L urine )

Application of Dipstick (Albustrix) in Proteinuria
• Albustrix: reagent strips are firm , plastic strips with reagent system
(Tetra-bromophenol blue at pH 3.0 is yellow) at one end . The reagent is an
indicator system and citrate buffer.
• Positive test results : The indicator changes to a shade of green in presence
of Albumin , globulin hemoglobin ,Bence-Jones protein and mucoproteins.
• +1 color block represents ≈ 30 mg of albumin/dL
• Detection limit of Dipstick = 200 -300 mg of Protein/ L
• Benign Proteinuria = 300 mg of Protein/day
• Pathological Proteinuria = 300-1000mg of Protein/day
• Glomerular proteinuria : >1000mg of Protein/day

Specificity of Albustrix
Albustrix
are not
affected
by urinary
Turbidity
X-ray contrast
media
(e.g.Uroselectan)
Quaternary
ammonium
compounds
(e.g. Cetavlon) Most drugs
and their
metabolites
preservatives
which may
affect other
tests.

Precautions for the use of an Albustrix
➢Container for urine : clean, free from contaminants [disinfectant, detergents
(ammonium compounds)]
➢Acid used as a preservative reduces the sensitivity.
➢Do not touch reagent area on the strip.
➢Recap bottle tightly as soon as strip has been removed.
➢Do not leave strip in urine ,or hold in or pass through urine stream ,to avoid
risk of dissolving out reagents.
➢Read a strip in a bright white light.
➢coloured fluorescent lighting may interfere with readings.
➢ Hold strip near to colour chart when making readings.
✓Markedly alkaline urine can give false positive reactions.

Differentiation between protein and a radio-opaque substance
• Uroselectan: used in X-rays of the urinary tract ,a radio-opaque substance.
• In X-rays of the urinary tract ,a radio-opaque substance such as Uroselectan
is used, and if urine is collected following this test false positive reaction
(Pseudo-albuminuria) will be given by sulphosalicylic acid .
• Differentiation between protein and a radio- opaque substance:
Substance Heat test
(boiling test)
Sulphosalicylic
acid
Albumin
Protein Positive(+) positive(+) positive(+)
Radio-opaque
substance
(Uroselectan)
Negative(-) positive(+) Negative(-)

Indications for quantitation of proteinuria
• Diagnosis of nephrotic syndrome: Nephrotic syndrome is a triad of edema ,
hypoalbuminemia and proteinuria (>3g/day) . 24 hours urine protein,
creatinine clearance and sodium should be measured for planning of
appropriate treatment. .
• Prognosis of progressive renal diseases : Proteinuria is a marker for assessing
progressive loss of renal disease , diabetic nephropathy , chronic
glomerulonephritis ,reflux nephropathy. Treatment that reduce proteinuria
(like anti-hypertensive drugs) decrease rate of progression.
• Diagnosis of early diabetic nephropathy : Early stages of diabetic
nephropathy are characterized by increase in glomerular filtration rate (GFR),
microalbuminuria and hypertension.

Applications of Quantitative proteinuria
❖Proteinuria is a biomarker for assessing the progressive loss of renal functions
in renal diseases :
1. Diabetic nephropathy
2. Chronic glomerulonephritis (glomerular permeability)
3. Reflux nephropathy
❖Treatment that reduce Proteinuria (like anti-hypertensive drug)decease the
rate of progression .

Principle of estimation of urinary proteins by Turbidimetry method
❖The estimation of urinary protein is based on the principle of turbidimetry.
❖ Turbidity causes the decrease in the intensity of incident beam of light as it
passes through a solution of particles which do not show tendency to settle.
The measurement of this decrease in incident light beam that is caused by
scattering ,reflectance and absorption of light is called turbidimetry.
❖Urinary proteins are precipitated using 3% trichloroacetic acid.
❖The turbidity is then read at 620nm and compared with turbidity produced in
a standard protein solution(0.5mg/ml) treated similarly.

Procedureofestimationofurinaryproteinsbyturbidimetrymethod
Label three test tubes as Blank , standard and unknown .Then make following additions.
Mixed and read optical density of Blank , standard and unknown colorimetrically at 620nm
(red filter).
Calculation of Urinary protein (mg/dL) using Beer and Lambert ’s law :
OD U –ODB /ODs –ODB x conc. of std (mg/ml) x volume of std x 100 /volume of urine
Urinary protein (mg/dL) : OD U –ODB X 0.5 X 1.O X 1OO/1.0
OD S –ODB
Blank(B) Standard(S) Unknown(U)
Distilled water 1.0 ml - -
Standard protein solution
(0.5mg/ml)
- 1.0ml -
Urine sample - - 1.0ml
3% trichloroacetic acid 4.0ml 4.0ml 4.0ml

Reference interval and values of urine protein
Criteria Urinary protein excretion in 24hr.
Conventional units SI units
Adult 1-14 mg/dL 10-140 mg/L
< 100mg/day < 0.1 g/day
Pregnancy < 150mg/day < 0.15 g/day

Comparison of Turbidimetry and Nephelometry
(methods for estimation of urinary protein):1

Comparison of Turbidimetry and Nephelometry
(methods for Quantitative analysis of urinary protein):2

Urinary protein: Creatinine ratio
• Urinary protein: creatinine ratio in nephrotic syndrome = > 3.5 :1
• Urinary protein: creatinine ratio gives better predictability of chronic kidney
disease (CKD) .

Urinary Albumin : creatinine ratio
• Urinary Albumin : creatinine ratio in males : < 23mg /gm of creatinine
• Urinary Albumin : creatinine ratio in females : < 32 mg /gm of
creatinine
• Patients showing higher values on one occasion are considered as
microalbuminuria.

Applications of measurement of urinary protein
❖Application of measurement of urinary protein may be carried out to:
1. Establish renal disease
2. Define nature of renal disease
3. Define the degree of renal dysfunction
4. Monitor the response to treatment

Methods for estimation of plasma protein concentration :
Kidney and Liver Function Tests based Upon Protein Metabolism
1. Estimation of plasma protein concentration is based on chemical
composition:
A. Peptide bonds:
i. Biuret method :–peptide bond of proteins +copper (Cu2+) + alkaline pH –
violet color complex
ii. Ultra- violet absorption by peptide bond : Spectrophotometer
micro gram protein =(optical density at 215nm- optical density at 225nm)x144
B. Nitrogen content :weight contribution in protein by nitrogen 16%(kjeldahl
method for N content) =weight of protein =N X 6.25

Ultra-violet absorption by peptide bond of a protein

Biuret Test :Kidney and Liver Function Test Based Upon Protein Metabolism

Principle of Estimation of Serum Protein by Biuret method
❖When serum is treated with Biuret reagent, the peptide bonds of protein
react with cupric ions in alkaline medium to form a violet color complex.
• The absorbance of this complex is measured colorimetrically at 530 nm.
• A standard protein solution(6mg/ml) is treated similarly & the color
intensities are compared with a standard of known concentration against
the blank.
• Biuret reagent: Sodium Potassium Tartrate, Copper Sulphate, Potassium
Iodide & Sodium hydroxide

Procedure of Estimation of Serum Protein by Biuret method
• Label three test tubes as Blank (B) ,Standard (S) ,Test (T)
• Pipette reagents as follows :
• Mix & incubate at 37 ᴼC or room temperature for 10 mins. Read optical density at
540 nm /green filter setting zero with blank.
• Concentration of protein in sample (g /dL) = OD Test (T) – OD blank (B) X 6
OD Standard (S) - OD blank (B)
BLANK(B) STANDARD(S) TEST(T)
Distilled water 100 μL - -
Protein standard (6 g /dL) - 100 μL -
Sample - - 100 μL
Biuret reagent 1 ml 1ml 1ml

Serum Proteins in normal and abnormal kidney (Renal) functions
Serum protein type Concentration ( physiological )
Total serum protein 6.5-7.5gm%
Serum albumin 3.5-5gm %
Serum globulin 2.5-3.5gm %
Serum fibrinogen 200-500 mg%
Albumin /globulin(A/G ) 2:1 to 1.5: 1
Renal diseases : Serum albumin decreases (due to proteinuria), A/G deceases.
Chronic liver diseases → Serum albumin (half life 2 days) decreases.
Cirrhosis → hypoalbuminemia and hyperglobulinemia→ A/G deceases.

Clinical conditions associated with increased total serum proteins and
globulins :Clinical applications of Determination of serum protein :1
• An increase in serum total proteins may occur in dehydration .Both albumin
and globulin are increased due to hemoconcentration.
• Increased serum total proteins may be found in multiple myeloma and clinical
conditions associated with high globulin concentrations .
• Clinical conditions associated with high globulin concentrations include:
1. Advanced liver disease
2. Multiple myeloma
3. Chronic infections such as:
a. tuberculosis
b. rheumatoid arthritis
c. subacute bacterial endocarditis
d. lupus erythematosus disseminates etc.

Hyperproteinemia
❖Hyperproteinemia(elevated serum protein concentration) is seen in the
following conditions.
a. Hemoconcentration in dehydration due to inadequate water intake or
excessive water loss as in severe vomiting, diarrhea & Addison’s disease.
b. Disease of reticuloendothelial system also give rise to hyperproteinemia,
frequently due to increase in globulin fraction.
c. In multiple myeloma, total protein concentration is elevated due to
increased synthesis of gamma globulin. Gamma globulins are also increased
in infections such as Tuberculosis & Kala azar.

Clinical conditions associated with decreased total serum proteins :
Clinical applications of Determination of serum protein :2
• Decrease in serum total proteins : is always due to low albumin levels
(Hypoalbuminemia) , accompanied either by no increase in globulins or
an increase in globulins so that the A/G ratio is changed .
• Low serum albumin may be due to :

Clinical conditions associated with Hypoalbuminemia :
Clinical applications of Determination of serum protein
• Hypoalbuminemia (Low serum albumin) may be due to :
a. A heavy loss of protein/albumin from blood in urine (as in nephritis, nephrotic
syndrome, Diabetes Mellitus).
b. Insufficient intake of proteins in the food (malnutrition).
c. Malnutrition disorders when protein intake is diminished.
d. Malabsorption of amino acids from the alimentary tract(as in steatorrhea) .
e. Impaired protein synthesis as a liver disease due to excessive liver damage
(as in cirrhosis of liver).
f. Increased catabolism of proteins (as in fever).
g. Hemodilution due to water intoxication or salt retention.
h. Loss of protein from blood from skin in severe burns.

