II) Chemical Examination of the Urine

Mostafa Sabry Abdullah, B. Pharm. Sci.Teaching Assistant, College of Pharmacy, Al-Azhar University, Assiut, Egypt.Section No.2

Constituents of Urine

1- organic ;

Nonprotein Nitrogenous compounds (N.P.N)

Nonprotein Non-Nitrogenous organic compounds

2- Inorganic ;

chloride, phosphate ,sulphate, carbon

B) Abnormal constituents of urine

1. Sugar

2. Protein

3. Ketone bodies

4. Bilirubin

and bile

5. Blood

6. UT stone

A)Normal constituents of urine

COLLECTION SPECIMENS OF URINE

• First morning sample;-(concentrated urine) chemical constituents , casts and crystals

• Random specimen;- Routine chemical screening, microscopic examination.

• 24 hours urine sample quantitative estimation of proteins, sugars, electrolytes ,

• first DROPS of urine NOT COLLECTED ;-

since it contains urethral and prostatic secretions which may be required if investigating urethra and prostrate.

• Midstream urine specimen is THE BEST PORTION.

1-Organic constituents of urine;-

A)Normal constituents of urine

 A- Non protein nitrogenous compounds (N.P.N.):-

They are usually the end products of protein metabolism in the body,

This substance includes:

1- Urea

2- Ammonia

3- Uric acid

4- Creatine

5- Creatinine

6- Amino acid s

1- Urea

It is the main solid components of urine.

Normal rang about 2O-40 gm/day.

It constitutes about 85% of the total urinary nitrogen.

Formation ; it is the end product of protein metabolism . Urea is formed in the liver from ammonia through urea cycle and excreted in urine by the kidneys.

The serum level of urea is about 20-50 mg/dl.

This value increase highly in kidney DIS. and decreased highly in liver disease.

physiologically

increased ;-

after high protein intake.

decreased ;-

after low protein intake .

Pathologically

increased;-

In protein catabolism as

in fever , D.M.

decreased ;-

in liver & kidney diseases.

Detection: It can be detected by Sod. Hypobromite Na HBr ---eff..

Eff….

2- Ammonia NH3:-Its normal urine level is about 0-7 gm/day.

-60% of this level is derived from deamination of amino acids mainly glutamine.

Glutamine by Glutaminase Glutamic acid + NH3.

-40% of its level is derived from deamination of the amino acids in the renal tissue itself.

Its secretion in urine

depends on urine PH and the urine PH depends on the food type. SO THAT , physiologically ;-

-Acid forming food tend to increase the output NH3 as Whole milk , egg, Yogurt. -Alkali forming food tend to decrease the output of NH3, as lemon ,tomato.

Pathologically

Acidosis=increase NH3 output as in case of D.M.

Alkalosis= decreased NH3 highly in urine and may be absent in urine.

Its serum level about 20- 110 micro g/dl:

Ammonia is a toxic substance for nervous tissue so that liver change it into urea through urea cycle

If it is highly increased this indicate liver disease and may cause hepatic coma.

Detection: It is detected by Nessler reagent with Na HCO3 ---) red colour.

3- Creatine:-

It is formed by interaction 3 amino acids glycine, arginine and methionine.

The normal urine level : is very small amounts about 0-0.2 gm/day

Clinical significance: -

around 95 %of creatine in the human body is stored in skeletal muscle.

increased physiologically in:-

1-Childrens 2-periodically in females.

3- After labor 4-During the last days of pregnancy

Increased Pathologically in :-

fever, muscle destruction, D.M.

4- Creatinine:-•It is the anhydride form of creatine •Its estimation is used as a kidney function test due to;it is totally excreted in urine.it is of endogenously produced. Its level isn’t affected by type of food. Its level is maintained with narrow normal range. The normal urine level: 1-1.8 gm/day in male.0.7-1 gm /day in females. This difference is due to ; the higher muscle bulk in male than females. Biosynthesis:

Detection:

It is detected by NAOH + Sod.picrate --) orange colour.

Clinical significance:

A) CK: - Creatine kinase enzyme = creatine phospho-kinase enzyme which catalyze formation of creatine phosphate

It present in 3 forms in serum,:-

1-CK.MM: - Derived from skeletal muscles and its serum level highly increased in muscle diseases “muscle atrophy”

2-CK-MB:-Derived mainly from Heart muscles its level highly increased in recent myocardial infarction

3-CK.BB:- Derived from Brain and its serum level increased in Brain cell damage.

B) Serum level of Creatinine is about 0.2-8mg/dl this level increased highly in kidneys dysfunction.

WHAT IS THE DIFFERENCE ?

• CREATINE

• CREATININE

• CAROTENE

• KERATINE

• CARNITINE

5-Uric acid:-• It is the principle end product of

purine metabolism in human.

