TEAM FOUR

Activity 12

Urine is a typically sterile liquid by-product of the body secreted by the kidneys through a process called urination and excreted through the urethra.

Urine is made of water and waste. 96% of it is water and 4% of it is waste such as urea and salts.

Normal fresh urine is clear and transparent. On standing, it may become turbid due to precipitation of phosphates. Also bacteria, blood, sperm, crystals, or mucus can make urine look cloudy.

also called Carbamide, the diamide of carbonic acid. Its formula is H2NCONH2. Urea has important uses as a fertilizer and feed supplement, as well as a starting material for the manufacture of plastics and drugs. It is a colourless, crystalline substance that melts at 132.7 C (271 F) and decomposes before boiling.

Urea is the chief nitrogenous end product of the metabolic breakdown of proteins in all mammals and some fishes. The material occurs not only in the urine of all mammals but also in their blood, bile, milk, and perspiration. In the course of the breakdown of proteins, amino groups (NH2) are removed from the amino acids that partly comprise proteins. These amino groups are converted to ammonia (NH3), which is toxic to the body and thus must be converted to urea by the liver. The urea then passes to the kidneys and is eventually excreted in the urine.

Agriculture

More than 90% of world production of urea is destined for use as a nitrogenrelease fertilizer. Urea has the highest nitrogen content of all solid nitrogenous fertilizers in common use. Therefore, it has the lowest transportation costs per unit of nitrogen nutrient.
Chemical industry Urea is a raw material for the manufacture of many important chemical compounds, such as Various plastics, especially the urea-formaldehyde resins. Various adhesives, such as urea-formaldehyde or the urea-melamineformaldehyde used in marine plywood. Explosive Urea can be used to make urea nitrate, a high explosive that is used industrially and as part of some improvised explosive devices.

Medical use Urea-containing creams are used as topical dermatological products to promote rehydration of the skin. Like saline, urea injection is used to perform abortions. Urea is the main component of an alternative medicinal treatment referred to as urine therapy. The blood urea nitrogen (BUN) test is a measure of the amount of nitrogen in the blood that comes from urea. It is used as a marker of renal function.

 Mix 10 ml of urine and 5ml of baryta mixture (Phospates, sulfates, urates, hippurates, are precipitates) Filter off the ppt. Evaporate the filtrate to a thick syrup. Filtrate contains Chloride, Creatinine, Salts, pigments and urea Extract the syrup with 95% alcohol and filtrate. Filtrate contains Urea with some pigments. Boil with animal charcoal, dissolve in a cool place. Allow the filter to crytallize.

ISOLATION OF URIC ACID from urine

EXPERIMENT
Add 50 cc. of concentrated hydrochloric acid to 500 cc. of urine and set aside in a cool place for 24 hours.

Pour off the liquid from the crystals of uric acid which adhere to the side of the vessel.

Collect the crystals, dissolve them in the smallest possible amount of boiling water, boil with a little boneblack, and filter the solution hot. On cooling, colorless crystals of uric acid are obtained. Examine them under the microscope and sketch the crystals.

(MUREXIDE TEST)

TEST FOR URIC ACID

Uric acid tests are tests that are done to measure the levels of uric acid in blood serum or in urine.

PURPOSE
evaluate the blood levels of uric acid for gout and to assess uric acid levels in the urine for kidney stone formation
to monitor patients already diagnosed with kidney stones, help measure the level of kidney functioning.

DESCRIPTION
URIC ACID BLOOD TEST
performed on a sample of the patient's blood, withdrawn from a vein into a vacuum tube. The procedure, which is called a venipuncture, takes about five minutes.

URINE TEST
requires the patient to collect all urine voided over a 24-hour period, with the exception of the very first specimen. The patient keeps the specimen container on ice or in the refrigerator during the collection period.

NORMAL RESULTS Blood Test Male: 2.1-8.5 mg/dL; female: 2.0-6.6 mg/dL. Values may be slightly higher in

Urine Test

250-750 mg/24 hours

ABNORMAL RESULTS

The critical value for the blood test is a level of uric acid higher than 12 milligrams per deciliter (about 3.4 ounces).