Renal disorders and A/G ratio
• Hypoproteinemia is often due to decreased serum albumin
concentration.
❖Normal Serum Albumin : Globulin ratio( A/G ratio): 2:1 to 1.5: 1
• The condition in which gamma globulin fraction is elevated ,
A/ G ratio is reversed.
• In renal disorders, there is a loss of albumin whereas globulins
are retained, thus A/G ratio is reversed.

Oedema and total serum/plasma proteins
• A reduction of total serum/plasma proteins is one of the causes of oedema .
• It may take place when total serum/plasma proteins are <5g/ dL and
albumin<2.5g/dL.
• In this condition water from blood passes into tissue spaces. Plasma proteins
may also be decreased in acute or chronic hemorrhage/myxedema .
Acute or chronic hemorrhage

Normal Serum protein electrophoretic pattern
Percentage (%) g /dL
Serum Albumin 55-65 3.5 -.4.7
Serum  1 globulins
(-1 Antitrypsin, ,TBG)
2- 4 .2 - .3
Serum  2 globulins(haptoglobin ,
ceruloplasmin, -2macroglobulin)
6-12 .4 - .9
Beta globulins( β-lipoproteins) 8-12 .5 – 1. 
Gamma globulins
(immunoglobulins)
12-22 .7 – 1. 5

Protein electrophoretic pattern in normal Kidney and Liver functions

Bence-Jones proteins
❖Presence of Bence-Jones proteins (Macroglobulin) : seen in urine of Multi Myeloma
(plasmacytoma, leukemia , Hodgkin’s disease , Lymphosarcoma) patients.
❖Bence-Jones proteins are  globulins with the light chains(κ orλ)of immunoglobulins
and are produced abnormally →monoclonal light chains produced plasmacytoma.
❖Being small molecular weight(44,000) proteins ,they are easily filtered through
normal glomerulus and excreted in urine.
❖Heat Test for Bence-Jones proteins:
When the urine is heated , at 45⁰C they start precipitating and at 60 ⁰C there is
maximum precipitate.
Bence-Jones proteins precipitate or coagulate at temperature between 45-60⁰ C . At
80 ⁰C these proteins start redissolving, redissolve completely at 100 ⁰C . Precipitate
reforms cooling .
Thus , these proteins soluble at temperature < 45⁰C & >60⁰C when heated.
❖It is detected by immunoprecipitation/serum protein electrophoresis(presence of
M band in gamma globulin region).

Concentration of Urine for electrophoretic detection of Bence –
Jones proteins using polyacrylamide hydrogel:1
• As there is usually insufficient protein in the urine for detection by
electrophoretic technique , the urine must be concentrated beforehand.
• Polyacrylamide hydrogel/Lyphogel : concentrates biological fluid in
5hr.
• Mechanism of action of Polyacrylamide hydrogel : each pellet
expands in aqueous solution to absorb 5 times its own weight of water
and low molecular weight substances as salt ,while excluding and other
substances of molecular weight of 20000 or more.

Procedure for Concentration of Urine for electrophoretic detection of
Bence -Jones proteins using polyacrylamide hydrogel:2
1. 10ml urine in a test tube + 1.48g Lyphogel
2. Cover the test tube with parafilm and leave for 5hr.or more.
3. Remove the gel pellets from the solution with forceps
4. Any fluid still adhering to swollen cylinders should be drain back into the
concentrate by touching them to side of the tube.
5. As 1g of Lyphogel absorb 5 ml water , this technique will remove 8 ml water.
6. Apply the concentrate of urine to an equilibrated electrophoretic strip along
with a fresh specimen of patient’s serum if possible.
7. If Bence-Jones protein is present ,the abnormal sharp protein band will
migrate between the beta(β) and gamma(γ )positions during electrophoretic
analysis.

Diagnosis of Multi Myeloma: Bence-Jones proteins in urine by
electrophoresis
Multiple myeloma: malignant proliferation of plasma cells usually in bone marrow.
This disease is associated with presence of urinary Bence-Jones proteins( 50-60% cases).
Presence of M band in gamma globulin region in serum protein electrophoresis is suggestive of
Multiple myeloma in remaining cases .

Micro-albuminuria
❖Micro-albuminuria :
• It is also called minimal albuminuria or pauci-albuminuria.
• It is identified ,when small quantity of albumin is seen in urine (30-300mg/day).
Normal albumin excretion: 2.5-30 mg/dL, Macroalbuminuria: > 100 mg /dL
• The test is not indicated in patients with overflow proteinuria (positive dipstick) .
• Early morning midstream sample is preferred .
• It is early indication of nephropathy in patients with Diabetes mellitus and
hypertension .It serves as a signal of early reversible renal damage.
• Confirmation of microalbuminuria : by overnight urine collection and calculation of
albumin excretion rate (> 20g/min).
• Detection of microalbuminuria : radial immunodiffusion or by enzyme linked
immunosorbent methods.
• Management of microalbuminuria : administration of ACE inhibitors decrease the
rate of microalbuminuria.

Protein selectivity index
❖With severe dysfunction in glomerular permeability ,lager protein molecules
like Transferrin get excreted in urine .
• Albumin /IgG clearance = (urine IgG X serum Albumin)/(urine Albumin x
serum IgG) x100
• Transferrin/IgG clearance= (urine IgG X serum Transferrin)/(urine Transferrin x
serum IgG) x100
• Transferrin/IgG clearance indicates highly specific proteinuria.
• In adults ,measurement of Protein selectivity index is of no apparent benefit.
• In children ,the minimal change in nephropathy causes selective proteinuria.

Test for detection of Urinary tract infection
• The common organisms which can cause urinary tract infections : Escherichia
coli , Enterobacter , Klebsiella ,Proteus species etc. These bacteria contain
enzyme which reduce the nitrates in the urine to nitrite.
• In Urinary tract infection ,test for Urinary Nitrites and Leukocyte esterase is
positive.
❖Strip for detection of urinary Granulocytic leucocytes : Granulocytes contain
esterase which catalyze the conversion of indoxyl carboxylic acid ester (from
reagent area on multistrix) to indoxyl. Indoxyl is oxidized to blue color .
• Test is performed on uncentrifuged urine at room temperature.
❖Strip for detection of urinary Nitrites: this is rapid and indirect method for
the early detection significant but asymptomatic bacteriuria.
▪ The urine must be incubated in the bladder for at least four hours hence early
morning urine specimen is preferred.

Amino acids as the Abnormal
constituents of urine and aminoaciduria

Amino acids as the Normal organic constituent of urine
❖Amino acids as the Normal organic constituent of urine:
➢Amino acids :
1. Normally very little is excreted urine .
2. Increased excretion (aminoaciduria) is abnormal. Aminoaciduria may also
occur secondary ,to other diseases ,such as in renal diseases when damaged
tubules are unable to reabsorb amino acids.

Color reactions of amino acids
Test Positive test results with Amino acids
Ninhydrin test All α-amino acids(except proline ,hydroxy proline)
Sanger’s test (1-fluro-2,4,
dinitro-benzene reagent
forms yellow colored complex with all α-amino
acids)
Xanthoproteic Test Phenylalanine ,Tryptophan , Tyrosine
Modified Million’s Test Tyrosine
Cole’s Aldehyde Test Tryptophan
Nitroprusside test Cysteine
Lead acetate Test Cysteine
Sakaguchi test Arginine
Histidine Test Histidine

Ninhydrin test for detection of urinary amino acids:1
• Qualitative Ninhydrin test for detection of urinary amino acids :
• Other applications of Ninhydrin test include :
1. Quantitative estimation of amino acids in cases of aminoaciduria(intensity of
purple color can be read at 570 nm and compared with a known standard).
2. Used as a to spray for detection of amino acids in urine on paper chromatogram/
Thin layer chromatogram(TLC).
Test observation Inference
2ml urine + 0.5 ml 1% ninhydrin
→ Boil for 1 minute →cool.
Purple color
Proline and
hydroxy
proline forms
yellow
colored
complex.
Given by all α-amino acids
Principle of test : all α-amino acids react
with Ninhydrin(pH 4 - 8) and by oxidative
decarboxylation form CO2 + NH3+ Aldehyde
Reduced ninhydrin reacts with liberated
NH 3 forming a purple colored complex
(Rheumann’s purple).

Ninhydrin test for detection of urinary amino acids :2
TLCfordetectionofaminoacidsandpeptides

Quantitative estimation of amino acids using Ninhydrin
reagent
Principle : all α-amino acids (except proline ,hydroxy proline) react with Ninhydrin
(pH 4 - 8) and by oxidative decarboxylation form CO2 + NH3+ Aldehyde. Reduced
ninhydrin reacts with liberated NH 3 forming a purple colored complex
(Rheumann’s purple). Intensity of purple color can be read at 570 nm and
compared with a known standard.
Heat in the boiling water bath for 10 minutes . Cool to room temperature
(25ᴼC±5ᴼC). Read O.D. at 570nm (yellow filter).
Amino acids in urine (mg/dL)= O.D Test/ O.D standard x 10
Test Standard Blank
Working ninhydrin reagent(ml) 1.0 1.0 1.0
Urine (ml) 1.0 - -
10mg/dL Cysteine standard - 1.0 -
Distilled water(ml) - - 1.0

Principle of Circular chromatography for separation
and identification of urinary amino acids
• Principle: liquid chromatography system is used to separate urinary amino
acids.
• Whatman No.1 filter paper is used as a supporting medium . Cellulose in
Whatman No.1 filter paper makes an ideal support medium where water is
adsorbed between cellulose fibers and forms a stationary hydrophilic phase.
• A small amount (2 -3 μl) of urine sample is applied on Whatman No. 1 circular
paper and it is applied on Whatman No.1 circular paper.
• It is introduced to liquid-liquid system containing n-butanol, acetic acid and
water.
• The various amino acids get separated according to their respective Rf values.
• The separated amino acids are stained by ninhydrin reagent which reacts with
α-amino acids to form violet color complex.

