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EXPERIMENT 15: URINE ● oxidation of urea to ammonium

● urine becomes ammoniacal in odor and
- Filtrate from the blood alkaline in pH

- Serves as a medium for the excretion of the ff: ➔ In ALKALINE urine, the ff precipitate:

➔ Salts ◆ Calcium phosphate [Ca3(PO4)2]

➔ Acids ◆ Calcium carbonate (CaCO3)

➔ Bases ◆ Magnum phosphate [Mg3(PO4)2]

➔ Waste products of metabolism ➔ In ACIDIC urine, the ff precipitate:

➔ Other toxic materials ◆ Uric acid (C5H4N4O3)

◆ Sodium urate (C5H3N4NaO3)

COLLECTION & PRESERVATION OF URINE SAMPLE ➔ Specific Gravity: range of 1.015 - 1.025

- studying quantitative and qualitative urine: examine the 24 ➔ Constituents:


- Bladder is emptied at 8:00 am and the urine is discarded

- Sample: urine taken from this time to 8 am the next day
1. Urea 1. Chlorides
2. Uric acid 2. Phosphates
4. Ammonia

➔ Volume: of 24 hour urine is 1000-1500 ml. ➔ These precipitates are the primary components of kidney
➔ Color: stones.

◆ Yellow - due to urochrome/urobilin pigments

◆ Amber - due uroerythrin pigments 2. DETECTION OF CREATININE

➔ When urine is allowed to stand for sometime, exposed to - Creatinine - breakdown product of Creatine phosphate
air: (CP)

◆ Uric acid may crystallize and settle out since it is

only slightly soluble in water or acid solution A. Nitroprusside test (Weyl)
◆ It becomes less acidic than fresh specimens 1) 5 mL urine + sodium nitroprusside
● It may become alkaline after standing [Na2[Fe(CN)5NO]
due to bacterial decomposition of urea 2) Alkalinize with NaOH
to ammonia
● Result: Ruby red ppt = presence of creatinine
➔ When urine is freshly voided:
- Color fades (turns into yellow) in normal urine if
◆ It has a pH of 6 (slightly acidic) added with acid
● due to Na2HPO4 and NaH2PO4

➔ When urine is allowed to stand after sometime w/o B. Picric acid Reaction (Jaffe)
1) 5 mL urine + picric acid (C6H3N3O7)
◆ Oxidation:
2) Alkalinize with NaOH
● Result: Red ppt = due to formation of tautomer 2) Filter off the earthy phosphates and small amount of
of creatinine picrate. magnesia mixture to the filtrate.

- Normally turns yellow when acidified. 3) Warm the solution.

- If it does not turn yellow, acetone ● Result: Magnesia mixture precipitates alkali phosphates (Na
bodies are present and K salts).

- Glucose gives a similar color (when heated) 4) The alkali phosphates of Na and K separate.

3. DETECTION OF PIGMENTS - Alkali phosphates are larger in amount compared to Earthly

phosphates with a ratio of 2:1
- Perform the Ammoniacal Zinc chloride test.

1) 2 mL urine + 1 mL ammonium hydroxide (NH4OH) → let

stand then filter --- PATHOLOGIC CONSTITUENTS---

2) Add 2 drops of zinc chloride (ZnCl2) to the filtrate 7. ALBUMIN

● Result: green fluorescence = presence of urobilin a) Coagulation Test

1) Boil urine

--- INORGANIC PHYSIOLOGICAL CONSTITUENTS --- ** filter if urine is not clear

4. SULPHATES - If urine becomes cloudy, it may be due

to phosphates.
a) Detection in inorganic sulphuric acid:
2) Acidify with very dilute acetic acid (3-4 drops) to
5 mL urine + acetic acid [CH3COOH] + Barium chloride (BaCl2)
dissolve the phosphates.
● Result: white ppt of barium sulfate (BaSO4) formed
3) Warm.

● Result: Phosphates should dissolve.

- If precipitate still forms, albumin is present
b) Heller’s Ring Test
1) 5 mL urine + nitric acid (HNO3) + silver nitrate (AgNO3)
Urine + 5 mL conc. HNO3
● Result: White ppt of AgCl formed.
● Result: White ring (coagulated protein) = presence of
2) Add excess NH4OH. albumin

● Result: silver chloride (AgCl2) is dissolved


**Normal amt of chlorides eliminated in 24h: 10-15 grams - Perform Benedict's Test : Semi quantitative test.

● Usually eliminated in the form of NaCl PROCEDURE:

1) 8 drops of urine + 5 mL Benedict’s reagent

6. DETECTION OF PHOSPHATES 2) Boil for 2 mins.

PROCEDURE: 3) Set aside to cool

1) 10 mL urine + NH4OH → warm. ● Result - Color depends on the quantity of glucose

● Result: Precipitation of earthly phosphates (Ca and Mg ○ Red - conc. amt. of glucose
salts) occur.
○ Yellow - glucose substantially present
- The earthy phosphates / phosphates of Ca and ○ Green - little glucose present
Mg separate.
○ Blue - no color change; no glucose present Ketonuria - presence of these substances in urine


- Perform the Benzidine test. PROCEDURE: Perform the Nitroprusside Test.

PROCEDURE: 1) 2 mL urine + 3 drops of 5% sodium nitroprusside

1) Heat 3 mL of urine to boiling. 2) Alkalize with NaOH.

2) Cool. Treat with 3 mL of sat. benzidine (C12H12N2) in ● Results: ruby red color = presence of acetone
glacial acetic acid.
3) Add 0.5 mL of acetic acid. Observe further.
3) Add 1 mL of 3% hydrogen peroxide (H2O2).
● red color persists = ketone bodies are present.
● Result: blue/green color = presence of blood

10. BILE

- Perform the Gmelin's Test.


1 mL of urine + HNO3. DON’T mix them!

● Result: Colored rings (blue, green, violet, red and reddish

yellow) = presence of bile pigments.

Bilirubin is normally ABSENT in urine because bilirubin is converted to

urobilinogen in the intestines and then absorbed in the bloodstream.
The absorbed pigment is then oxidized to urochrome or urobilin
which makes its way to the kidneys(responsible for urine color).

Presence of bilirubin indicates obstruction of the flow of bile from the

gallbladder so that the bile is absorbed in the blood and bilirubin goes
to the kidneys.


Acetic acid decomposes so rapidly with the formation of acetone, that

the usual test for acetone are also given for aceto-acetic acid.

Test for ketonuria = tests for either acetone or aceto-acetic acid/both.

Ex: Nitroprusside test

Ketone bodies are brought about by increased lipid metabolism

wherein the utilization of lipids is incomplete. These bodies are:

➔ Acetoacetic acid (diacetic acid),

➔ Acetone

➔ Beta hydroxybutyric acid.