PRINCIPLE OF URIC ACID TEST

Principle

Uricase
Uric Acid + O2 + 2H2O ----------- Allantoin + CO2 + H2O2 POD 2H2O2 + 4-Aminoantipyrine + TBHBA ------- Chromagen + 4H2O

Uric Acid is oxidized by Uricase to allantoin and hydrogen peroxide. TBHBA + 4-aminoantipyrine + hydrogen peroxide, in the presence of peroxidase, produces a colored chromagen that is measured at 520nm. The color intensity at 520nm is proportional to the concentration of Uric Acid in the sample.

(NITROPRUSSIDE TEST)

CREATININE

Creatine is an important compound produced by the body. It combines with phosphorus to make a highenergy phosphate compound in the body. Creatine phosphate is used in skeletal muscle contraction. PURPOSE

The creatinine test is used to diagnose impaired kidney function and to determine renal (kidney) damage.

NORMAL RESULTS
Adult female: 0.5-1.1 mg/dL Adult male: 0.6-1.2 mg/dL Adolescent: 0.5-1.0 mg/dL Child: 0.3-0.7 mg/dL Infant: 0.2-0.4 mg/dL

Newborn: 0.3-1.2 mg/dL.

ABNORMAL RESULTS Two to 4 mg/dL indicate the presence of impairment of renal function. Greater than 4 mg/dL indicates serious impairment in renal function.

PRINCIPLE OF CREATININE TEST

creatininase Creatinine +H20 ------------ creatine

creatinase
Creatine + H2O ------------- sarcosine + urea sarcosine oxidase

Sarcosine + O2 + H2O -------------- glycine + formaldehyde + H2O2

POD H2O2 + 4-aminoantipyrine/HTIB --------------- H2O + dye

ascorbate oxidase
Removal of ascorbate: 2 L-ascorbate + O2 + H2O ---------------- 2 Ldehydroascorbic acid + 2 H2O

POD

H2O2 + 4-aminoantipyrine/HTIB --------------H2O + dye

ascorbate oxidase

Removal of ascorbate: 2 L-ascorbate + O2 + H2O ---------------- 2 L-dehydroascorbic acid + 2 H2O

G. Inorganic Physiological Constituents

Inorganic Constituents of Human Urine Phosphates (H2PO4-, HPO42-, PO43-): Amount in urine varies with the amount of parathyroid hormone in the body parathyroid hormone increases the quantity of phosphates in urine. The regulation of urinary phosphorous excretion is principally dependent on regulation of proximal tubule phosphorous reabsorption. A variety of factors influence renal tubular phosphate reabsorption, and consequent urine excretion. The normal weight of this inorganic constituents is 2.0 g/l.

Sulphates (SO42-): Sulphates are derived from amino acids. The quantity of sulphates excreted in urine varies according to the quantity and type of protein in the persons diet. Sulfate is a major anion in the urine that has significant affinity for cations and modulates the availability of cations for reacting with other anions in the urine. It thus is an important factor of urinary supersaturation(4) for various crystals or stones. The normal weight of this inorganic constituents is 1.5 g/l.

Chloride (Cl-) : Amount in urine varies with dietary intake. An indicator of fluid balance and acid-base homeostasis, it is involved in maintaining osmotic pressure, proper body hydration, and electric neutrality. In the absence of acid-base disturbances. The normal weight of this inorganic constituents is 9.0 g/l.

CLINICAL INFORMATION

Phosphorus imbalance refers to conditions in which the element phosphorus ispresent in the body at too high level (hyperphosphatemia) or too low level (hypophosphatemia).

disorder characterized by growth retardation

X-linked hypophosphatemic Rickets due to kidney defects in phosphate reabsorption and Vitamin D metabolism.

Hemochromatosis - is an inherited disease in which too much iron builds up in your body. Iron is a mineral found in many foods. Your body normally absorbs about 10 percent of the iron in the food you eat. If you have hemochromatosis, you absorb more iron than you need. Your body has no natural way to get rid of the extra iron. It stores it in body tissues, especially the liver, heart and pancreas. The extra iron can damage your organs. Without treatment, it can cause your organs to fail.