Procedure of Circular chromatography for separation and identification of urinary
amino acids
1. Add sufficient quantity of n-butanol ,acetic acid and water mixture in the circular container
cover it with an airtight lid.
2. Perform the experiment next day ( i.e. after 18 to 24hr.) so that becomes saturated with
the vapors of organic solvents.
3. Mark 5 points on the inner circle of Whatman No.1 circular paper for application of
sample. By using glass capillaries apply one drop each of the amino acid solutions on the
point of application.(If necessary ,heat the solution to dissolve the amino acids).
4. Make the slit at the center of circular filter paper . Attach the paper wick and introduce the
paper wick along the circular filter paper to liquid –liquid system.
5. Allow the solvent to run until it reaches near the periphery of the circular paper. It takes
about 20-30 minutes .
6. Remove the paper and mark the solvent front .
7. Dry it in hot air oven (at 100ᴼ C) for 5 to 10 minutes .
8. Spray with ninhydrin solution.
9. Mark the spots of separated amino acids and determine their Rf values.

Circular chromatography for separation and
identification of urinary amino acids

Phenylketonuria
• Phenylketonuria : in is an inborn error of metabolism , where the body is
unable to converted phenylalanine to tyrosine by deficiency /mutation in
enzyme Phenyl alanine oxidase.
Phenylalanine Tyrosine
• Diagnosis :Phenyl pyruvic acid + FeCl3 → green or blue color(spot test as color
fades in a few minutes to a yellowish color).
• Accumulation of Phenylalanine in the blood ,urine and CSF. Transamination
converts Phenylalanine into phenyl pyruvic acid which is excreted in the urine
as early as two or three weeks after birth.
• Management of Phenylketonuria : dietary control to prevent the infant from
becoming mentally retarded.
• Phenistrrix(sensitivity 8-10mg/dL) :thereagentareaisimpregnatedwithferric
ammoniumsulphate,magnesiumsulphateandcyclo-hexylsulphanicacid.[in30second:
greyishgreen→phenylpyruvicacidpositive, white/cream→phenylpyruvicacid
negative,brownish→ingestedp-aminosalicylicacid(PAS)].ReplacementbyGuthrietest.

Diagnosis of Phenylketonuria
Guthrie test(Bioassay using Bacillus subtilis)

Clinical symptoms of Tyrosinemia II

Signs and symptoms of amino aciduria:1
Aminoaciduria Mutated/absence
ofenzyme
Clinical
manifestations
Molecule
inblood
Substancein
urine
Management
PhenylketonuriatypeI Phenylalanine
hydroxylase
Seizure,Phenyl
alaninehypertoria
Phenylalanine Dietarypyruvate Restrictionof
DietaryPhe
Hypertyrosinemia
typeI
Fumaric
acetoacetate
hydroxylase
Mentalretardation,
Hepatorenaldamage
Tyrosine,
Methionine
Tyrosine,PHPPA Restrictionof
Phenylalanine,
Tyrosine
Alkaptonuria Homogentisicacid
hydroxylase
Arthritis Homogentisic
acid
Homogentisicacid Restrictionof
Phe,Tyr
HomocystinuriatypeI Cystathioninebeta
synthase
Mentalretardation,
Ectopialentis
Homocysteine,
Methionine
Homocysteine Cysteine,
Methionine
HomocystinuriaII Methyltransferase Mentalretardation Homocysteine Homocysteine Folate
Homocystinuria
typeIII
MethyleneTHFA
reductase
Mentalretardation Homocysteine Homocysteine Folate
Histidinemia Histidase Speechdefect,
mentalretardation
Histidine,
Alanine
Imidazolepyruvic
acid
Restrictionof
Histidine

Signs and symptoms of amino aciduria:2
Aminoaciduria Mutated/absenceof
enzyme
Clinicalmanifestations Molecule
inblood
Substancein
urine
Management
/treatment
Cystinuria transporter Urinestones cysteine cysteine Alkalizationoffurine
Hartnup’s disease Transporter(Trp
rejectionbyboweland
renal-tubular
epithelium)
Pellagra,MR,ataxiaand
othercerebellarsigns
Tryptophan Trp,Tyr,Phe,His,
Leu,Ileu,Ala ,Ser,
Gln,Val,lowbasic
aminoacids
lowprotein,
Niacin
Cystathioninuria Cystathionase MR,talipes,deafness Cystathionine Cystathionine Notrequired
Maplesyrup
urinedisease
Branchedchainketo
aciddecarboxylase
Mentalretardation,
acidosis,maplesyrup
odorofurine
Val,Leu,Ileu
ketoacids
Val,Leu,Ileu keto
acids
Restrict Val,
Leu,,Ileu
Methylmalonic
aciduria
MethylmalonylCoA
mutase
Mentalretardation,
ketosis,hypotonia
Methyl
malonic acid
ketonebodies
Methylmalonic
acid,
VitaminB12
Hyperprollinemia
TypeA
Proline
dehydrogenase
Familialnephritis,,renal
hypoplasia,deafness
Proline Proline,Hydroxy
proline,Glycine

Signsandsymptomsofaminoaciduria:3(ureacyclerelated)
Aminoaciduria Mutated/absenceof
enzyme
Clinical
manifestations
Molecule
inblood
Substanceinurine Management
Citrulinuria Argininosuccinate
synthetase
Mental
retardation(MR),
vomiting,seizure
Citrulline,
Ammonia
Citrulline,
Methionine
High Arg,
low protein
diet
Arginosuccinic
aciduria
Arginosuccinate
lyase
MR,Vomiting,
trichorhexis,ataxia
nodosa,coma
Arginosuccinic
acid ,
citrulline
Arginosuccinate
, citrulline
Argininoprotein,
lowprotein diet
Argininemia Arginase Spastic diplegia Arginine,
Ammonia
Arginine,
Ornithine
low protein
diet
Hyperornithinemia Ornithine
decarboxylase
Vomiting,lethargy Ornithine,
Ammonia
Ornithine,Lysine,
Ammonia
lowproteindiet
OTC deficiency
(generalized)
Ornithine
transcarbamolyase
Convulsion,
lethargy,vomiting
Ammonia Oroticacid,Uracil,
Glutamine
lowproteindiet
CPSIdeficiency
(Glycine)
Carbamoylphosphate
synthetaseI
Vomiting,lethargy Glutamine,
Ammonia
Glutamine lowproteindiet

Inborn errors in amino acids transport system in renal tubules
Hartnup disease Cystinuria
Renal Aminoaciduria is seen in :Inborn errors in amino acids transport system in renal
tubules e.g. Hartnup disease ,cystinuria .

Reducing substances as the

Reducing substances found in urine
❖Reducing substances found as abnormal constituent of urine include :
• Glucose : which appears in urine with or without hyperglycemia. Glycosuria without
hyperglycemia is termed as renal Glycosuria.
• Fructose : is found in urine :
1. After ingestion of Fructose- rich food such as fruits , honey etc.
2. In liver disease.
3. In essential Fructosuria due to defective /absence of fructokinase.
4. In hereditary Fructose intolerance which characterized by elevated serum fructose
Fructose-1-phosphate due to deficiency Aldolase B.
• Lactose : appears in urine during lactation and weaning.
• Galactose :excreted in urine in galactosemia ,in which the conversion of galactose to
glucose is impaired .
• Pentoses: appears in urine:
i. After ingestion of large amount of cherries ,prunes and plums.
ii. In Essential Pentosuria.
• Homogentisic acid: is excreted in urine in Alkaptonuria ,an inborn error of Tyrosine
metabolism.

Glycosuria
❖ Normal urine contains small amount of glucose(2-20mg/dL) which can not be
detected by routine biochemical tests.
❖Excretion of detectable amount of reducing sugar in urine called glycosuria. It may
be benign or pathological.
❖Glucose appears in urine when plasma Glucose levels exceed the renal threshold
(Hyperglycemic Glycosuria).
❖ Glycosuria without hyperglycemia (blood glucose levels normal) is due to defective
renal reabsorption and is called renal Glycosuria. It reflects the inability of renal
tubules to reabsorb glucose because of specific tubular lesions and subsequent
lowered renal threshold ,glucose appears in urine after ingestion of food.
❖Renal Glycosuria is usually benign condition and but may be seen in :
▪ Late pregnancy(Non-pathological Glycosuria due to lowered renal threshold ,lactose
may be present in urine.)
▪ Wilson’s disease ,Nephrotic syndrome ,heavy metal poisoning , cystinosis→ tubular
defects that result in decreased ability to reabsorb glucose.

Alimentary glycosuria
❖Alimentary glycosuria :
• Due to intake of large amount glucose /carbohydrates following the intake of
low carbohydrate diet for about a weak.
• Body’s tolerance for glucose is decreased.
• Sugar will pass from intestinal tract into blood stream faster than liver can
remove it.
✓In the cases of transitory glycosuria ,blood sugar level returns to normal once
the person is out of that specific condition which causes hyperglycaemia .

Hyperglycemic Glycosuria
❖Glycosuria with Hyperglycemia occurs in the following conditions :
1. Diabetes Mellitus (the most common cause),myocardial infarction ,brain tumors
2. Endocrinal hyperactivity(hyperthyroidism ,Hyperpituitarism , Hyperadrenalism, Cushing's
syndrome etc.).Injection of these hormones can also produce Glycosuria.
3. Severe liver disease or whole organ disease of Pancreas
4. Infections ,cerebral hemorrhage
5. Anesthesia
6. Asphyxia
7. Stress and anxiety (due to increased epinephrine /glucocorticoid secretion →Transient
Glycosuria →excessive secretion of Cortisol/ thyroid hormone due to emotional stress).
8. Surgical removal of the pancreas
9. Alloxan induced diabetes
10. Administration of phlorizin, an inhibitor of the renal tubular reabsorption of glucose
❖Hyperglycemic Glycosuria occurs when blood glucose level exceeds renal threshold
(175 - 180mg/dl).The renal tubules cannot reabsorb all the filter glucose.

