Urinalysis for detection of normal inorganic and organic constituents

An illustrative presentation on urinalysis for detection of normal inorganic and organic constituents for medical, dental , pharmacology and biotechnology students to facilitate easy-learning.

1. Dr. Rohini C Sane
Urine analysis (Urinalysis): Normal inorganic and organic constituents
Ammonium phosphomolybdate (canary yellow).

2. 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.Clearancetests
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)

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

5. Specimen collection for Standard urine analysis (urinalysis):1
Type of urine sample Application in clinical practice
More concentrated urine sampleFirst voided midstream (avoid initial portion of the
urine steam)morning sample in a clean, dry and wide
mouth plastic / glass bottle with screw cap tops.
Suitable for qualitative analysis of urine.
Urine specimen collection for
infants and young children
In disposable collection apparatus(a plastic bag with
an adhesive backing about the opening to fasten it to
child so that he/she void directly in a bag). Avoidfecal
contamination.
Random Urine samples (collected
randomly during the day and are some
times so dilute due to increased fluid intake)
suitable for urgent routine examination to get general
idea of expected pathological condition but tend to
givefalse positive picture of patient health.
Post prandial urine sample (collected
2hr. after lunch or dinner are the best)
detection of glycosuria
Urine samples collected for 24hours quantitative analysis of urine

6. Specimen collection for Standard urine analysis (urinalysis):2
Type of urine sample: First voided
midstream morning sample in a clean,
dry and wide mouth plastic bottle with
screw cap tops.
Urine specimen collection for infants and young
children: In disposable collection apparatus( plastic
bag with adhesive seal and opening to fasten to
child so that he/ she voids into bag) , avoid fecal
contamination

7. Specimen collection for Standard urine analysis (urinalysis):3

8. Urine samples collected for 24hours
•Useful for quantitativeanalysisof urine.
•Needs careful collection.
•Stoppered during collection to preserve the urine sample as bacterial
contamination during the collection period may affect the constituent to be
analysed.
•Preferablyshould be stored at 2ᴼ -8ᴼ C in a refrigerator(cool and dry) .
•Certain preservatives should be added if chemically unstable compounds to be
analysed.
•Type of preservative for urine : depends on analyte to be determined.
•The most commonly used preservativefor urine: 10% solution of Thymolin
isopropanol (5 ml for 24 hr. urine sample/one small crystal per 100ml of urine).

9. Expected changes in the composition of stored urine at room temperature
Lysis of red blood cells by hypotonic urine
Decomposition of cast
Bacterial multiplication
Decrease in glucose due to bacterial growth
Formation of ammonia from urea by action of bacteria and the nature of urine changes to
alkaline

10. Preservation of urine specimen for urinalysis
•All the specimens for routine analysis should be examined while fresh (within
1hr. after collection).
•When urine is to be kept for longer than one hour before analysis ,avoid
deuteriation of chemical , cellular material and multiplication of bacteria ,it
should be stored at 2ᴼ -8ᴼ C in a refrigerator (cool and dry conditions).

11. Preservatives for 24 hours Urine sample
PreservativesConcentration Application and limitation for urinary examination
Concentrated
HCl
10ml /24hr.
Urine(20ml2NHCl
24 hr. urinary sample preservation for VMA, Steroids , adrenaline,
noradrenaline, quantitative analysis (e.g. ammonia , urea , calcium).
Toluene
(best and all
round)
2ml/100ml urineas a physical barrier as it floats on the surface of urine. Good for chemical
constituents . It is not effective if bacteria are already present urine.
interferes protein estimation by sulphosalicylic acid.
Boric acid5g/120 ml urine General preservative
Thymol One small crystal
per 100ml of
urine
Inhibits bacterial and fungi growth. May interfere with acid precipitation
test for protein . preserves sediments, interfere with reducing sugars and
acetone reactions . suitable for estimation electrolytes , amylase, urea,NH
3.
Formalin 1 drop /100mlPreservation of formed elements. May precipitate protein.
Chloroform5ml/100ml of
urine
Forms upper layer . It causes no changes in the characteristics
of the cellular sediment. Interfere the test for sugars .
Commercial
preservation tablet
1 tablet /30ml
urine
tablets release formaldehyde. Concentration of formaldehyde
is controlled , so that it may not interfere.

12. Normal daily excretion of solutes
Compound Total daily urinary excretion under normal dietary intake
Sodium 100 –200 mmols 2–4 g
Potassium 50 –70mmols 1.5 –2 g
Magnesium 4–8 mmols 0.1 –0.2 g
Calcium 1.2 –3.7 mmols 0.1 –0.3 g
Phosphate 20 –50 mmols 0.7 –1.6 g
Chloride 100–250 mmols 110 -125 mEq
Bicarbonate 0––50 mmols 9-16 g
Sulphate (inorganic) 40 –120 mequ 0.6 –1.8 g
Sulphate (organic) 0. 06–0.2 g
urea 15 –30 g
Creatinine 1 –2g 0.3–0.8 g
Uric acid 0.5–0.8 g 0. 08 –0.2 g
Ammonia 30 –75 mequ 0. 04 –1 g
Amino acids 0. 08 –0.15 g

13. Quantity of a constituent excreted per 24hours:1
constituent Quantity of
constituent excreted
per 24hours
constituent Quantity of
constituent excreted
per 24hours
Sodium 2–4 g Urea 6–18 g
Potassium 1.5 –2 g Creatinine 0.3–0.8 g
Magnesium 0.1 –0.2 g Creatine 60-150mg
Calcium 0.1 –0.3 g Uric acid 0. 08 –0.2 g
Inorganic phosphate 0.7 –1.6 g Ammonia 0. 04 –1 g
Chloride 9-16g Amino acids 0. 08 –0.15 g
Bicarbonate 9-16 g Hippuric acid 0.1-1.0 g
Sulphate (inorganic) 0.6 –1.8 g Ketone bodies 3-15mg
Sulphur 0.7-3.5 g Iodine 50-250μg
Indican 0.4-2mg Arsenic <50μg
Oxalic acid 15-20mg Lead <50g

14. Quantity of a constituent excreted per 24hours:2
constituent Quantity of constituent
excreted per 24hours.
Purine bases 7-10 mg
Allantoin 20-30mg
Coproporphyrins 60-280μg
Phenols 0.2-0.5 g
Vitamins small quantity
Hormones small quantity
enzymes small quantity
These values generally vary with diet.