Electrolyte Imbalance(Chlorides) Electrolytes are essential components of body fluids, such as blood and urine. Typical electrolytes found in the body are sodium, calcium, potassium, chloride. Electrolytes help regulate water distribution throughout the body and they play a major role in the acid-base balance, or pH, of the body.

An electrolyte imbalance is usually caused when the level of water found in the body is altered. This can be due to dehydration, excessive sweating, diarrhea, fever, vomiting, or a disorder of an organ or gland . Eating disorders may also result in electrolyte imbalances. Imbalances are more likely to develop in older people, since the kidneys function less effectively as the body ages. A laboratory test performed on blood or urine is used to diagnose an electrolyte imbalance.

Chlorides
Chloride is an important electrolyte and works to ensure that your body's metabolism is working correctly. Your kidneys control the levels of chloride in your blood. Therefore, when there is a disturbance in your blood chloride levels, it is often related to your kidneys.

Chloride Test
Procedure:

1.

Place one mL of urine in a test tube and acidify with 2 drops of nitric acid (HNO3).

2.

Add 2 drops of silver nitrate (AgNO3).

Chloride Test
Principle:

AgNO3 reacts with the chloride in urine to precipitate AgCl which produces a white precipitate. The appearance of which indicates end point.

Positive Result: white precipitate

What are the common signs/symptoms when the Chloride test is done?
Prolonged vomiting

Prolonged diarrhea
Weakness

Fatigue
Respiratory distress

A decreased level of urine chloride can be seen with Cushings syndrome.

Cushings Syndrome

Disorder of Adrenal Gland

Cushings Syndrome
Chloride levels also affect acid-base balance. This mineral is absorbed from the intestines and excreted primarily by the kidneys.

A urine chloride test may be performed along with a blood or urine sodium when evaluating the cause of low or high blood chloride levels. The doctor will look at whether the chloride measurement changes mirror those of the sodium. This helps the doctor determine if there is also an acid-base imbalance and helps to guide treatment.

Hyperchloremia
Hyperchloremia also occurs when too much base is lost from the body (producing metabolic acidosis) or when a person hyperventilates (causing respiratory alkalosis).
*Respiratory alkalosis is a condition marked by low levels of carbon dioxide in the blood due to breathing excessively.

An increased level of blood chloride (called hyperchloremia) usually indicates dehydration.

H. Pathological Constituents

Glucose
Glucose is a prime commodity of the body. It is the source of energy for all cells. This is why glucose is never present in the urine. Glucose that passes through the filters of the kidney (glomeruli) and bound to be passed out with urine are reabsorbed by the kidney tubules and returned to the blood. This reabsorption of glucose keeps this valuable substance out of the urine.

Glucose Test
Procedure:

1.

Place 1 mL of Benedicts reagent in a test tube and add 2 drops of urine.

2.

Boil for 1 to 2 minutes.

Glucose Test (Benedicts Test)

Principle:

Reduction of Cupric (blue) to Cuprous (brick red) by the reducing sugars.

Positive result: Brick red ppt. ++++ Green/Greenish yellow/orange ppt.

Related Diseases
Urine usually contains a very small amount of glucose. The urine of a healthy individual contains about less than 0.1% glucose, which cannot be detected by the regular urine tests. Urine usually does not contain a high amount of sugar, as the kidneys reabsorb all the filtered glucose and return it to the bloodstream.

There is a renal threshold for glucose and it is approximately 160 to 190 mg/dL. If the level of blood sugar exceeds this threshold level, the kidneys become overwhelmed and fail to reabsorb it efficiently. In such a case, urine can contain it in significant high amounts. This condition is medically known as glycosuria or glucosuria.

ALBUMIN (Hellers Ring Test)

Hellers Ring Test

used to clinically detect the presence of albumin in urine.

the presence of albumin is indicated by the formation of at the junction of the solution and concentrated nitric acid.

PRINCIPLE
concentrated acids remove the hydration shell around the protein molecules and help in precipitation

PROCEDURE
Place 1ml of conc. HNO3 in a test tube.

Incline the tube and pour urine slowly on one side so as to from a layer over it.