• It is derived from exogenous purine (dietary) or endogenous (tissue purine).

• Uric acid is one of the normal antioxidant.

Normal urine level about 0.7-1 gm/day.

Clinical significance:

A) The urine level highly increased in

I-Leukemia. II-sever liver diseases. III-Gout.

B) Serum level is about:-

4 - 8.5 mg/dl in male.

2.7 - 7.3 mg/dl in females.

Detection: it is detected by phsphotungestic acid + NaOH ------ blue colour.

6- Amino acids:-It is urine level is very small "150

- 200" mg/day.

Pathologically ;-Its high level in urine called "Amino aciduria” which occur due to:-

1-Terminal liver disease: due to failure of deamination of amino acids.

2- Kidney disease: due to inherit tubular defect in reabsorption of amino acids

B) Non nitrogenous organic compounds

1- Carbohydrates:

Present in very small undetected amounts

eg;- Glucose - pentoses especially after high fruits intake.

2- Lipids:-

Very small amounts of short chain fatty acids.

3- Organic acids:-

Mainly glucouronic acid, which present in conjugation with other substances e.g. six hormones.

Organic acids also may present as citric acid, lactic acid and oxalic add

Normal levelURINE

• UREA---20-40 GM/DAY

• AMMONIA 0-7 GM/DAY

• CREATINE 0-0.2 GM/DAY

• CREATININE 0.7-1.8 GM/DAY (F,M)

• URIC ACID 0.7-1 GM/DAY

• AMINO ACID 150-200 MG/DAY

SERUM• UREA SERUM 20-50 mg/dl.

• NH3---20- 110 micro g/dl.

• CR .NN. 0.2-8mg/dl

• URIC A. 2.7-8.5 mg/dl (F,M)

2.Inorganic constituents of urine:-

A)Normal constituents of urine

A- Chloride: CI-

Normal urine level about "10-15" gm/day which come mainly from diet.

Clinical significance: Chloride urine level decrease;-

1- In prostatic obstruction.

2- In pneumonia due to Cl retention in exudates.

3- In sever vomiting and diarrhea.

4- In severe burns.

5 - In some kidney diseases associated with CI retention.

6- In Addison disease "Hypocortisolemea"

N.B:- Chloride and urea are the main two substances which affect SP.G of urine.

Detection: sliver nitrate with dilHCO3-----) white ppt.

B- Sulphates:-

It is derived from oxidation of the oxidation of sulpher present in sulpher containing AA. which are "cysteine & methionine". which come mainly from diet.

Sulphates present in urine in three forms.-

1- In organic sulphates:

As Na ,K ,mg. sulphates which act about 90% of total sulphates.

2- Etherial sulphates :- as indican.

3- Neutral sulphates :- e.g. cysteine and thiosulphates.

normal urine In level about 0.08-2 gm/day.

C- Phosphates ;-•Normal level is about 1-2 gm/day.

•It is derived from oxidation of phosphor protein of diet e.g casein & milk.

•It may be divided into:-

1- Alkaline phosphates : Na& K phosphates" 65 " %

2- Earthy phosphates : Ca2+&mg phosphates" 35" %.

• The PH of urine depends on the ratio between acidic "NaH2 P03" and basic "Na2 HP03" phosphates.

• Phosphate excretion in urine depends on:-

1- Phosphate content of diet.

2- Ca2+ and phosphate metabolism.

3- The body need to phosphate bone fracture and during lactation.

Clinical significance:

Phosphates are increased in URIN as the following:-

1- In case of rickets in children . VIT.D

2- In case of osteomalicia in adults.

3- In case of hyperparathyroidism.

Phosphates are decreased in the following cases;-

1- During pregnancy and lactation.

2- During III of rickets by vitamin D. which increase Phosphate reabsorption.

3- During healing of major fracture.

4- During administration of Ca2+.

5- In case of hypo para thyroidism.

D)Na , K• Na+: 2-4 g/day and K+: 1.5 - 2 g/day.

• It depend on Nature OF diet, during fasting urinary excreting of K > Na due to tissue breakdown.

Urine level of inorganic comp.;-

 CHLORIDE ; 10-15 GM/DAY

SULFATE; 0-2GM/DAY

PHOSPAHTE; 1-2 GM/DAY

NA ,K ; 1.5-2GM/DAY

B) Abnormal (Pathological) constituents of urine.

B) Abnormal (Pathological) constituents of urine

1. Sugar

2. Protein

3. Ketone bodies

4. Bilirubin and bile

5. Blood

6. UT stone

1- Sugars :-

1- Glucose ~ glucosuria

2- Fructose ~ fructoseuria.

3- Pentoses ~ pentosuria

4- Lactase ~ lactosuria ~ specially during lactation and in infants.

1-Glucose :-

Normally less than 0.1 gm/day.