15. Physical examination of urine

16. Physical examination of urine
Physical examination of urine include:
1.The 24 hours urinary output (volume)
2.Appearance
3.pH
4.Color
5.odor
6.Specific gravity
7.Osmolarity

17. Volume as a physical characteristic of urine: 1a
❖Normal urine output : 600-2500 ml per day with an average about 1200 ml
per day.
❖Urine output is influenced by
•Fluid intake
•External temperature
•Diet
•Beverages
•Coffee
•Tea
•Alcohol (has diuretic effect)

18. Volume as a physical characteristic of urine: 1b
Condition Observation
Polyuriaincreased excretion of urine ( >3000 ml/ 24 hr.) seen in Diabetes Mellitus,
Diabetes Insipidus ,excess water intake, intake of diuretics(caffeine ,
alcohol),cardiac failure
Oliguriadecreased excretion of urine (<400 ml/ 24 hr.)seen in fluid deprivation,
excess fluid loss as in hemorrhage , neurogenic shock, dehydration,
obstruction in the urinary tract, Acute nephritis ,early stages of
glomerulonephritis ,cardiac failure, fever, diarrhea
Nocturia increased excretion in urine at night occurs during early stages of renal
disease followed by oliguria (decreased in urine volume).
Anuria complete cessation of urination is seen in terminal stages of renal
disease/failure (<100 ml/ 24 h),acute tubular necrosis, bilateral renal
stones, surgical shock.

19. Volume as a physical characteristic of urine: 1c
❖Volume as Physical characteristics of urine :
Conditions associated with increased
volume of urine
Conditions associated with
decreased volume of urine
Excessive water intake (may be psychological ) Dehydration , fluid deprivation
Increased salt intake Excessive Sweating
Chronic renal disease( due to tubular damage or
osmotic diuresis)
Kidney damage(e.g. Acute
glomerulonephritis, obstruction in
urinary tract)
Diabetes mellitus (due toosmotic diuresis ) Low blood pressure shock
Diabetes insipidus Edema of any etiology
Diuretic therapy Antidiuretic hormone therapy

20. Appearance ofurine as Physical characteristic of urine:2a
Appearance ofurine as Physical characteristic of urine:2
Condition Appearance of urine
Normal Clear,Transparent when fresh
AbnormalTurbid (Presence of Phosphates / Pus cells/ bacteria/Chyle /
Obstruction of Lymphatics in the Urinary tract due to
Filariasis, may be due to fat particles in an individual with
nephrotic syndrome)

21. Appearance ofurine as Physical characteristic of urine:2b
Normal urine Turbid urine

22. pH as a Chemical characteristics of urine:3a

23. pH as a Chemical characteristics of urine:3b
Chemical characteristics of urine :
Reactions to litmus ofnormalurine: acidic , average pH 6(5.5-8.0)
Titrable acidity = 250 -700 ml of 0.1 N acid /HCl ,25 mequ of H
+
❖Urine becomes acidic in following conditions :
a)protein rich diet (due to increase in phosphates and or sulphate)
b)Metabolic or respiratory Acidosis or renal tubular acidosis
c)Fever
❖Urine becomes alkaline in following conditions :
1.On long standing urea →ammonia (decomposition by urease -bacterial activity)
2.After meals (postprandial Alkaline tide)
3.Metabolic or respiratory alkalosis (blood alkaline →urine alkaline)
4.Vegetables containing Citric & Tartaric acid →conversion to bicarbonate
5.Urinary tract infection

24. Color ofurine as a physical characteristic of urine: 4a
Color ofurine Conditions
Amber /straw Normal ( presence of urochrome , urobilin , uroerythrin , hematoporphyrin
–to a minor extent)
Dark /greenish Yellow Jaundice (presence of bile pigments) ,Riboflavin/vitamin B complex intake
,high fever
Black Alkaptonuria ,Malignant Melanoma , formic acid poisoning
Smoky Red Hematuria (presence of blood) due to renal stones ,cancer some injury or
disease of urinary tract of kidney ,Rifampicin
Brownish Red hemoglobinura(presence of hemoglobin),myoglobin
Dark brown presence of methemoglobin
Port wine presence of porphyrins
Darkens on long standing Alkaptonuria(presence of homogentisic acids)
Milky urine Chyluria (fat in urine)
Darkening of pale yellow color of urine indicates presence of concentrated urine or presence of another pigment.