Allow to stand for sometime and a white ring is produced.

Basis of Hellers Test

Proteins get denatured when acid is

added and this forms a white coagulum which is slightly yellow in color because of nitro- derivatives of proteins given by aromatic amino acids.

Formation of white ring

CLINICAL USE
Use to detect the presence of albumin in urine.

Proteinuria means the presence of an excess of serum proteins in the urine. The excess protein in the urine often causes the urine to become foamy, although foamy urine may also be caused by bilirubin in the urine, retrograde ejaculation, pneumaturia (air bubbles in the urine) due to a fistula.

There are three main mechanisms to cause proteinuria: Due to disease in glomerulus Because of increased quantity of proteins in serum (overflow proteinuria)

Due to low reabsorption at proximal tubule (Fanconi syndrome)

This proteinuria in most of the cases is due to glomerulonephritis induced by hepatitis C, for example, these are cryoglobulinemic, membranoproliferative glomerulonephritis associated with HCV infection that were first published by Doctor Cruzado from Barcelona in the American Journal of Transplantation.

BILE PIGMENTS (Gmelins Test)

Gmelins Test
a test for bile pigments (as in the urine) that is made by carefully mixing the solution to be tested with nitric acid containing some nitrous acid and that shows a positive result when a series of colors appears at the juncture of the solution and the acid.

PRINCIPLE

Bile pigments are oxidized by acid to give different colors.

PROCEDURE
Place 1ml of conc. HNO3 and upon it superimposed 1 ml of urine.

Play of colors produced. It shows that bile pigments in urine were present.

CLINICAL USE
Use to detect the presence of bile pigments in the urine.

RELATED DISEASES
Jaundice is a yellowish pigmentation of the skin, the conjunctival membranes over the sclerae (whites of the eyes), and other mucous membranes caused by hyperbilirubinemia (increased levels of bilirubin in the blood). This hyperbilirubinemia subsequently causes increased levels of bilirubin in the extracellular fluid.

Yellow discoloration of the skin, especially on the palms and the soles, but not of the sclera and mucous membranes (i.e. oral cavity) is due to carotenemia - a harmless condition important to differentiate from jaundice.

Jaundice of the skin caused by hepatic failure.

A 4-year-old boy with icteric (jaundiced) sclera which later proved to be a manifestation of hemolytic anemia due to G6PD deficiency following f ava bean consumption.

Terminology
BILE ACIDS

chemicals made by the liver and put into bile

natural detergents made from cholesterol. synthesized in the hepatocyte contain both hydrophobic (lipid soluble) and polar (hydrophilic) faces emulsification of lipid aggregates solubilization and transport of lipids in an aqueous environment

Terminology
PETENKOFERS TEST presence of bile salts consists in the addition to the suspected liquid of a drop of a strong solution of cane sugar and then of sulphuric acid If bile salts are present a bright cherry-red colour is developed.

Procedure
Place 2 ml of urine in a test tube and add 2 drops of 5% sucrose. Incline the tube and run 1 ml of conc. H2SO4 on the side.

Shake and note down the result.

Principle
this red reaction which was formed at the point of contact shows the presence of bile acids.

Since, it was proven by Pettenkofer that sucrose can be converted to fat invitro; bile acid took its action. Thus, the addition of the chemical sulphuric acid shows the positive result on the bile acid s presence by the formation of a red color at its point of contact by the reaction of cholic group of bile to the sulphuric acid

Clinical Use

Terminology
ACETONE TEST a test for the presence of ketonuria NITROPRUSSIDE TEST A test for cystinuria in which sodium cyanide and then nitroprusside are added to urine. reacts with ketones

Procedure
Mix 2 ml of urine and 2 drops of 5% freshly prepared aqueous sodium nitroprusside Alakalinize with NaOH

Note what color is produced

Add 0.5 m Hac and make further observations

Principle
Nitroprusside also reacts with ketones and is used in tests for ketone bodies in urine. The proprietary Acetest tablets contain a mixture of glycine, sodium nitroprusside, disodium phosphate, and lactose. In the presence of glycine, acetoacetate or acetone forms a blue-purple colour.

Clinical Use