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Activity 26: Physical Properties of Urine

URINE

Sterile, liquid by-product of the body that is secreted by the kidneys.

Cellular metabolism generates numerous by-products that require elimination from the bloodstream.

These by-products are eventually expelled from the body in a process known as micturition.

COLOR – straw to amber

*Colorless to straw – indicates low specific gravity and large quantity

*Amber – indicates high specific gravity and small quantity

Abnormal (Pathological Coloration)

Reddish amber – increased in urobilinogen

Brownish yellow/green – indicate bile pigments

Red to smoky brown – due to blood and blood pigments

Milky – due to large amounts of pus, bacteria/fat

Brownish black – indicate melanin

ODOR – aromatic

- depends on diet and drugs used

Cloudiness:

-freshly voided is clear (usually) but cloudy sometimes due to the following sediments:

• Amorphous phosphates – form white cloud/precipitate

• Amorphous urates – form a white/pink cloud of sediment

• Epithelial cells and mucus – when large inamounts

• Pus – makes urine turbid

• Fat – render urine turbid

• Bacteria - produce uniform cloudiness

pH

-4.8-7.5 (Average: 6)

24 Hr. Specimen – are less acidic than freshly passed and may become alkaline after standing

Pathological Significance:

• Acidity - increased in acidosis, fevers, diet with excess proteins

• Alkaline – in chronic cystitis and urine retention due to decomposition of urine in bladder

SPECIFIC GRAVITY (24 Hr. Specimen)

-1.015 to 1.025

COMPOSITION-H2O: about 1000-1500g-Solids: about 55-70g in 24 hours• Nitrogenous organic compounds• Non-nitrogenous organic substances• Inorganic salts• Traces of Fe, Cu, Zn, Cl..etc.

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Average volume: 1-2 L a day

ABNORMAL

Polyuria = a condition of excessive production of urine

Oliguria = decreased in quantity

Anuria = total suppression of urine

1. Why does urine become turbid on standing?

flocculate of molecules composed of nucleoprotein mucoid

precipitation of calcium phosphate

decomposition of urea to form ammonia

fat globules or pus cells infection in the urinary tract

2. Explain the alkaline fermentation of urine.

Ammoniacal fermentation

- the conversion of the urea of the urine into ammonium carbonate, through the growth of bacteria.

- CON2H4 + 2H2O (NH4)2CO3

- Note: Whenever urine is exposed to the air in open vessels for several days it undergoes this alkaline fermentation.

3. What is the importance of determining the specific gravity of urine?

rough estimate of the total solids in the urine provide information of the state of hydration of the

patient - if highly concentrated, the patient is dehydrated determine the presence of renal disease helps distinguish between renal failure and dehydration determines the concentrating ability of the kidneys

4. Why must the examination be done on a 24-hour specimen?

if the complete examination of the constituents of the urine is needed

notably better than that exceeding 24-hours because: - microorganisms may grow- convert urea to ammonium carbonate giving a more

pungent ammoniacal odor than normal- nitrogenous constituents are altered by the loss of

nitrogen as volatile ammonia

5. What are the substances responsible for the normal color of urine?

urochrome – gives the normal yellow color is due to a pigment metabolite arising from the body's destruction of hemoglobin.

uroerythrin – urinary pigment that gives a redish yellow color to deposits of urates.

urobilinogen – when oxidized gives a darker or deeper yellow color

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6. Outline the origin of the color of urine and feces.

Heme

Biliverdin

Mesobilirubinogen

Bilirubin

Hemoglobin

Stercobilinogen(L-urobilinogen)D-Urobilinogen

Stercobilin(L-urobilin)

D-Urobilin

+ Protein (Globin)

+ 2H (reduction)

+2H

-2H (auto-oxidation)

+2H

-2H

-2H Urobilin

Activity 27: Constituents of Urine

Urine Constituents

A.1. Sulfate – white ppt.

A.2. Chloride – white ppt.