Glycosuria ~ presence of sugar in urine

Glucosuria ~ presence of glucose in urine

Physiological glucosuria :-

- Alimentary ~ after high carbohydrate intake.

- Emotional ~ due to liberation of adrenaline.

- Pregnancy ~ due to inc. glomerular filtration rate.

Pathological glucosuria:-

• Glucosuria associated with hyperglycemia as in D.M & hyper-pituitarism.

• Glucosuria with normal blood glucose level as congenital low renal threshold ( D. innocence)

Detection:

a) Fehling:-• Boil 2 ml of urine in a test tube (A).

• Boil 2 ml Fehling reagent in another tube (B).

• Add tube A TO B and mix allow to stand without further healing ~ red yellow or green ppt.

 b) Benedict:-2 ml of urine +2 ml Bindict (alkaline

solution & Cu S04) and heat to boil and allow to cool ~ red or green ppt. acc. To conc.

2- Proteins(Proteinuria = 30 mg/dl)

• Under normal conditions ;-urine contains non detectable amounts of protein.

• It is characteristic for all acute and chronic kidney diseases.

• N.B:- The most excreted protein is albumin due to

1-the low M.W and 2- high plasma conc. of albumin compared with other protein called ~ Albuminuria.

Other proteins in urine:-

1- Bence Jones protein: Which is abnormal type of globulin, "light chain of immunoglobulin" present in urine of patient suffering from multiple myeloma "malignant plasma cell".

2- Haemoglobinuria :- as in malaria & haemolytic anemia.

3- Myoglobinuria: In case of massive muscle damage e.g In burns and sever electric shock.

A-Physiological causes:

1- Sever muscular exercises.

2- High protein diet.

3- During pregnancy.

4- Standing for long time

Orthostatic albuminuria".

N.B;-

B- Pathological causes

1- pre-renal due to ;-

-renal vein congestion, C.H.F.

- Liver diseases,- Hypertension

2- Renal albuminuria: in the case

- Glomerulonephritis.

- Pyelonephritis.- T.B of kidney

3- Post renal albuminuria: Due

to lower UTI e.g. Cystitis.EDEMA STRAT FROM EYE………………. KIDNEYEDEMA STRAT FROM ABDOMEN….. LIVEREDEMA STRAT FROM LEG ……………….HEART

Causes of Albuminuria

Detection.-

a) Heat coagulation test:-

5 ml of urine & heat the upper part of the tube ~ agglutination is formed in the upper part.

b) Heller's test:

3 ml concHNO3 + 3 ml of urine on side of T.T ~ white ring at the junction indicate albumin.

c) Sulpho Salicylic acid test:

6 drops of 20% SSA (aqueous) on 5 ml urine ~ if clouds ~ albumin.

3- Ketone bodies :-

three types of Ketone bodies: 1-acetoacetic acid,

2-B-hydroxy butyric acid and

3-acetone.

• The first two are normal products of fatty acids catabolism.

• Acetone is 2ry product result by decarboxylation of acetoacetic acid.

Its urine level less than 18 ng/day.

• They are formed in the liver and are destroyed or used by the extra hepatic tissues.

• Ketonurea may be expected to occur in ;- the acidosis & D.M & starvation.

Detection:

Rothera's test:-• Make complete saturation of 2

ml urine with ammonium or sodium sulphate to remove any interfering substance.

• Add 3 drops of freshly prepared Sod. nitro prusside.

• Add 3 ml Cone. ammonium hydroxide.

• Mix and allow to stand undisturbed ~ characteristic permanganate colour (Ring).

4- Bilirubin and bile: Any failure of the liver to excrete bile

cause inc. amount of Bilirubin in the body fluid produce jaundice.

Bile is elevated in hepatocellular disease and biliary obstruction.

It is appear in urine in case of obstructive jaundice & haemolytic diseases. It give urine greenish brown colour.

Detection:

Sulpher test :-Add a few powder of sulpher on the

surface of 3 ml of urine

If give ppt ~ Bile salt is present.

5- Blood "Haematouria: -

in case of':-

1- Urinary Bilharziasis.

2- Glomerulonephritis.

3- Traumatic or malignant tumor.

N.B ;-

IF the blood in the last drop of urine…… source is bladder

IF the blood in whole urine …………………… source is kidney

IF the blood in the first portion of urine.. …source is urethra

6- Urinary tract stones :-

Recent studies indicates that 10% of adult males suffer from stones at least once at life.

The most common stones are Ca - oxalate & Ca - phosphates & urate.

but Mg & NH4 - phosphates and cysteine stones are rarely common.

Chemical Examination of Urine Samples

• 1. Test for Glucose: .

• 2. Test for Albumin: .

• 3. Test for Ketone Bodies .

• 4. Test for Bile Salts: .