Qualitative Biochemical Test for Glycosuria
Presence of reducing sugar in urine is referred as Glycosuria and can be
detected by Benedict ’s Test .
Benedict ’s Test for Glycosuria:
5ml Benedict’s reagent +
8 drops urine → bring the
reaction mixture to boil.
Observe the change of
color.
Color
precipitate(Green/
Yellow/ Orange/ Red )
Glucose present .Color of precipitate
indicates severity of Glycosuria .
Semi-Quantitation of urinary sugar
(approximate concentration):
0.5gm% →Green, 1.0gm % →Yellow,
1.5gm % →Orange ,
2.0 gm % →Red
False positive Benedict ’s Test: with all reducing substances in urine (Ascorbic acid,
Homogentisic acid ,Hippuric acid , Glutathione , Galactose) .
Detection of Glycosuria can be done using Clinitest / clinistrix / Glucostrix (dipsticks).

Benedict ’s Test
Composition of Benedict’s reagent : Sodium citrate , sodium carbonate and copper sulphate .
Sodium citrate prevents precipitation of cupric hydroxide.

Principle of Glucose oxidase-peroxidase method for
urinary Glucose estimation
❖Principle: Glucose oxidase (GOD) oxidizes the specific substrate
β-D-glucose to gluconic acid & hydrogen peroxide is liberated.
Peroxidase enzyme acts on hydrogen peroxide to liberate nascent
oxygen (O). Nascent oxygen (O) then couples with 4 - amino antipyrine
& phenol to form red quinonimine dye. The intensity of color complex is
directly proportional to the concentration of glucose present in serum.
The intensity of color is measured using green filter (530 nm) &
compared with standard treated similarly.
❖Normal range for urinary Glucose (GOD-POD method): 2-20 mg/dL

Procedure for Glucose oxidase-peroxidase method for urinary
Glucose estimation
Removal of interfering substances adding activated charcoal to urine.
Filter through a fine filter paper.
Dilute 5ml urine to 50ml with distilled water in a volumetric flask (1:10 dilution).
↓
↓
Blank(B) Standard (S) Urine (U)
Distilled water 20μL - -
Glucose standard (1mg/ml) - 20μL -
Diluted Urine - - 20μL
Glucose reagent
(glucose oxidase,
peroxidase, 4-amino
antipyrine and phenol)
3.0 ml 3.0 ml 3.0 ml
*
*
Mix thoroughly and keep tubes at 37ᴼC for 15 minutes . Read colorimetrically
at 530nm /green filter. Urine Glucose (mg/dL)=ODU- ODB/ ODS-OD B X1000

Reducing and non-reducing substances in urine
Sugars
Glucose
Fructose
Lactose
Galactose
Pentoses –ribose , ribulose
Non-carbohydrates
Homogentisic acid
Salicylates
Ascorbic acid
Glucuronides of drugs
 Positive Benedict’s Test (5 ml Benedict’s reagent + 0.5 ml urine)→ boil for 2 minutes→
colored precipitate →indicates Mellituria.

Differential diagnosis of Reducing substances in urine:1
To denote the excretion of specific sugar the suffix “ uria” is added to the
name of sugar.
Glucosuria : Glucose (the most common sugar)excreted in urine
Lactosuria : Lactose excreted in urine
Fructosuria : Fructose excreted in urine
Glycosuria : any Reducing sugar excreted in urine(used incorrectly to denote
Glucose excreted in urine)

Differential diagnosis of Reducing substances in urine:2
Glucosuria Fructosuria Pentosuria
(Xylosuria)
Galactosuria Non-
carbohydrates
Reducing
substances
Diabetes Mellitus,
Transient Glycosuria,
Renal Glycosuria,
Alimentary Glycosuria
Benedict’s Test:
Positive
Deficiency of fructokinase,
Hereditary Fructose
intolerance i.e. Fructose
not metabolized &
excreted in urine
(Aldolase B deficiency)
Seliwanoff’s test : positive
(red color)
Xylosuria:
Deficiency of
xylitol
dehydrogenase
or xylulose
reductase
Bial ’s orcinol test:
positive
Deficiency of
galactose -1-
phosphate
uridyl
transferase
Mucic acid
test: positive
Glucuronides
of drugs,
Salicylates,
Ascorbic acid ,
Homogentisic
acid
Benedict’s
Test: Positive
Lactosuria: normal reducing sugar in urine, elevated in Third trimester of pregnancy and lactation,
lactose intolerance .Methyl amine Test positive

Causes of Fructosuria
❖Causes of Fructosuria: (Fructose excreted in urine)
a. Physiological Fructosuria (harmless condition): found in the urine
eating fruits ,honey and jams . If glucose is found as well it is indicative
of diabetes.
b. Hereditary Fructose intolerance: Fructose not metabolized due to
Aldolase B Deficiency(elevated serum fructose & fructose -1P)
excreted in urine.
c. Deficiency of fructokinase
➢ Fructosuria is harmless condition ,but it must be identified and not
wrongly classified as diabetes.

BiochemicaltestsfordetectionofUrinaryGlucoseandfructose
Molisch’s test: 2ml urine +
1drop of alcoholic alpha-
naphthol solution. Mix . Add
2ml conc.H2SO4 from side of
test tube (without shaking).
Glucose Fructose BothGlucoseand fructoseare
carbohydrates.Carbohydrate
undergooxidationwithconc.H2SO4
toformfurfural.Furfural reactswith
α-naphthol toformviolet complex.
Violet ring at
the junction of
two layers .
Violet ring at
the junction of
two layers
Seliwanoff’s test: 3 ml of
Seliwanoff’s reagent
(Resorcinol and concentrated
HCl) + 0.5 ml of urine→ mix →
heat for 30 seconds(up to
boiling point).
Nocolor appearance
of cherry red
color on
cooling
Fructose/ketosesformsfurfuralwith
conc.HCl fromSeliwanoff’sreagent.
FurfuralreactswithResorcinol to
formcherryredcolor.Aldose
(Glucose)don’t’formfurfuralwith
conc.HCl.
Benedict’s Test ColorPrecipitate ColorPrecipitate Glucoseandfructosearereducing
sugarspresentinurineofDMor
Fructosuriapatientsrespectively.
TheyreduceCuSO4 toCu2O.
Fehling’s Test: 2ml Fehling’s
reagent + 2ml urine. Mix. Boil.
Red
precipitate
Red
precipitate

Principle of Molisch’s test
Carbohydrate undergo oxidation with conc.H2SO4 to form furfural . Furfural reacts with
alpha -naphthol to form violet complex .

Seliwanoff’s test for detection of Urinary fructose
Seliwanoff’s test distinguishes aldoses(Glucose)and ketoses(Fructose). Positive
test is suggestiveofFructosuria(Fructose excreted inurine) . Fructose appears in urine
of patient’s with hepatic disorders.

Composition of Fehling’s reagent used for detection of
reducing urinary sugars
➢Fehling A solution : 7% copper sulphate
➢Fehling B solution : Sodium potassium tartrate and potassium hydroxide
➢Fehling reagent is prepared by mixing equal volume of Fehling Aand Fehling B
solution (1ml each).

Fehling’s Test for detection of urinary reducingsugars

Barford's test distinguishes between urinary monosaccharides and
disaccharides : 1
Barford'stestdistinguishesurinarymonosaccharidesanddisaccharides:qualitative
biochemicalTest)
2ml Barford's reagent
(Copper acetate and
glacial acetic acid) +2ml
urine→ Boil and allow it to
cool .
Glucose/
fructose
Lactose/
Maltose/
Sucrose
Barford's test is used to
distinguish between urinary
monosaccharides and
disaccharides .
Only monosaccharides
reduce cupric ions from
Barford's reagent to
cuprous oxide in weakly
acidic medium .
Disaccharides don’t reduce
Barford's reagent.
Scanty red
precipitate
sticking to
sides of test
tube.
No
precipitate

Barford's test distinguishes between urinary monosaccharide and
disaccharides:2
Barford's test is used to distinguish between urinary monosaccharides and
disaccharides .

Lactosuria
• Lactosuria: Lactose excreted in urine.
• Lactose is second most common reducing sugar in urine.
❖Physiological Lactosuria(harmless condition) :
1. Third trimester of pregnancy and during lactation . This is temporary
condition , Lactosuria disappears upon cessation of lactation . In pregnancy,
it is important to distinguish Lactosuria from glucosuria .
2. Urine of 3 or 5 day old infants: as their digestive systems are not fully
developed and there is deficiency of enzyme lactase.
❖Pathological Lactosuria : children and adults who are deficient in intestinal
lactase.
✓Methylamine Test : A biochemical Test for detection of urinary lactose

Methylamine Test for detection of urinary Lactose
Methylamine Test : Qualitative Test for detection of urinary lactose
Methylamine Test : A biochemical Test for detection of urinary lactose :
5ml of Methyl amine in sodium hydroxide + 1 ml of urine →mix →heat at 56 C
for 30 mins→ red color indicates presence of lactose in urine.