A.3.b. Alkali Phosphate –white ppt.

1 2 3.b.

3.a. Triple Phosphate

What is triple phosphate?

- Ammonium magnesium phosphate

Sources of phosphate in urine:

- breakdown of tissue, foods rich in phosphoproteins, phospholipids and nucleoproteins

A.4.a. Addition of 1 ml. of ammonium thiocyanate - Formation of red color

A.4.b. With 5 drops of potassium ferrocyanide- Blue solution

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B.1. Urea Crystals

How does urea become a component of urine?

It is the pricipal and product of protein metabolism and constitutes about 80% - 90% total nitrogen excretion.

B.2.a. Uric Acid Crystals

B.2.b. Result: Formation of Black spot

Uric acid - Reducing property

How does it become a component of urine?

-Uric acid is the final end product of purine oxidation in the body

3.a. Weyl’s Reaction –Formation of Red color

3.b. Jaffe’s Reaction – Dark orange - red solution

How does creatinine become a component of urine? - As a product from metabolism in the muscle tissues.

4. Obermeyer’s Test –blue color

How does it become a component of urine?

- C8H6NOSOK -The potassium salt of indoxylsulfate (indican) found in urine is a result of bacterial action on tryptophan in the bowel.

5.a. Hippuric Acid Crystals

6. Oxalic Acid Crystals

Odor:

Irritating odor

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8. Purine Bases – Cloudy sol’n w/ white ppt.

7. PigmentsResult – White ppt.

What pigments are usually present in urine?

- Urochrome, urobilinogen, and uroerythrin

C.PATHOLOGICAL Result

1 . Glucose brick red ppt.

2. Albumin White ring b/n two

liquids

3. Acetone Red – purple

4. Blood Blue green color

Albumin (Heller’s Test) – white ring

Acetone (Legal’sTest) – Red – purple

Glucose – brick red ppt.

Blood – bluegreen color

D. Uric CrystalsBlood casts

Mucus threads

Hyaline casts

Granular casts

Granular casts

Epithelial casts

Pus casts

Tyrosine Cholesterol Epithelial cells

Epithelial cells

Squamouscells

Leukocytes

Uric Acid Calcium oxalate

HippuricAcid

Ammonium-magnesiumphosphate

Calcium phosphate

Cystine

Crystals Cells and Casts

What are the different microscopic constituents of urine?

Questions:

1. What pathological condition is indicated by?

a. Glucose-normally cannot be detected in urine-its presence in appreciable amount is termed as glycosuria-alimentary glycosuria (glycuresis) occurs after intake of CHO-pathological conditions of the kidney such as glomerulonepthritis-increased amount of glucose in blood is called hyperglycemia-hyperglycemia and glycosuria is found in: diabetes mellitus

b. Albumin – its presence is called albuminuria; properly termed as proteinuria.

2 types:

Functional - no diseased organ and the excretion is only slight and temporary due to strenuous exercises, cold baths, fever

Pathological – maybe either renal, pre-renal and post renal

c. Ketones (end product of fatty acid metabolism) in urine – made up of:-Aceto-acetic acid-Butyric acid-Hydroxybutyric acid-Acetone

•the first three are normal products of fat metabolism•they are produced in the liver•are utilized or destroyed in the extrahepatic cells

**as long as production compensates destruction, no Ketonuria is produced; but when production is too great- ketonemia then ketonuria develops

d. Blood – its presence is called hematuria.•Found whenever there is lesion in the kidney or any part of the urinary tract•Hemoglobinuria –presence of hemoglobin in urine due to:

destruction of RBC liver cannot change all the hemoglobin into bile pigments

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2. What is meant by creatinine coefficient and what is its significance?