25. Color ofurine as a physical characteristic of urine: 4b
Normal urine straw colored( due to
presence of urochrome)

26. Color ofurine as a physical characteristic of urine: 4c
Alkaptonuria Chyluria

27. Odor ofurine as a Physical characteristic of urine:5
Odorofurine Conclusion
Aromatic (and on long standing
an ammoniacal due to urea
splitting bacteria )
Normal of freshly voided urine(due to
presence of volatile organic acids)
Aromatic Presence of organic acids
Fruity Presence of acetone (severe Diabetes
Mellitus-ketoacidosis)
Maple syrup or burnt sugar Maple syrup urine disease
An ammoniacal Presence of Urea splitting organisms
Unpleasant odor(foul smell)Presence of Gram negative organisms

28. Specific Gravity of urine as a physical characteristic of urine: 6a
❖The simplest of tubular dysfunction is assessed by measurement of Specific
Gravity of early morning urine. Specific Gravity is an indication of osmolality.
❖SpecificGravityofurine depends on concentration of solutes whereas osmolarity
depends on the number of osmotically active particles .Hence in cases proteinuria ,
the Specific Gravity ofurine is elevatedsignificantly,but osmolarity is mildly
elevated .
❖The earliest manifestation of renal disease may be due to difficulty in concentrating
the urine (even when blood urea is normal in some cases)
❖Determination of specific gravity : using urinometer/ urine strips for specific gravity.
❖Specific gravity of at least one sample /specimen should exceed 1.020
❖Maximum specific gravity < 1.020 indicate impaired renal function.
❖Specific gravity of urine<1.003 : suggestive of Diabetes insipidus.
❖Abnormal constituents of urine which elevate specific gravity : Glucose, protein

29. Specific Gravity ofurine as a physical characteristic of urine: 6b
❖Specific gravity of urine can vary widely depending on diet, fluid intake and renal
function. Specificgravity can also be determined by urine strip method .
❖Normal range of Specific Gravity of early morning urine: 1.010-1.020 (measured using
urinometer)
❖Normal range of Specific Gravity of 24 hour specimen urine: 1.015-1.025 (measured
using urinometer)
❖FixedSpecific Gravity of urine:observed in chronic renal failure (SpecificGravity: 1.010)
Conditions associated with Increasedspecific gravityConditions associated with Decreased specific gravity
1.Diabetes mellitus (polyuria) 1.Diabetes insipidus (polyuria)
2.Nephrosis 2.Chronic Nephritis /tubular dysfunction
3.Perspiration 3.Inadequate Water intake /water depletion
4. Steatorrhea
The earliest manifestation of renal damage may be inability to produce concentrated urine.

30. Urinometer for the measurement of the Specific Gravity of early morning urine:6c
Specific gravity test facilitates the determination of concentrating and dilution power of kidneys .It is useful to
indicate presence of renal defect where Blood urea is within normal limits in some cases .

31. Procedure for Specific gravity determination using urinometer:6d
1.Mix urine well and fill the container three forth full of urine.
2.Removeallfoamusingaroughfilterpaper.
3.Float urinometer in the urine .Rotate it carefully so that it can be prevented
from touching bottom or sides of container.
4.Notethespecific gravity reading from the scale.
5.If thequantity ofurineissmalldiluteurine(1:5or1:10).determine specific
gravity of diluted sample , multiply last two digits of recorded specific gravity
by dilution factor (5 or 10)
➢Recorded specific gravity 1.003
➢If urine dilution =1:5 (or 1:10)
➢Corrected specific gravity = 1.015(1.003x5) / (for dilution1:10→=1.030
1.003x10)
➢For each gram of albumin per 100 ml of urine specific gravity is increased by
0.003.

32. Temperature correction for the specific gravity of urine:6e
❖The specific gravity of urine is measured using urinometer .
❖Urinometer is calibrated at 15
0
C or at 20
0
C and hence temperature correction
is applied for room temperature as follows:
a.For 3
0
C rise in room temperature ,add 0.001 to the observed specific gravity.
b.For 3
0
C fall in room temperature ,subtract 0.001 from the observed specific
gravity.
•Temperature difference = room temperature (
0
C) -15
0
C
❖Corrected specific gravity =
Observed specific gravity + (0.001 x temperature difference /3)

33. Specific gravity measurement of urine using Refractometer :6f
❖Specific gravity measurement using Refractometer requires few drops of
urine.
❖Principleof Refractometer:
•The refractiveindexof a solution is related to content of dissolved solids
present. It is a ratio of the velocity of light in air to ratio of the velocity of light
in solution.
•The ratiovariesdirectlywiththenumberofdissolvedparticlesinsolution.
•Although the instrument measures the refractiveindexof a solution ,scale
reading is generally calibrated in terms of specific gravity for human urine and
serum.

34. Specific gravity measurement of urine using Refractometer :6g

35. Calculation for concentration of Total solids in urine
•Units of Total solids in urine = g/L
Concentration of Total solids in urine = last two digits of specific gravity x2.66*
g/L
*2.66is (Long’s coefficient).

36. Colligative properties
•Colligative property : Any property that depends on the number of these
particles.
•Colligative properties include:
1.Boiling point
2.Freezing point
3.Vapour pressure
4.Colloid osmometer

37. Osmolarity of urine and serum:1
•The concentrating activity of renal tubules is regulated by osmoreceptorswhich
are sensitive to changes in the solute concentration rather than to the changes
in the specific gravity of the filtrate.
•The simultaneous determination of urine and serum osmolarity is a
considerably more accurate way of measuring the concentrating ability of the
tubules.

38. Osmoreceptors in the hypothalamus
Osmoreceptors in the hypothalamus that are sensitive to changes in the Osmotic pressure of
the blood influence the synthesis and secretion of ADH.

39. Osmolarity of urine and serum:2
•Osmolarity: is a measure of the concentration of free particles in a solution .
These particles may be ions or unionized molecules.
•Osmolarity = molarity x number of particles (ions or unionized molecules)
resulting from ionization.
•The osmometer : capable of determining the osmolarity of a solution called a
colligative property.
•Types of instruments for clinical use:
1.Freezing pointosmometer
2.Vapour pressureosmometer
3.Colloid osmometer

40. Freezing point osmometer
Principle of Freezing point osmometer : the measure the osmotic pressure of a
solution by freezing point depression. A depression in the freezing point of a solution of
0.000186 below that of water( taken as 0ᴼ C) is equivalent to milliosmole (mOsm) of
osmotic activity per liter.
Working:
1.The serum/ urine specimen inserted thermistor probe and stirring wire is lowered
into the cooling bath .The serum/ urine sample is supercooled.
2.The is stirred gently during the cooling step.
3.When thegalvanometerreadingindicatesthatsufficientcoolinghasoccurred, the
stirrer is violently agitated to initiate crystallization.
4.The galvanometer movement changes direction as the heat of fusion is released.
5.The temperature at the sample probe remains relatively constant for 2 to 3 minutes
(equilibrium period). The temperature is freezing point of the solution and the
reading are displayed in milliosmoles.