BiochemicaltestsfordetectionofUrinaryLactose
Molisch’s test: 2ml urine + 1drop
of alcoholic alpha naphthol
solution. Mix . Add 2ml
conc.H2SO4 from side of test tube
(without shaking).
Violet ring at
the junction of
two layers
Lactose is a carbohydrate.
Carbohydrate undergo oxidation
with conc.H2SO4 to form furfural.
Furfural reacts with alpha -
naphthol to form violet
complex .
Seliwanoff’s test: 3 ml of
Seliwanoff’s reagent + 0.5 ml of
urine→ mix → heat for 30
seconds(upto boiling point).
No color ketose forms furfural with conc. HCl
from Seliwanoff’s reagent. Furfural
reacts with Resorcinol to form
cherry red color. Aldose (Lactose)
don’t’ form furfural with conc. HCl .
Benedict’s Test Color Precipitate Lactose is reducing sugars present in
urine during Lactosuria .Fehling’s Test: 2ml Fehling’s reagent
+ 2ml urine. Mix and boil.
Red precipitate

Osazone formation for detection urinary sugars Glucose ,
Fructose , Lactose and Maltose
• Osazones are yellow crystalline compounds formed when reducing sugars
( i.e. sugars containing reactive aldehyde and ketone groups) are heated with a
mixture of phenyl hydrazine hydrochloride ,glacial acetic acid and sodium
acetate.
• Different sugars form Osazones with the characteristic crystalline structure to
be observed under microscope.
• Properties of Osazones of Glucose and Fructose : insoluble in hot water
(therefore separate out during boiling)
• Properties of Osazones of Lactose and Maltose : soluble at higher
temperature (therefore they crystallize only on cooling after boiling)
• Clinical application of Osazone formation : is important for identification of
sugars present in the urine i.e. Glycosuria

Osazone preparation for identifying reducing sugars excreted
during Glycosuria
Test(preparations
ofOsazone)
Lactose Maltose Glucose
/fructose
Inference
5drops of glacial
acetic acid + 2
knife points of
sodium acetate +
1 knife points of
phenyl hydrazine
hydrochloride.
Mix well. Then
add 5ml of urine.
Keep boiling
water bath for 30
minutes.
Lactose Osazone
appear like a
lady powder
puff ,having thin
hair like
projections with
a dense center.
Maltose
Osazones
appear
sunflower
shaped with flat,
broad needles
arranged like
the petals of
sunflower.
Osazones of
Glucose/
Fructose
appear bundles
of hay.
Osazones of
Monosaccharide
(Glucose /Fructose) are
identical and insoluble at
high temperature.
Glucose /fructose can
only be distinguished
with the help of
Seliwanoff’s and Rapid
furfural Test. Osazones of
of Lactose and Maltose
are soluble at high
temperature and
therefore crystallize out
on cooling.

Osazones for identifying reducing sugars excreted during Glycosuria
Lactose Maltose Glucose /fructose
Lactose Osazone appear
like a lady powder puff,
having thin hair like
projections with a dense
center.
Maltose Osazones appear
sunflower shaped with
flat , broad needles
arranged like the petals of
sunflower.
Osazones of Glucose/
Fructose appear
bundles of hay.
In Sucrose ,carbon atom 1and 2 are involved in glycosidic linkage are
not freely available for the reaction . Hence sucrose cannot form
Osazones .

MicroscopicanalysisofGlucosazone,Fructosazone,Lactosazoneand
Maltosazone
/ Fructosazone

Galactosuria
❖Galactosuria(very rare condition) : galactose excreted in urine due to
deficiency of galactose -1-phosphate uridylic transferase which catalyzes
galactose -1-phosphate to glucose -1-phosphate .
❖In Galactosemia there is accumulation galactose in brain tissue which results
in physical deterioration and mental retardation in infant .
❖Physiological Galactosuria(harmless) : after taking large amounts of galactose
or lactose
❖ Mucic acid test for detection of urinary galactose /lactose :
urine+ concentrated nitic acid → boil → formation of crystals of Mucic acid
❖Ortho- toluidine test for detection of urinary galactose
❖Management of Galactosuria: Galactose/Lactose free diet as early as
possible after birth.

Mucic acid test for detection of urinary galactose and Lactose

Ortho- toluidine test for detection of urinary Galactose
Test Observation Interpretation
5ml ortho-toluidine reagent in a
test tube + 0.5ml urine → boil for
10 minutes in water bath.
Green color
No green color
Galactose present
(if Lactose and Glucose
absent in urine. Both
Lactose and glucose react
with ortho-toluidine
reagent) .
Galactose absent
(original color of reagent
persist).
Galactose is found in the urine only very rarely , only satisfactory means of
conforming its presence is by chromatographic analysis. Thin layer
chromatography of various sugars is quicker and more sensitive.

Essential Pentosuria
❖Essential Pentosuria (harmless condition) : L-Xylulose excreted in
urine due to deficiency of any of two enzymes( Xylitol dehydrogenase
or Xylulose reductase).
❖ Alimentary Pentosuria : due to ingestion of cherries , berries , plums,
honey ,fruits and jams .
❖Bial’s orcinol test for detection of urinary pentoses :
5ml of Bial ’s orcinol reagent + 0.5ml of urine →mix→ heated to boil →
green color →positive test .

Bial’s orcinol test for detection of urinary pentoses
Essential Pentosuria (harmless condition) : L –Xylulose excreted in urine due to deficiency of
any of two enzymes (Xylitol dehydrogenase or Xylulose reductase).

Results of qualitative tests for reducing sugars
Reducing
sugar
Molisch's
test
Benedict’s
Test
Seliwanoff’s
test
Methylamine
test
Bial’s
test
Yeast
fermentation
test
Glucose + + - - - +
Lactose + + - + - -
Fructose + + + - - +
Pentose + + - - + -
Galactose + + - - - -
Confirmatory Test : Thin layer chromatography

Thin layer chromatography of reducing sugars:1
Sugar Characteristic color
with Aniline –diphenyl
amine locating agent
Rf
Lactose grey 0.14
Galactose grey 0.34
Fructose pink 0.39
Glucose grey 0.42
Pentoses Grey brown > 0.5

Thin layer chromatography of reducing sugars:2
←Solvent front
1. Lactose
2. Glucose
3. Glucose & Galactose
4. Normal urine
5. Urine containing Glucose
and Galactose
6. Fructose
7. Pentose
← Point of application
1 2 3 4 5 6 7
Schematic diagram
Solventfront

Non-carbohydrates Reducing substances
• Non-carbohydrates Reducing substances : many drugs isonicotinic acids ,
para-amino salicylates , penicillin , cephalexin , nalidixic acid are excreted as
conjugates of glucuronic acid.
• Glucuronic acid : In alkaline condition , glucuronic acid is released ,which is
a powerful reducing agent→ so Benedict’s Test positive.
• Salicylates : Salicylic acid is conjugated with Glycine and excreted in urine.
This can give Benedict’s Test positive. This test becomes negative when drug
is withdrawn.
• Ascorbic acid or vitamin C : a powerful reducing agent→ so Benedict’s Test
positive.
• Homogentisic acid : is an intermediate in the catabolism of phenylalanine
and Tyrosine . Alkaptonuria is inborn error of metabolism .
Homogentisic acid → Benedict’s Test positive(yellow precipitate). This can be
distingshed by positive Ferric Chloride Test.

Ketone bodies as the Abnormal
constituents of urine and ketonuria

Ketone bodies : Acetone ,Acetoacetic acid and β-hydroxyl
butyrate
Ketone bodies
(with relative percentage)
Chemical formula formation
Acetone
(2-4 %)
CH3COCH3 Acetone formed in the body
from Acetoacetic acid by loss
of carbon dioxide .
Acetoacetic acid
(18-20%)
CH3COCH3COOH Acetoacetic acid is formed in
the liver in Diabetes Mellitus,
starvation and increased
oxidation of fat to provide
energy.
β-hydroxyl butyrate
(76-78%)
CH3CHOHCH3COOH β-hydroxyl butyric acid
formed from the reduction
of Acetoacetic acid.

Ketonuria
Ketone bodies : Acetone ,Acetoacetic acid , β-hydroxyl butyrate
Ketosis : Presence/accumulation of Ketone bodies in blood .
Ketonuria : Presence /excretion of Ketone bodies in urine.
Concentration of ketone bodies in urine (physiological ): 5-15mg/day
Qualitative tests for detection of urinary Ketone bodies :
1. Rothera’s Test
2. Gerhards Test :
❖Ketostrix are available for rapid test of ketone bodies .
❖Causes of ketosis :
a. Inadequate carbohydrate in diet.
b. A defect in carbohydrate metabolism (the body tends to metabolize
increasing amounts of fatty acids .Due to this, the other intermediary
products such as ketone bodies also increase in blood).

Clinicalconditionsassociatedwithketosisandketonuria
❖Clinical conditions associated with ketosis and ketonuria include :
1. Prolonged fasting
2. Impaired carbohydrate metabolism(pregnancy and Diabetes Mellitus)
3. Starvation
4. Fever
5. Persistent/prolonged vomiting
6. Von Gierke’s Disease
7. Alkalosis
8. Anorexia
➢Detection of ketonuria in a patient with Diabetes Mellitus is of great
significance since a change in insulin dosage or other management is often
indicated.

Diagnosis of ketonuria
.
Since in most instances of ketonuria,
acetone, Acetoacetic acid and beta-hydroxy
butyric acid, all are excreted in urine
simultaneously ,
a test which determines one of these
components is generally satisfactory for the
diagnosis of ketonuria.

Rothera’s Test for detection of urinary Ketone bodies:1
Rothera’s Test : a qualitative biochemical test for detection of urinary Ketone bodies
Excessive excretion of ketone bodies occurs in a state of ketosis which is seen in:
a. Uncontrolled Diabetes Mellitus
b. Starvation
c. Severe vomiting
Saturate 3ml Urine with solid
Ammonium sulphate. Then
add 3drops freshly prepared
Sodium nitroprusside
solution. →shake .Layer
with3ml concentrated
ammonium solution on the
top .
Violet/purple ring at the
junction of 2 layers
Indicates the presence of
acetone or acetoacetic acid
or both . Sodium
nitroprusside reacts with
acetone or acetoacetic acid
to form violet compound.
β- hydroxyl butyric acid →
Rothera’s Test negative

Rothera’s Test for detection of urinary Ketone bodies:2
During the periods of gastro-intestinal disturbances ,acute infections, stress or surgery
and whenever routine management does not control disease ,the urine of all diabetic patients
should be tested for ketone bodies.

Gerhardt’s Test for detection urinary Acetoacetic acid
Gerhardt’s Test for detection urinary Acetoacetic acid: QualitativeBiochemicaltest
Salicylic acid and Salicylates → Gerhards Test false positive
Excessive excretion of ketone bodies occurs in a state of ketosis which is seen in:
a. Uncontrolled Diabetes Mellitus
b. Starvation
c. Severe vomiting
3ml of urine + 10% Ferric
chloride dropwise.
If phosphates present ,
precipitate of ferric
phosphate is formed ,it
should be filtered off. Then
add few drops of ferric
chloride added .
Bordeaux red /purplish color Acetoacetic acid present.
Acetoacetic acid reacts with
ferric chloride to form ferric
acetoacetic acid .