Creatinine – the anhydride of creatine•creatinine bears a direct relation to the muscle mass of the individual expressed as CREATININE COEFFICIENT (amount of creatinine in mg excreted in 24 hours per kg of body weight)•average adult male – coefficient is 20 – 26; female is 14 –22

** Significance: decrease value indicates muscle wasting due to prolonged negative nitrogen balance; seen in starvation, diabetes, muscle dystrophy

3. Why is lactose found in urine of lactating women?

Lactosuria appears after the 3rd month of pregnancy and increases in incidence up to the 35th week and following delivery

-chiefly of mammary origin even prior to lactation-caused by high level of lactose of mammary origin in the maternalhoodstage-lactose is excreted by glomerular filtration and is not reabsorbed by the tubules so that all the filtered lactose appears in the urine

4. What is the significance of large amounts of indican in urine?

Indican – salts of indoxyl sulfate derived from indole which in turn arises from the action of putrefying bacteria on tryptophan and CHONS containing it.

-occurs in large intestines if any indole is absorbed -it undergoes a series of detoxification transformations in the liver and indoxyl is formed-it is conjugated with sulfate and neutralized to yield a salt -indican is eliminated in the urine and can be detected by OBERMEYER’S TEST-an increase in the amount is indicative of increased putrefaction in the large intestines

5. What is phosphaturia and is its significance?

-normally kidneys excrete between 1 and 5 grams of phosphoric acid in the form of phosphates but at times it becomes excessive . Such abnormal excretion and precipitation is called PHOSPHATURIA

2 types of phosphate excreted:

a. Alkaline phosphate – those combined with alkali metals like Na, K, and NH4+b. Earthy phosphate – combined with earth metals like Ca, Mg

SIGINFICANCE:-the amount is increased in hyperthyroidism-in the early stages of pulmonary tuberculosis-diseases on the bones like rickets/osteomalacia-index for the extent of phosphate metabolism

Activity 28: Substances in Blood

BLOOD

Specialized bodily fluid that delivers necessary substances to the body's cells – such as nutrients and oxygen – and transports wasteproducts away from those same cells

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Erythrocytes (Red Blood Cells) -7-8um diameter-Biconcave disc-without nuclei-lifespan: 120 days

Leukocytes (White Blood Cells)-Have nucleus-Most live for a few hours to a few days

Thrombocytes (Platelets)-2-4 um diameter-no nucleus-Lifespan: 5-9 days

Blood performs many important functions within the body:

•Supply of oxygen to tissues•Supply of nutrients such as glucose, amino acids, and fatty acids•Removal of waste such as carbon dioxide, urea, and lactic acid•Immunological functions, including circulation of white blood cells, and detection of foreign material by antibodies•Coagulation•Regulation of body pH•Regulation of core body temperature

A. Inorganic substances

1. Reagent: Ash + dil. HCl + potassium ferrocyanideResult: prussian blue solutionConstituent of blood present: ironEquation: 4Fe+3 + 3Fe(CN) -4

6 Fe4[Fe(CN)6]3

2. Reagent: blood + water + heat to boilingResult: redish brown solution

Reagent: dil. HNO3 + AgNO3

Result: white ppt. formedConstituent of blood present: chlorideEquation: AgNO3 + Cl AgCl + NO3

3. Reagent: dil. HNO3 + ammonium molybdate

Result: formation of yellow brown ppt.

Constituent of blood present: phosphate

Equation: H2PO4 + 12MoO4 + 3NH4 + 22H3O (NH4)3PO4.12MoO4 + 34H2O

4. Reagent: ammonium oxalate

Result: formation of white ppt

Constituent of blood present: calcium

Equation: Ca+2 + C2O4 CaC2O4

B. Hemoglobin

Blood exposed to air: dark red color

Conclusion: formation of oxyhemoglobin took place upon exposure of blood to air

C. Glucose in Blood

occurs after the separation from coagulated proteins

Reagent: Benedict’s reagent

Result: brick red ppt.