41. Components of Freezing point osmometer
•Cooling module : an insulated tank contains the thermostatically controlled
cooling bath . The bath is usually filled with a mixture of ethylene glycol or
water. It is maintained at –7 ᴼC.
•Operating head : controls the vibration coil, stirring rod and temperature
probe.
•The stirring rod: vibrates back and forth in order to stir the sample and to
ensure that the cooling process is uniform. When the sample is supercooled
the stirring rod vibrates violently and causes the initiation of seeding process in
which the crystal are formed.
•The thermistor: is an electronic component of metal oxide ,encapsulated in
glass . Its electrical resistance varies with temperature. The device determines
the temperature of a solution by varying its resistance.
•Measuring system: contains a galvanometer for measuring small increments of
current . It is used to show the direction of current flow in a Wheatstone
bridge .

42. Principle and Components of Freezing point osmometer
Components of Freezing point osmometer

43. Calculation of Osmolarity of urine/serum using Freezing point
Freezing point
0.00186
Example :
Freezing point = -60
-60
0.00186
Osmolarity of urine /Serum =
Osmolarity of urine /Serum =
Osmolarity of urine/ Serum = 322

44. Osmolarity ofurine as a physical characteristic of urine: 7
❖Osmolarity of urine of a normal individual is variable depending on the state of hydration.
Osmolality decreases with excessive fluid intake(as low as 50 mosm/kg) and increases with
restricted fluid intake( up to 850 mOsm/kg) .
❖It is measured with osmometer based on the depression in freezing point of the sample
(a colligative property) .
❖Normal range of Osmolarity of urine( average fluid intake): 300 -900mosmol (milliosmoles) /Kg
❖Normal range of Osmolarity of plasma : 285-300millimoles/Kg
❖Normal ratio of Osmolarity of urine: Osmality of plasma= 2.4: 1 or more
❖Urine without any Proteins or high with molecular weight substances : Osmolarity of
urine= 800 mOsm/kg and Specific Gravity of urine= 1.020
❖Normal range of Osmolarity of random urine sample :600mOsmol(millimoles)/Kg and it
increases to 850 mosmol /Kg after fluid restriction
❖Measurement of Osmolarity of urine helps to assess renal tubular function.
❖Patients with deficiency of ADH (central Diabetes insipidus)or a deceased response to
ADH (Nephrogenic diabetes insipidus) →Osmolality of urine < 300mosmol(millimoles)/Kg

45. Chemical examination of urine

46. pH as a Chemical characteristic of urine
Chemical characteristics of urine :
Reactions to litmus ofnormalurine: acidic , average pH 6(5.5-8.0)
Titrable acidity = 250 -700 ml of 0.1 N acid /HCl ,25 mequ of H
+
❖Acidity increased in :
a)protein rich diet (due to increase in phosphates and or sulphate)
b)Metabolic or respiratory Acidosis or renal tubular acidosis
c)Fever
❖Urine becomes alkaline in following conditions :
1.On long standing urea →ammonia (decomposition by urease -bacterial activity)
2.After meals (postprandial Alkaline tide)
3.Metabolic or respiratory alkalosis (blood alkaline →urine alkaline)
4.Vegetables containing Citric & Tartaric acid →conversion to bicarbonate
5.Urinary tract infection

47. Reactions to litmus of urine
pH status Interpretation
Normal urine pH pH range: 5.5 –6.5
Low pH –acidic urineHigh protein diet→produce sulfuric acid, phosphoric acid , acidosis
High pH –alkaline urineDiet rich in vegetables →organic acids present in vegetables are
converted to bicarbonates in body, alkalosis , urinary tract infection
Place a drop of urine by using a pasture pipette/glass rod on the red and blue litmus paper.

48. Interpretation of kidney function Tests for acid-base status
Metabolic acidosis : is a characteristics complications of renal disease.
Causes of Metabolic acidosis related to renal diseases :
a)by accumulation of phosphates, sulphates and non-protein nitrogenous
substances in blood during renal disease.
b)Acidic anions such as phosphate and sulphates buffered by cations which cannot
be recaptured and returned to blood in exchange for hydrogen ions by the
tubules.
c)Failure of the renal tubular mechanism for secretion of hydrogen ions and for
formation of ammonia .
d)Depletion in serum bicarbonate (alkali reserve).
e)Reabsorption of sodium is defective ,which causes hypokalemia and severe
dehydration may occur ,secondary to the electrolytic depletion.
f)Potassium can be excreted by tubular secretion as well as by filtration hence
chronic renal failure, Serum potassium may be normal or slightly elevated.