Salicylates
• Salicylates and acetoacetic acid give similar color in Gerhard’s Test .
• They can be differentiated by their behavior on heating .
• If urine is boiled ,acetoacetic acid loses carbon dioxide and is converted
into acetone .
CH3COCH3COOH →CH3COCH3 + CO2 ↗
• Acetone will not give a positive reaction with ferric chloride after boiling.
• Salicylates ,on the other hand are unaffected by boiling.
• If the heating is carried out after adding the ferric chloride ,the color due
acetoacetic acid will disappear ,while the color due to salicylate
persists.

Bile salts and bile pigments
as the Abnormal constituents of urine

Clinical application of urinary bile salts ,bile pigment
and urobilinogen detection
• Clinical application of urinary bile salts ,bile pigment and urobilinogen
detection : useful in diagnosis of jaundice.
• Icterus or jaundice : yellow pigmentation of the skin ,conjunctiva and mucous
membrane by the abnormal increase of bilirubin in blood (serum). This may
be observed in pre-hepatic, hepatic and post-hepatic conditions.
• Differential diagnosis of jaundice by urinalysis:
Type of jaundice Urinary bile pigments Bile salts urobilinogen
Pre-hepatic absent absent Very high +3 to +4
Hepatic Present trace to +4 present Increased +2
Post-hepatic Present +2 to +4 present Presentormaybe absent

Biochemical Tests for detection of urinary Bile salts
Hay’s Sulphur flower Test:
Take 2test tubes (test, control)
Test: 5 ml of urine +sprinkle
dry Sulphur flower on the
surface of the urine.
Control : 5 ml of water +
sprinkle Sulphur flower.
If bile salts is present in
urine , Sulphur flower
sink to the bottom .
In water they do not sink.
Bile salts present (Obstructive
jaundice).
Bile salts reduce surface tension
of urine and hence Sulphur
flower sink to the bottom .
Sulphur flower float if Bile salts
absent(normal/ Physiological).
Petenkoffer’s test : 2ml urine +
1 drop cane sugar solution +2
ml conc. H2SO4
Violet ring at the junction
two liquids.
Canesugarformsfurfuralwithconc.
H2SO4.Furfuralcombineswithbile
saltstoformvioletringcomplex.
BilesaltsandBilepigments:normallyabsentinurinebutwheneverthereisobstructiontotheirpassagee.g.
obstructivejaundice,theyareregurgitatedintogeneralcirculationandexcretedinurine.
Inhemolyticjaundice,excessivebilepigmentsareformedandtheirexcretioninurineisalsoincreased.
Qualitative Test for detection of urinary Bile salts:

Hay’s Sulphur powder Test for detection of urinary bile salts
Sulphurparticlesinktobottom:Bilesaltspresent,Sulphurparticlesremainfloating:bilesaltsabsent

Functions of Bile Salts in Cholesterol solubility:2

Direct and indirect bilirubin in differentiation of Jaundice

Gmelin’s and Fouchet’s test in urine : Liver function tests based
upon bile pigment metabolism
❖Type of Urinary bilirubin is a conjugated bilirubin.
1. Gmelin’s test :
Urine in Kahn’s tube layer it with conc HNO3 → rock the test tube to achieve
maximum contact between two layers→ play of colors at the junction of two
layers (yellow –bilirubin /green –biliverdin/red –Bilifuschin /violet -bilicyanin )
2. Fouchet’s test :
Urine + Barium chloride + Magnesium sulphate → bile pigments adsorbed on
Barium sulphate →filter sing Whatman's filter paper →add Fouchet’s reagent *
→pista green color
➢ Trichloro acetic acid of Fouchet’s *reagent causes de-adsorption of bilirubin
from Magnesium sulphate & Ferric chloride oxidizes bilirubin into biliverdin. .

Biochemical Tests for detection of urinary Bile pigments (Bilirubin)
Gmelin’sTest:TakeKahn’stube.To
it,add3mlconc.HNO3. Thenadd
equalamountofurineslowlyfrom
sideoftesttube.Incaseofnormal
urine,thistestnotbeperformed.
Aplayofcolorsblue,green,
yellow,violetorred.
conc.HNO3oxidizesbilirubinto
multicoloredBiliverdin,Bilifuschin,
Bilicyanin.
Fouchet’s Test:5mlUrine+ 1ml
MgSO4 + 1mlBaCl2 →filter.
MgSO4 +BaCl2→BaSO4(adsorbs
bilepigments).AddFouchet’s
Reagentonprecipitate.
Yellowtogreenishblue
precipitate.
Fouchet’scontainsTCAandFeCl3.
TCAreleases theabsorbed
pigmentfromBaSO4andFeCl3
oxidizesbilirubintogreencolored
biliverdin.
Interpretation of presence of bilirubin in Urine : Hepatitis ,Obstructive jaundice
Qualitative Test for detection of urinary Bile pigments (Bilirubin) :

Concentrated HNO₃
Gmelin’s Test
Urine
Bilirubin –yellow ,Biliverdin –green ,Bilifuschin –Red , Bilicyanin -violet
Gmelin’s test in urine : Liver function test based upon bile pigment metabolism

Fouchet’s Test for detection urinary Bile pigments (Bilirubin):1
Interpretation of presence of bilirubin in Urine : viral ,alcoholic ,toxic and drug
induced Hepatitis ,Obstructive jaundice.

Fouchet’s test in urine : Liver function test based upon bile pigment
metabolism :2
pista green color→

Urinary Bile pigments : Urobilinogen
❖Normal urine contains traces of Urobilinogen (one of the Bile pigments).
• Urobilinogen →oxidation on long standing urobilin→ deepening of color of
urine.
• Normal concentration of urine Urobilinogen = < 4 mg /day(trace)→
Schlesinger test negative/weakly positive .
• Interpretation of presence of increased Urobilinogen in Urine : hepatitis ,
intravascular hemolysis.
• Hemolytic jaundice : Schlesinger test positive for urinary Urobilinogen.
• Hepatocellular jaundice/ obstructive jaundice : Schlesinger test negative (as
low urine Urobilinogen).

Biochemical tests for detection of urinary Urobilinogen
1. Schlesinger test
2. Ehrlich’saldehydeTest
3. Watson and Schwartz’s test
4. Reagentstripmethod(para-dimethyl aminobenzaldehyde=Ehrlich’s reagent)

Schlesinger Test for urinary Bile pigments : Urobilinogen
❖Synthesis of urobilin in human body
Urobilinogen
↓ auto oxidation
Urobilin
❖Schlesinger's test for Urobilin detection:
1.Zinc acetate +alcohol → mix
2. Urine + iodine → mix
①+② →filter →green fluorescence
Schlesinger's test results Interpretation
No green fluorescence Obstructive jaundice
Green fluorescence Hemolytic jaundice

Ehrlich’s aldehyde test ( Liver function tests based upon bile
pigment metabolism):1
❑Synthesis of urobilinogen in human body:
Bilirubin
↓ reduction (intestinal bacteria )
Urobilinogen
❑Ehrlich’s test for Urobilinogen detection :
Urine +Di-methyl aminobenzaldehyde →pink color
Ehrlich’s test results Interpretation
Faint pink Normal
No red color Obstructive jaundice
Distinct red color Hepatic /hemolytic jaundice ,hepatitis

Ehrlich’saldehydeTestfordetectionof urinaryUrobilinogen:2
Positive Ehrlich’s aldehyde Test for detection of urinary Urobilinogen
Hepatic /hemolytic jaundice ,hepatitisNormal urine

Ehrlich’saldehydeTestfordetectionof urinaryUrobilinogen:3
5ml of fresh post-prandial sample
of urine in a test tube + 0.5ml
Ehrlich reagent (para-dimethyl
amino benzaldehyde)→ Mix by
inversion. Allow it to stand for 5
minutes.
Distinct Pink
color(the
intensity directly
proportional to
concentration of
urinary
urobilinogen).
Urobilinogen present in
urine in excess quantity.
Urobilinogen present in
urine reacts with para-
dimethyl amino
benzaldehyde to form
pink color complex.
Ehrlich’s aldehyde Test for detection of urinary Urobilinogen: a biochemical qualitative test
False Negative results of Ehrlich’s aldehyde Test (for detection of urinary Urobilinogen) observed
in:
1. Urinary tract infection(nitrites of bacterial origin)reduce Urobilinogen to urobilin.
2. Antibiotic therapy(as intestinal bacteria i.e. E.coli which produce urobilinogen are killed
by antibiotic therapy).

Ehrlich’s aldehyde Test for detection of urinary Urobilinogen :4
by reagent strip method
(by reagent Strip method) specific for urobilinogen)
Test area is impregnated with Ehrlich reagent(para-dimethyl aminobenzaldehyde)
or methoxybenzene diazonium tetra fluoroborate.