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Questions:

1. Inorganic constituents of blood and its function

Calcium – plays a role in blood clotting

Phosphorus– for the maintenance of c id-base balance and calcium equilibrium

Chlorine – in the form of NaCl- plays a role in osmotic pressure

Magnesium – 18 parts magnesium gives rise in rate vasodilation and hyperirritability of the nervous system

Iron – essential component of hemoglobin, muscle myoglobin

Copper – important in bone formation and cellular respiration

2. Structure of the Hemoglobin molecule

◦ conjugated CHON with a Mw of 68000

◦ made up of CHON “globin” to which heme groups are attached

◦ globin contains high amount of histidine, lysine and little of isoleucine (for the specificity of hemoglobin)

◦ its buffering property and the CO2 carrying capacity

◦ iron containing pigment “heme” is specially for the pigmentary property

3. Methemoglobin and its color

a derivative in which iron is in the ferric state

produced by the oxidation of hemoglobin as when potassium ferricyanide is added to blood

color: red-brown

4. Carboxyhemoglobin and its color

CO combines with heme portion of hemoglobin to form carbon monoxide hemoglobin called also carboxyhemoglobin and carbonyl hemoglobin

Color: bright cherry red

5. Importance of blood sugar determination

a. important in the detection of diabetes mellitus and hyperglycemia

b. to diagnose toxic or inflammatory condition of the liver due to:

◦ galactosemia◦ presence of lacic acid◦ acidosis◦ thymine deficiency

c. adjust insulin dosage

d. understand effects of various foods

Activity 29: Tests for Blood

A. Guaiac Test

-bluish green color

B. Benzidine Test

-Bluish green color

C. Hemin Test

-Red brown rods

Hemin Test – red brown rods

Guaiac Test and BenzidineTest – bluish green color

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1. What is the chemical name and structural formula of HEMIN?

IUPAC Name:chloro[3,7,12,17-tetramethyl-8,13-divinylporphyrin-2,18-dipropanoato(2−)]iron(III)

Synonyms: Ferriheme chloride, Ferriprotoporphyrin chloride 2. Describe a method on detecting blood stains on cloth.

Hemin Test

-this test is quite satisfactory and the technique is simple and gives equally reliable result with fresh blood from clothes on stains as long as that it is not been exposed to the sun or high temperatures for a long period of time

Activity 30: Plasma

Plasma

- straw colored liquid

- A watery, protein rich fluid that forms the matrix of the blood

- 91.5% water and 8.5% solutes, most of which are proteins

A. Preparation of Oxalated Plasma

-light yellow viscous fluid

B. Separation of Plasma Proteins and Fibrinogen

1. Plasma + distilled water + saturated ammonium sulfate sol.

= light yellow solution with bubbles suspended

2. Precipitate + water and perform the Biuret test

= purple solution

3. Filtrate from 2 + water + saturated ammonium sulfate sol.

= cloudy light yellow solution with bubbles and white ppt.

4. Precipitate + water and perform the Biuret test

= purple solution

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5.Filtrate from 4 contains albumin + distilled water and perform the Biuret test

=purple solution

6.Heat the remaining filtrate from 4=cloudy solution with white ppt.*Substance precipitated: albumin

C. Serum1.Serum – clear yellow fluid2. Fibrin – color: red

- appearance: thread like appearance3. Millon’s Test – red color

Questions:

1. What is the difference between plasma and serum?

Basis Plasma Serum

1. How to be obtain? When whole blood is mixed with an anticoagulant

When blood is allowed to clot

Fluid part of blood thatcontains water soluble solutes

Fluid portion left after blood has clotted

2. Components Composed of proteins, fibrinogen and clotting factors

Doesn’t contain fibrinogen

3. Preference Preferred in hematology Preferred in clinical chemistry

2. What are plasma proteins? Give the function of each.

3. What is meant by icterus index? What is its significance?

Icterus index – calculation of bilirubin in plasma or serum. For the determination of latent jaundice, symptom of liver disease characterized by yellow skin due to deposition of bile pigments.