49. Metabolism of Ammonia
NH
3→ NH
4⁺ ion ( exist ammonium ion at p H 7.4)
I .FormationofAmmonia:
a )Transamination & Deamination of biogenic amines ,NH
2group of Purines
& Pyrimidine
b) Urea →bacterial urease →NH
3
II . Transport & storage :
Conc of serum ammonia →10 -20 micro gram /dl
Efficienttransport&immediate utilization of ammonia for urea synthesis
Ammonia is transported as Glutamine & Alanine (Glucose –Alanine cycle)
No free Ammonia in serum under physiological conditions

50. Formation of ammonia in the human body

51. Sources of Blood Ammonia
❑Blood Ammonia : is an index of ureasynthesis by liver.
❑Sources of ammonia in human body :
1.Transamination & deamination
2.Nitrogenous material by bacterial action in gut
3.Kidney hydrolysis of glutamine by glutaminase
4.Pyrimidine catabolism

52. Functions of Ammonia
❖Waste products of nitrogen
❖Synthesis of compounds like
a)Non essential amino acids
b)Purines
c)Pyrimidine
d)Amino sugars
e)Asparagine
f)NH₄⁺(acid base balance )

53. Toxicity of Ammonia
❖SymptomsofToxicity of Ammonia
•Marginal elevation –toxic to brain
•Slurring of speech
•Blurring of vision
•Tremors
•Coma
•Death
Biochemistry of Toxicity of Ammonia
NH
3↑
↓
Glutamate ↑
↓
α-KGA (intermediate of TCA CYCLE ) ↓
↓
TCA ( impairment of TCA ) ↓
↓
ATP in brain ↓

54. Hyperammonemia
(a) Genetic defects in enzymes of Urea cycle (neonates)
↓
Mental retardation
(b) Acquired Hepatitis (alcoholism-defective urea synthesis)

55. Urea cycle

56. Metabolic disorders of Urea cycle-Hyperammonemia
Type of Hyperammonemmia Defective Enzyme
Hyper ammonemmia I CPS I
Hyper ammonemmia II Ornithine Transcarboxylase
Hyper ammonemmia III
Citrullinemia
Arginosuccinate synthtase
Hyper ammonemmia IV
Arginosuccinic aciduria
Arginosuccinase
Hyper ammonemmia V
Hyperargininemia
Arginase

57. Clinical application of determination of Ammonia in urine
•Urinary Ammonia : Under normal dietary conditions , urinary ammonia
derived from dietary amino acids.
•Determination of Ammonia in urine : gives measure of the ability of renal
tubules to produce ammonia in a state of acidosis.
•Concentration of Ammonia in Urine : 0.03 -0.08 gm/100 ml of urine
•TiterableAcidity of urine :20-50 mequ/L
Conditions associated with increased
Urinary acidity
Conditions associated with decreased
Urinary acidity
High protein diet Metabolic alkalosis
Diabetic ketoacidosis Respiratory alkalosis
Starvation

58. Qualitative Test for urinary Ammonia
Ammonia is a normal inorganic constituent of urine.
Qualitative test for detection of urinary Ammonia:
Test Observation Inference
3ml urine + 3ml of 5%NaOH
→Heat
When fumes start appearing
, hold the filter paper
premoistened with 3-4 drops
of phenolphthalein indicator
solution close to the mouth
of the test tube.
Pinkspot appears on
the filter paper
which disappears
quickly.
Salts of ammonia are
unstable and on heating
in alkaline solution
decompose to form
vapors of ammonia
which being alkaline
turns phenolphthalein
pink .

59. Estimation of Blood Ammonia as KFT and LFT
❖Estimation of BloodAmmoniaby Micro Diffusion Method :
Blood ( arterial )+ K
2CO
3→Ammonia released →titration with HCl
❖Interpretation of Estimation of Blood Ammonia
Normal rangeof Blood Ammonia →15-45 microgram/100 ml
Elevated of Blood Ammonia:
a.Cirrhosis (250 microgram /100ml )and or
b.Development of collateral circulation→portocaval anastomosis
c.Parenchymal hepatic disease
❖Increasedblood Ammonia →Hepatic coma due to CNS complications
❖Estimation of BloodAmmonia may be helpful to exclude or diagnose hepatic failure
in patients with unexplained stupor andcoma .
•Blood Ammonia estimation is indicated neonates suspected to have urea cycle
disorders and in organic acidurias .

60. Precautions for serum /plasma ammonia estimation
•Fasting arterial blood sample
•Use vacutainers ,blood to be withdrawn until it is full.
•Partial filling allows entry of air
•Glutamine in the specimen is a source of ammonia contamination →this can
be avoided by placing the sample in ice and centrifuging to separate to
plasma /serum
•Carryout assay as soon as possible
•EDTA / Heparin can be used as anticoagulants
•Enzymatic assay (with Glutamate dehydrogenase is done by photometry or
by ammonia selective electrode)

61. Renal compensatory mechanism in metabolic and
respiratory acidosis
Renal becomes secondary compensatory mechanism in metabolic acidosis .
Whereas in respiratory acidosis , renal becomes primary compensatory
mechanism to establish homoeostasis in acid –base balance.
Compensatory renal mechanism in metabolic and respiratory acidosis:
1.Proton -sodium (H
+
-Na
+
)exchange increased
2.Bicarbonate (HCO
-
3)reabsorption increased
3.Ammonia (NH
3)formation increased

62. Renal compensatory mechanism in metabolic and
respiratory alkalosis
Renal becomes secondary compensatory mechanism in metabolic alkalosis .
Whereas in respiratory alkalosis , renal becomes primary compensatory
mechanism to establish homoeostasis in acid –base balance.
Compensatory renal mechanism in metabolic and respiratory alkalosis:
1.Proton –sodium (H
+
-Na
+
)exchange decreased
2.Bicarbonate (HCO
-
3)reabsorption decreased and its excretion increased
3.Ammonia (NH
3)formation decreased
4.Potassium (K
+
) excretion increased →hypokalemia
5.Increased Retention of chloride ions (Cl
-
)

63. Renal Mechanism of Acid-Base balance
1. Elimination of non volatile acids ,Lactic acids, H₂SO₄buffered with cations
(Na⁺ ) are removed by glomerular filtration
2.(Na⁺ ) ↔ H⁺ across tubular membrane to prevent loss of Na⁺
•H⁺ →secretion ,NaHCO₃ recovery
•Loss of Na ⁺ is prevented by :
a)Bicarbonate mechanism
b)Phosphate mechanism
c)Ammonia mechanism
3.HCO³⁻ reabsorption
4. NH ₃ production