Comparison of types of Jaundice
Criteria Hemolytic /prehepatic jaundice Hepatic (hepatocellular)
jaundice
Obstructive jaundice
Causes hemolysis of RBC –drugs and
toxins ,malaria ,incompatible
blood transfusion ,
hemoglobinopathies,
antibodies ,Gilbert or CN
Syndrome
hepatic cells affected –infection,
viral hepatitis, toxin ,liver
poison/toxic hepatitis,
intrahepatic cholestasis
obstruction to flow of bile-
gallstones ,tumors strictures,
narrowing of bile duct as a
result of surgery/carcinoma
of pancreas , extrahepatic
cholestasis
Liver function Normal Abnormal Normal
Serum Bilirubin
(Van Der Bergh Test )
free or unconjugated bilirubin
increased /indirect positive
(2-5 mg % )
conjugated/direct or
unconjugated-biphasic
Positive (50 mg% )
conjugated/direct
positive
(20 mg% )
Bilirubin(urine)
(Fouchet's’s test)
Negative Positive (+ ) Positive (+ + +)
Urobilinogen (urine)
(Ehrlich test )
Increased /positive Increased /normal Negative /clay color stool
Urobilin (Urine)
(Schlesinger Test)
increased/positive varied negative
Bile Salts (Urine)
(Hay’s Test)
negative positive (+ ) positive (+ +)

Comparison of retention and regurgitation Jaundice
Test Hemolytic /prehepatic /retention jaundice Obstructive/ post hepatic
/regurgitation jaundice
SerumBilirubin
(VanDerBerghTest)
free or unconjugated bilirubin increased /indirect
positive(2-5 mg % )
Conjugated bilirubin/direct
Positive (20 mg% )
TotalSerumBilirubin Increased Increased
unconjugated bilirubin Increased -
Conjugated bilirubin - Increased
Fouchet's ‘s test
(urine bilirubin )
Negative ( unconjugated bilirubin bound to albumin is
water insoluble and not filtered by glomerulus)
Positive (+ + +) conjugated bilirubin is
water soluble
Urobilinogen (urine)
(Ehrlich test )
Increased /positive Negative/normalinviral hepatitisandcirrhosis
.Absentinextrahepaticcholestasisbecausebilirubin
doesn'tentertheintestine
Urobilinogen (fecal )
(Ehrlich test )
Increased /positive Lowinviral hepatitisandcirrhosis.absentin
extrahepaticcholestasis→claycolorstool
Urobilin (Urine)
Schlesinger Test)
increased/positive negative
Bile Salts (Urine)
Hay’s Test
negative positive (+ +)present in cholelithiasis

Porphyrins and porphobilinogen
❖Porphyrins:
▪ Complex iron free cyclic substances.
▪ Represent tetrapyrrole ring ,which consists of four pyrrole rings linked by
methene bridges.
▪ Are intermediates in the biosynthetic pathway of heme.
▪ Produced in bone marrow and liver(main sites of biosynthesis).
▪ Those synthesized in bone marrow are intermediates in biosynthesis of
hemoglobin.
▪ Those synthesized in liver and other tissue are intermediates in biosynthesis
of myoglobin.

Porphobilinogens
❖Properties of Porphobilinogen, Uroporphobilinogen ,
Coproporphobilinogen, Protoporphobilinogen :
▪Colourless substances.
▪Oxidized forms→ corresponding porphyrins(red pigments)
which exhibit fluorescence when observed /viewed under
UV- light.
➢Urine which contains large amount of porphyrins may have
port wine colour.

Porphyria
❖Types of Porphyria:
1. inherited of Porphyria:
a) Erythropoietin : enzymes deficiency occurs in erythrocytes
b) Hepatic : enzymes deficiency lies in the liver
2. Acquired of Porphyria

Disorders of porphyrin metabolism

Autosomal recessive
Autosomal dominant
Erythrodontia
RBC -FLUORESCENCE
Liver ,skin fluorescence

1. Acute intermittent Porphyria
• Age : after puberty (Artist –Vincent , King George III –MAD )
• Deficient enzyme : Uroporphyrinogen synthase I (↑ ALA –no feed back
mechanism)
• No porphyria ,no photosensitivity
• Urinary Excretion: δ- aminolevilinate (ALA) & porphobilinogen (PBG)
• Exposure of porphobilinogen (PBG) to air darkens (formation of Porphobilin)
• Diagnosis : urinary concentration of PBG (darkens on exposure to air)
• Symptoms : intermittent severe Abdominal pain ,Vomiting ,cardiovascular
abnormalities, peripheral neuropathy, neuropsychiatric disturbances
(as ↓ Trp pyrrolase , ↑ Trp, 5 ′OH Tyramine)
• Treatment : Hematin →↓ ALA , ↓ PBG
• Adverse effects : Barbiturate (ppt on attack)→ ↑ ALA synthase
as cytochrome 450 →PBG↑, increase with carbohydrate diet & menopause.

2. Congenital erythropoietin Porphyria
❖Deficient enzyme : ↓ Uroporphyrinogen cosynthtase III
❖Excretion in urine : Uroporphyrinogen I & Coproporphyrinogen I
↓ oxidation ↓
Uroporphyrin I & Coproporphyrin I
❖Symptoms of Congenital erythropoietin Porphyria:
1.↑ Hemolysis
2. Erythrodontia (teeth)
3. Exposure of skin to sunlight → UV sensitization of porphyrin →absorbed and
emit red fluorescent light
4. Dermatitis ,scarring ,burning & itching of skin- ear & nose (Leprosy like),
(as ROS ↑ → accumulation of Porphyrin)
5. Port wine color urine (presence of uroporphyrin I and coproporphyrin I)

Acquired Porphyria
1. Exposure of body to toxic compounds (toys / Xerox ink)
Examples : Heavy metals ( e.g. Lead , Mercury) ↓ALA Dehydratase
Hexachlorobenzene ↓Uroporphyrin synthase I
Drugs (Griseofulvin) ↓ Ferro chelatase
Acute alcoholism
Therefore ↓Heme ,↑ALA synthase and Acquired porphyria associated with
anemia.
2. Liver diseases : infectious hepatitis and obstructive jaundice. Porphyrin
excreted is Coproporphobilinogen-I.
3. Blood dyscrasias : Leukemia ,pernicious anemia ,hemolytic anemia .
Porphyrin excreted is Coproporphobilinogen-I.
↓ALA Dehydratase
↓Uroporphyrin synthase I
↓ Ferro chelatase

Sign and symptoms of Porphyria
Hemolysis

Sign and symptoms of Congenital erythropoietin Porphyria

Procedure of Watson and Schwartz test to detect and
differentiate urinary urobilinogen from porphobilinogen:1
5ml of urine + 5ml Ehrlich’s
reagent → Mix. Allow it stand
for 10 minutes.+ 5ml saturated
sodium acetate . Mix. + 5ml
chloroform . Shake vigorously
and allow aqueous layer and
chloroform layer to separate .
If pink color develops then
follow following test procedure.
Pink layer + Butanol → Mix.
Appearance of pink color
(similar to Ehrlich Test) in
chloroform layer.
Appearance of pink color
(similar to Ehrlich Test) in
aqueous layer.
Appearance of pink color in
butanol layer . Color is easily
detected when viewed from
top of the test tube .
Indicates presence of
urobilinogen in urine.
porphobilinogen in
urine.
porphobilinogen in
urine.

Test results of Watson and Schwartz test to detect and
differentiate urinary urobilinogen from porphobilinogen:2

False negative results of Watson and Schwartz test (to detect
and differentiate urinary urobilinogen from porphobilinogen)
❖False Negative results of Watson and Schwartz test(for detection
of urinary Urobilinogen)observed in:
1. Urinary tract infection(nitrites of bacterial origin reduce Urobilinogen to
urobilin).
2. Antibiotic therapy (as intestinal bacteria i.e. E.coli which produce
urobilinogen are killed/destroyed by antibiotic therapy).

Determination of occult blood
• Occult = hidden
• Occult blood test : capable of detecting even minute quantity of blood
(which is not visible to eyes) in urine.
• Application of Occult blood test : helps to actually detect the free
hemoglobin from lysed red blood cells. In cases where all the red cells stayed
intact(and even the microscopic examination reveals the presence of red
blood cells) ,these tests may give negative test for blood.
• Normalurine:Occultbloodtestnegative(freehemoglobinandmyoglobinabsent)
• Hemoglobinuria and Myoglobinuria : Occult blood test positive
(a correlation must be made with microscopic examination).
➢Hematuria , Hemoglobinuria and Myoglobinuria can occur either individually
or together.

Hematuria (Presence of blood in urine)
❖Intact glomerulus doesn’t allow the passage of RBC. But with severe
glomerular damage/in some kidney diseases ,blood can be passed into urine .
Presence of blood(RBC) in urine is called hematuria .This can often be detected
macroscopically or if the condition is slight ,microscopic examination will reveal
intact blood cells [centrifuged normal urine→ 1or 2RBC per high power field
(hpf) should not be considered abnormal].
❖Thus detection of microscopic hematuria or RBC ,cast confirm glomerular
damage and is the earliest sign before the decrease in GFR.

Clinical conditions associated with Hematuria (presence of blood in urine)
1. Renal diseases :
• Injury /trauma of Urinary tract or kidneys (most common condition)
• Acute or chronic glomerulonephritis
• Acute infections
• Nephrotic syndrome
• Toxic damage to glomerulus
• Malignant hypertension
• Renal calculi
• Acute cystitis
• Tumors in ureter or bladder
2. Traumatic catherization
3. Menstruation
4. Bleeding disorders(Leukemia ,Thrombocytopenia , coagulation
factor deficiency, Sickle cell disease or trait, severe Scurvy)
5. Use of anti-anticoagulant drugs

Hemoglobinemia and Hematuria
Hemoglobinemia: i.e. presence of free hemoglobin in the blood.
Intravascular hemolysis
Release of free hemoglobin from destroyed red cells.
Binding of free hemoglobin to haptoglobin.
Release of excessive hemoglobin in circulation.
Haptoglobin binding capacity exceeded.
Filtration of free hemoglobin through glomerulus.
Reabsorption of free hemoglobin into renal tubular cells.
Loss of unabsorbed hemoglobin in urine.
hemoglobinuria

Hemoglobinuria
❖Hemoglobinuria: is presence of free hemoglobin in urine as a result of
intravascular hemolysis of RBC liberating hemoglobin in plasma . Above
certain level in blood , free hemoglobin is filtered in the glomerulus and
appear in the urine.
❖ Hemolytic conditions associated with Hemoglobinuria :
1. Incompatible blood transfusion
2. Sickle cell anemia
3. Thalassemia
4. Hemolytic anemia (no intact red cells but free hemoglobin present in urine)
5. Enteric fever
6. Malaria
7. Paroxysmal (induced by cold and stress)
8. Exposure to chemicals(sulfonamides ,arsenide)
9. Poisoning from snake venom ,spider bites and bacterial toxins

Color of urine containing hemoglobin
pH of urine Alkaline Color of urine containing hemoglobin
Acidic Normal or dark brown
Alkaline Pink or red
❖Hemoglobinuria should be suspected :
➢ When the test for occult blood is positive but no red blood cells are seen in
microscopic examination .
➢ When the degree of positive test does not correspond to number of red
blood cells in the microscopic examination.