64. Compensatory phase of acid-base imbalance:1
Metabolic acidosisRespiratory
acidosis
Metabolic
alkalosis
Respiratory
alkalosis
1.Primary mechanism:
Respiratory
2.p H ↓
3.Respiratory center
Stimulated ↑
4.CO₂ released
5.PlasmaH₂CO₃↓till
physiological ratio
value achieved
1.Primary mechanism
:Renal
2.H⁺↔Na⁺↑,
NH₃ Synthesis ↑
3. Reabsorption of
HCO₃⁻ ↑(renal
tubular cells )
4. Plasma
HCO₃⁻↑till
physiological ratio
value achieved
1. Primary mechanism
:Respiratory
2. p H↑
3. Respiratory
center inhibited
4. CO₂ retention↑
5. Plasma H₂CO₃↑
till physiological
ratio value
achieved
1.Primary mechanism:
Renal
2. H⁺↔Na⁺↓,NH₃
Synthesis ↓
3. Reabsorption of
HCO₃⁻↓ (renal
tubular cells )
4.Plasma
HCO₃⁻↓till
physiological ratio
value achieved

65. Compensatory phase of acid-base imbalance:2
Metabolic acidosisRespiratory
acidosis
Metabolic
alkalosis
Respiratory
alkalosis
1.Secondary
mechanism:
Renal
2.H⁺↔Na⁺↑,
NH₃ Synthesis ↑
3. Reabsorption of
HCO₃⁻↑(renal
tubular cells )
1.Secondary
mechanism :
Respiratory
2.Lung diseases-
Respiratory
mechanism –fails
1. Secondary
mechanism:Renal
2.H⁺↔Na⁺, ↓
NH₃ Synthesis ↓
3. Reabsorption
of HCO₃⁻ ↓(renal
tubular cells )
1.Secondary
mechanism:
Respiratory
2.Lung diseases-
Respiratory
mechanism –fails

66. Urinary findingsincompensatory phase of acid-base imbalance
Metabolic acidosisRespiratory
acidosis
Metabolic
alkalosis
Respiratory
alkalosis
1.pH acidic
2.Excretion of
NH₄Cl↑
3. Excretion of
NaH₂PO₄↑(by
renal tubular cells)
4.Titrable acidity ↑
1.pH acidic
2.Excretion of
NH₄Cl↑
3. Excretion of
NaH₂PO₄↑(by
renal tubular cells)
4.Titrable acidity↑
1.pH alkaline
2.Excretion of
NH₄Cl↓
3. Excretion of
NaH₂PO₄↓(by
renal tubular
cells )
4.Titrable acidity ↓
1.pH alkaline
2.Excretion OF
NH₄Cl↓
3. Excretion of
NaH₂PO₄↓(by
renal tubular
cells)
4.Titrable acidity ↓

67. Clinical conditions associated withacid-base imbalance
Metabolic acidosisRespiratory
acidosis
Metabolic
alkalosis
Respiratory
alkalosis
1.Diabetes Mellitus
2. Starvation
3. Lactic acidosis
4. Violent /Heavy
exercises
5. Ingestion of
acidifying salts
6. Renal insufficiency
retention of acids
7. Loss of HC0₃⁻ ( as
diarrhea , fistula)
1. Damage of CNS
2.Brain damage
3. Drug poisoning
4.anesthesia excess
5. Obstruction to escape of
CO₂
6. Impaired diffusion-:
a.Pneumonia
b.Pulmonary edema
c.Fibrosis
d.emphysema
e.Reduction of respiratory
surface
7. Blood flow ↓congenital
heart diseases
8.Loss of ventilation function(
as thoracic pressure ↑-cyst
,pulmonary cancer )
1.Excess loss of
HCl
2.Pyrolic
obstruction
3.intestinal
obstruction
4. pylori spasm
5.Alkali ingestion
6.X-ray irradiation
7.K⁺ loss →
K⁺ deficiency
1. Stimulation of
respiratory
center
2.CNS diseases –
Meningitis,
3. Salicylates
4. Hysteria
5.High altitude
6.Unjudious use
of respirator
7.Hepatic coma

68. Urinalysis for detection of Normal
Inorganic constituents

69. Normal Inorganic constituents of urine
❖Inorganic normal constituents of urine :
➢Ions:
1.Chloride(Cl
-
)
2.Sodium( Na
+
)
3.Potassium(K
+
)
4.Calcium(Ca
2+
)
5.Magnesium( Mg
2+
)
6.Ammonia (NH
3
+
-minor component of NPN)
➢Sulphate :
i.InorganicSulphur (SO
4
3-
completely oxidized form -4 % of total)
ii.Ethereal Sulphate (conjugated Sulphur)
iii.Neutral Sulphate (incompletely oxidized)
➢Phosphates ( PO
4
3-
) :
a)Alkaline Phosphates (Sodium ,Potassium)
b)Alkaline earth Phosphates (calcium ,magnesium)

70. Qualitative test for detection of Urinary chlorides
Chlorides are excreted mainly in the form of sodium chloride. The amount of
sodium chloride excreted in urine varies between 5 to 25 g/day depending on
dietary chlorides.
Silver nitrate test :Qualitative test for detection of Urinary chlorides
Test Observation Inference
3ml of urine + 1ml concentrated
HNO
3+ 1 ml AgNO
3solution
Curdy white precipitate Urinary chlorides are
precipitated as AgCl.
(concentrated HNO
3added
keeps phosphates in
solution)