Hematuria and Hemoglobinuria
❖Hematuria and Hemoglobinuria can be detected by three methods :
1.Chemical method (Benzidine test)
2. Microscopic method
3. Spectroscopy
Hematuria → suggestive of nephritis /post renal hemorrhage
Haemoglobinuria → is due to abnormal amount of hemolysis
❖Principle of Benzidine Test for detection of hematuria :
H2O2→H2O +(O) (peroxidase activity by heme)
Benzidine +(O) → Blue /Green Color complex
❖Occult Blood Test in urine can be performed rapidly using Hemostrix strips or
Occult Test tablets .

BenzidineTest for detection of hematuria for Hematuria and Hemoglobinuria:1
Benzidine Test for detection of hematuria for Hematuria and Hemoglobinuria :
(qualitative test and spot test):
In a clean test tube take 3ml
glacial acetic acid and dissolve a
pinch of benzidine powder in it.
Add 3ml of fresh H2O2 solution
→ divide reaction mixture into
two parts .
Test: 1st part + 1ml urine
→Heat . observe color change .
Control : 2nd part + 1ml distilled
H2O →heat
Blue /Green Color appears
immediately
Color disappears on
standing(spot test) to be
observed immediately.
No change of color
Ifbloodispresent,peroxidase
activityofhemeliberatesnascent
oxygenfromH2O2whichreacts
withBenzidinetoformblue/green
colorcomplex.
H2O2→H2O +(O) (peroxidase
activity by heme)
Benzidine +(O) → Blue
/Green Color complex
Changeofcolour incontroltest
indicatescontaminationof
reactionmixture.

Reports of occult blood test
Color Reports of occult blood test for presence of blood
Faint green Trace
Green +
Greenish Blue ++
Blue +++
Deep blue ++++

BenzidineTest for detection of hematuria for Hematuria and Hemoglobinuria:2
If blood is present , peroxidase activity of heme liberates nascent oxygen from H2O2 which
reacts with Benzidine to form blue/green color complex.

Absorption spectra for detection of urinary hemoglobin:1
Type ofhemoglobin Absorptionspectrafor
detectionofurinary
hemoglobin
Function/dysfunction
Oxyhemoglobin Bands appear between D
and E Fraunhofer’s lines .
Alpha(α) band is narrow,
dark and near to D line
(570 millimicrons).
Beta(β) band is broad ,faint
and near E line
(540 millimicrons).
Oxyhemoglobin is a normal form of
hemoglobin and carries O2 from lungs
to tissue and CO2 from tissue to lungs.
Methemoglobin
(Endogenouscyanosis ,
chemicalpoisoningdue
aniline,nitrobenzene,
antipyrine,sulphonamides)
A prominent band in the
red region (533
millimicrons).
1%normalhemoglobin,formedfromhemoglobin
insideRBCandreleasedinplasmaifhemolysis
occursindiseasestate.Conversionofferrousto
ferricdestroysitscapacitytocombinewithO2and
hencecannottransportoxygen.

Absorption spectra for detection of urinary hemoglobin:2
α β

Myoglobin
❖Myoglobin : a heme protein of striated muscle.
❖Function of myoglobin : facilitates the movement of oxygen within
muscle and serve as a reservoir of oxygen .
❖Excretion of myoglobin in urine : is filtered (molecular weight
17000) through the glomerulus and excreted in urine.
❖Toxicity of myoglobin: toxic to the renal glomerulus / tubules and in
large amounts it is associated with acute renal failure .

Clinical conditions associated with Myoglobinuria
❖Clinical conditions associated with Myoglobinuria (occurs due to
break down of muscles as in):
1. Subtle injury /Crush injuries of cardiac or skeletal muscle (release
myoglobin into circulation)
2. Burns / Electric shock/ heat stroke causing destruction of muscles
3. Malignant hyperthermia
4. Paroxysmal after exercise
5. Following myocardial infraction(due to release of myoglobin from
myocardium)

Comparison of hematuria , hemoglobinuria and
Myoglobinuria
Urinary finding Hematuria Hemoglobinuria Myoglobinuria
Color of urine Pink , red,
smoke
Clear pink ,red,
brown
Clear red/ brown
Urinary occult blood strip Positive dots Positive diffuse Positive diffuse
Urinary protein strip Renal +4
Non-renal +/ -
Positive
/negative
Positive
/negative
Urinary RBCs many occasional occasional
Urinary casts Renal RBC
Non-renal
none
hemoglobin Myoglobin
Comparison of Urinary findings:

Alterations in urine test results
Test Falsepositive Falsenegative
Specificgravity Contaminationduringcollectionandstorage None
pH Increasedwhilestandingduetoureaseproducing
microorganisms
Blood Hypochloritebacterialperoxidase Ascorbicacid,nitrites
Protein Fever,cells,bacteria,concentratedurine Dilutionofurine
Glucose Oxidizingagents Ascorbicacid
Ketonebodies Captopril,M-dopa Prolongedstanding
Bilirubin Rifampicin,chlorpromazine Sunlight,Ascorbicacid
Urobilinogen Alkalineurine,Sulphonamide Broadspectrumantibiotics,sunlight
Leukocyte
esterase
Oxidizingagents,Trichomonas Ascorbicacid,Tetracyclines,
cephalosporin
Nitrites Ascorbicacid,Mycobacterium

Urine analysis by strips
Screeningofurine
chemistryby multistrix:
fast,reliable,specificand
sensitive

Multistrix reagent strips for urinalysis:1
❖Multistrix reagent strips : are plastic strips with reagent areas which are
affixed on the strip. These different cellulose areas are impregnated with the
specific testing chemicals according to test.
Urinalysis for Test areas impregnated with chemical/s
Phenyl pyruvic acid Ferric ammonium sulphate, Magnesium sulphate ,
cyclohexyl sulphonic acid
Leucocytes Indoxyl carboxylic acid ester and a buffer
Nitrite p-arsanilic acid
Ascorbic acid Sodium phospho-12-molybdate buffered in an acidic
medium

Multistrix reagent strips for urinalysis:2
Urinalysis for Test areas impregnated with chemical/ reagent
pH Methyl red (pH 4.4-6.2 , bromothymol blue pH 8.0-9.6)
Specific gravity Polyelectrolytes, bromothymol blue
Protein Bromothymol blue
Glucose Glucose –peroxidase enzyme ,chromogen(KI)
Ketone Sodium nitroprusside (or sodium nitro ferric cyanide),
Glycine , a buffer
Bilirubin 2-4 dichloroaniline diazonium salt or 2-6 dichlorobenzene
diazonium tetrafluoroborate
Urobilinogen p-dimethyl amino-benzaldehyde
Occultblood
(Hb,Mb)
3,3’,5’5’-Tetramethyl benzidine peroxidase and
2,5-dimethyl-2-5-dihydroperoxyhexane

Mucopolysaccharides as the Abnormal
constituents of urine and Mucopolysaccharidoses

Types of Mucopolysaccharidoses:1a
Type of
Defective Enzyme/
Enzymes
Biochemical findings
( accumulated products
and their excretion in
urine)
Hurler’s syndrome
(MPSI)
The most severe form
α-L-Iduronidase
(β-galactosidase)
Dermatan sulphate and
Heparan sulphate
Enzyme replacement
therapy (ERT) is available.
Hunter’s syndrome
(MPS II)
Iduronate sulfatase
(β-galactosidase)
X-linked deficiency ,
degradation of dermatan
sulfate and heparan sulphate
are affected. No corneal
clouding . Enzyme
replacement therapy(ERT)
available.

Types of Mucopolysaccharidoses:1b
Type of
Clinical features
Hurler’s syndrome
(MPSI)
The most severe form
Clouding of cornea ,skeletal deformities ,
hepatosplenomegaly
Hunter’s syndrome
(MPS II)
No corneal clouding ,skeletal deformities ,
hepatosplenomegaly , milder course

Type of
DefectiveEnzyme/
Enzymes
( accumulated product/s and its
excretion in urine)
San Filippo Type A(MPS III) Heparan sulfamidase Heparan sulfate
San Filippo Type B
(MPS III)
N-Acetyl
glucosaminase
Heparan sulfate
San Filippo Type C
(MPS III)
Glucosamine-N-
acetyltransferase
Heparan sulfate
San Filippo Type D
(MPS III)
N-Acetylglucoamine-6-
sulfatase
Heparan sulfate
Moquino's syndrome
(MPS IV) Type A
Galactosamine
sulfatase/Galactose 6-
sulfatase
Keratan sulfate and
chondroitin sulfate
Moquino's syndrome
(MPS IV) Type B
Galactosidase Keratan sulfate

Type of
Clinical features
San Filippo (MPS III) Minimal somatic changes ,severe mental
retardation
Moquino's syndrome
(MPS IV) Type A
Severe bone changes ,may be mentally
retarded, Clouding of cornea ,aortic
regurgitation.
Moquino's syndrome
(MPS IV) Type B
Moquino’s Ulrich
Bone deformities ,mental retardation,
deafness

Type of
DefectiveEnzyme/
Enzymes
( accumulated product/s and
its excretion in urine)
Scheie syndrome
(MPS V)
α-L-Induronidase Chondroitin sulfate /
Dermatan sulphate
Maroteaux Lamy
(MPS VI)
N-Acetyl
galactosamine
sulfatase
Dermatan sulphate
SLY syndrome
(MPS VII)
Beta (β)-
Glucuronidase
Dermatan sulphate
Mucolipidosis
(MPS VIII)
β- Glucuronidase Dermatan sulphate and
Heparan sulphate
(MPS IX) Hyaluronidase Hyaluronan

Type of
Clinical features
Scheie syndrome
(MPS V)
Stiff joints ,coarse facies , cloudy cornea ,
may be mentally retarded

Clinical significance of urinary Melanin
• Melanin : a pigment of skin ,hair, and choroid of eye . It is derived from
Tyrosine.
• Normal urine : melanin absent.
• Urine of some patients with malignant melanoma : melanin and its
precursor melanogen present.
• Melanogen is readily oxidized to melanin after exposure to air.
• Urine containing melanin turns brown or black after standing.

Malignant melanoma
Melanogenisreadilyoxidizedtomelanin
afterexposuretoair.Urinecontaining
melaninturnsbrownorblackafterstanding.
Skin cancer

Urinalysis for detection of abnormal constituents

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