71. Clinical interpretation of silver nitrate test for urinary
chlorides

72. Clinical interpretation of urinary Calcium excretion
Under normal dietary intake , urinary calcium excretion accounts for about 15-
40% of total calcium , the remaining being excreted in the feces .
Average excretion Urinary Calcium : 200-300mg /day
Clinical conditions associated with urinary calcium excretion:
Increased urinary calcium excretionDecreased urinary calcium excretion
Hypervitaminosis D Tetany
Hyperparathyroidism
Renal calcium oxalate stones
Multiple myeloma

73. Clinical interpretation of urinary Phosphates excretion
Phosphates in urine are derived chiefly from the metabolism of phosphorous
containing foodstuffs, tissue components such as phosphoproteins,
phospholipids and nucleoproteins .
Quantity of phosphate excreted is extremely variable as it depends on the nature
of the diet.
Increased Excretion of Phosphates in urineDecreased excretion of phosphates in urine
Hyperparathyroidism Hypoparathyroidism
Bone diseases Pregnancy
Renal diseases
Diarrhea

74. Qualitative tests for detection of urinary Calcium and Phosphate
Test Observation Inference
10ml of urine + 3ml strong ammonia solution
dropwise. Boil. Filter .Discard filtrate. Pour 5ml of 1:5
hot acetic acid on the precipitate on filter paper .Collect
the solution in a test tube and divide into 2 parts and
perform test for (Ca
2+
) and ( PO
4
3-
) as follows :
1 stpart + 2 ml ammonium/potassium oxalate
Ammonium molybdate test for urinary phosphates:
2
ND
part + 1ml concentrated HNO
3+ 5ml ammonium
molybdate .Boil.
Gelatinous white
precipitate
(scanty)
White turbidity
Canary yellow
ppt
precipitate of Calcium
phosphate
Calcium present gives white
precipitate of calcium oxalate.
Phosphate present react with
ammonium molybdate in
presence conc. HNO
3to form
ammonium phosphomolybdate
(canary yellow).
Qualitative test for detection of urinary calcium:

75. Ammonium molybdate test for urinary phosphates

76. UrinaryExcretionofinorganicsulphates
Sulphur is ingested through chondroitin sulphate and Sulphur containing amino
acids (cysteine , cystine and Methionine).
Sulphur is metabolized and excreted in two forms viz ethereal sulphates and
inorganic sulphates .
Barium chloride test : Qualitative test for detection for Urinary inorganic
sulphates
Test Observation inference
Barium chloride test : 3ml of
urine + 1ml concentrated HCl
+ 1 ml Barium chloride
solution (2%)
Curdy white
precipitate
Inorganic sulphates are
precipitated as Barium
sulphates (concentrated
HCl added keeps
phosphates in solution)

77. Barium chloride test for detection for Urinary inorganic sulphates

78. Urinalysis for detection of
Normal organic constituents

79. Normal organic constituents of urine
❖Normal organic constituents of urine :
➢Urea
➢Creatinine
➢Creatine
➢Uric acid
➢Amino acids
➢Hippuric acids
➢Other substances :
•urobilinogen
•water soluble vitamins
•hormones
•enzymes

80. Urea as aNormal organic constituent of urine
❖Urea as a normal organic constituents of urine :
➢Urea:
1.is the end product of protein(amino acid) metabolism.
2.Normal daily urinary excretion of Urea= 30gm
3.Urinary excretion of Ureais increased in high protein diet, fever, Diabetes
Mellitus , Adrenal cortical hyperactivity.
4.Urinary excretion of Ureais decreased in terminal stages of liver diseases and
acidosis .

81. Qualitative tests for detection of urinary urea:1
Test Observation Inference
a. Specific urease Test:
3ml urine + a drop of
phenolphthalein + a pinch of
urease powder .Shake the
contents of the tube and
allow to stand for 10 minutes.
b. Hypobromite test :
3ml urine+ few drops of
sodium hypobromite solution
(freshly prepared).
The solution will develop
intense pink color after 10
minutes.
a marked effervescence is
observed which disappears
quickly.
Urea is hydrolyzed by urease
enzyme to form ammonium
carbonate which makes the
solution alkaline. Hence
phenolphthalein turns pink.
Urea decomposed by
hypobromite to release
nitrogen gas.

82. Qualitative tests for detection of urinary urea:2
Sodium HypobromitetestSpecific urease Test

83. Creatinineas a Normal organic constituent of urine
❖Creatinineas a Normal organic constituent of urine :
➢Creatinine:
1.istheendof purine metabolism .
2.is an excretory product formed during muscular activity from creatine
phosphate by non-enzymatic spontaneous dehydration(unhydrideof
creatine).
3.is purely endogenous and doesn’t depend on the dietary intake of proteins.
4.Normal daily urinary excretion of Creatinine= 1-2 gm.
5.urinary excretion of Creatinine is related to functioning of muscle mass and
more in men than in women.

84. Jaffe’sTestfor detection of urinary creatinine
Jaffe’s Test for detection of urinary creatinine: Qualitative tests
Test Observation Inference
3ml saturated picric acid solution +
3ml of 5 % NaOH solution. Mix and
divide into two equal parts :
1
st
part of reaction mixture + 2ml
urine→Mix
2
nd
part of reaction mixture + 2ml
distilled water
Orange red
colour
Yellow colour
Creatinine reacts with picric acid
in alkaline medium to form
creatinine picrate(orange colour ) .
Serves as control.

85. Qualitative test for detection of urinary creatinine by Jaffe’s Method

86. Creatine as a Normal organic constituent of urine
❖Creatine as a Normal organic constituent of urine :
➢Creatine:
1.Normally very little is excreted urine.
2.urinary excretion of Creatine occurs in children and more in women than in
men (more muscular mass).
3.urinary excretion of Creatine is increased in :
a)pregnancy
b)starvation
c)Diabetes Mellitus
d)fever
e)hyperthyroidism
f)muscular dystrophy

87. Uric acid as a Normal organic constituent of urine
❖Uric acid as a Normal organic constituent of urine (NPN=non-protein
nitrogen)
➢Uric acid:
1.isthechiefendof purine metabolism.
2.Normal daily urinary excretion of Uricacid= 0.7-1.0 g
3.Daily urinary excretion of Uricacidon purine free diet(decreased) = 0.1 g
4.Daily urinary excretion of Uricacid on high purine diet/Gout(increased)=2.0 g
5.Urinary excretion of Uric acid is increased in :
a)Gout
b)Leukemia
c)Liver diseases
d)Cancer (increased availability of purine for its catabolism to uric acid )

88. Qualitative tests for detection of urinary uric acid
•Make urine alkaline by adding 1ml of 2% sodium carbonate to 3ml urine in a
test tube and use alkaline urine for following tests:
Test Observation inference
1. Schiff’s Test:
Add 2-3 drops of AgNO
3
solution on filter paper . Add
5-6 drops urine (alkaline) on
the same filter paper. Warm
filter paper gently.
2.Benedict’s uric acid test :
3ml alkaline urine + 0.5 ml of
Benedict’s Uric acid reagent
→Mix the content by
shaking the test tube .
Greyish black spot develop
on the reaction area of filter
paper.
Blue color develops
In alkaline medium ,uric acid
reduces AgNO
3to metallic
silver.
Uric acid reduces phospho-
tungstic acid present in
Benedict’s uric acid reagent
to blue color tungstous salt.

89. Schiff’s Test and Benedict’s uric acid test for detection of
urinary uric acid
Benedict’s uric acid test Schiff’s Test
In alkaline medium ,uric acid reduces AgNO
3
to metallic silver.
Uric acid reduces phospho-tungstic acid
present in Benedict’s uric acid reagent to
blue color tungstous salt.

90. 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.

91. Ninhydrin test for detection of urinary amino acids:1
•QualitativeNinhydrin test for detection of urinary amino acids :
•Other applications of Ninhydrin test include :
1.Quantitative estimation of amino acids in cases of aminoaciduria.
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 Given positive by all amino acids.
Principleoftest: amino acids react with
Ninhydrin and by oxidative decarboxylation
form CO
2+ NH
3+ Aldehyde . Reduced
ninhydrin reacts with liberated NH
3
forming a purple colored complex
(Rheumann’s purple)

92. Ninhydrin test for detection of urinary amino acids:2
TLC for detection of amino acids and peptides
Quantitative
estimation of
amino acids in
cases of
aminoaciduria

93. Hippuric acid as a Normal organic constituent of urine
❖Hippuric acid is a Normal organic constituent of urine.
➢Hippuric acid :
1.isthedetoxification product of benzoic acid with glycine. The conjugation
of Benzoic acid (present as a food preservative and in fruits , vegetables)
with glycine occurs in the liver.
2.Normal daily urinary excretion of Hippuricacid= 0.1-1.0 g( average 0.7 g)
3.excretion of Hippuricacid is used as a liver function test.

94. Hippuric Acid Test: KFT and LFT associated with detoxification
❑HippuricAcid :
Benzoic Acid( Toxic )+ Glycine →Hippuric Acid
8 g Benzoic acid consumed in diet →3g Hippuric acid in urine
❑StepsofHippuricAcid Test:
1.Precipitation of Hippuric acid with Ammonium sulphate
2.Dissolve precipitate in water
3.Titration with NaOH

95. Qualitative test for urinary Hippuric acid
Test Observation Inference
3ml urine + 1ml of 5% NaOH+
dilute ferric chloride solution
dropwise
Cream colored precipitateFormation of cream colored
Ferric Hippurate
Qualitative test for detection of urinary Hippuric acid :

96. Clinical importance of Urinary amylase
•Human serum amylase (optimum p H ): 6.9-7.0
•Normal value Human serum amylase :800 units /dL
•Full activity of serum /urine amylase is displayed in presence of chloride
(cofactor),bromide and mono hydrogen phosphate ions.
•High serum amylase values : carcinoma of pancreas, renal failure ,mumps
•Variation in urinary amylase reflect alteration in serum amylase so long as the
kidneys are functioning normally.
•Normal urinaryexcretionofurinaryamylase: 50-300 Caraways units (C.U.)/24
hr. urine specimen.
•High values of both serum andurinary amylase : acute pancreatitis (at 24-48
hours sample after onset of attack). The rise starts within hour of onset of pain
and usually returns to normal in 4 to 8 days .
•Low values of both serum and urinary amylase : suggestive of liver disease.

97. Biochemical test for urinary Ethereal sulphates
•Ethereal sulphates consist of potassium salts of sulfuric acid esters of phenols
such as indoxyl , skatoxyl ,and cresol . These are detoxification compounds of
phenol and formed in the liver . Indoxyl and skatoxyl sulphates are formed by
putrefactive decomposition of Tryptophan in the intestine.
•Qualitative test for detection of urinary Ethereal sulphates :
Test observation Inference
5ml urine + 5ml Baryto mixture→
Filter. To clear filtrate ,add 2ml of
concentrated HCl . Boil for 2
minutes in a beaker and cool at
room temperature.
White turbidity Ethereal sulphates are
dissociated on boiling
with conc. HCl to liberate
inorganic sulphates
which form white
precipitate with Baryto
mixture.

98. Other substances as Normal organic constituent of urine
❖Other substances as Normal organic constituent of urine include :
1.Urobilinogen
2.Water soluble vitamins
3.Hormones
4.enzymes

99. Urine analysis by reagent strips

100. Thank you
Google Images