Qualitative AnalysisCHAPTER 1

Carbohydrates are polyhydroxy aldehydes or ketones or compounds, which yield them on hydrolysis. Carbohydrates are the main source of energy in all living organisms.

Simplest sugar, which cannot be hydrolyzed further.

They give two molecules of monosaccharides on hydrolysis.

They give 2–10 monosaccharide units on hydrolysis, e.g. Maltotriose, Raffinose.

They give more than 10 molecules of monosaccharides on hydrolysis.

They are subdivided as follows:

These tests are generally based on the following properties of carbohydrates:

Procedure: To 2 mL of carbohydrate solution 3 drops of Molisch's reagent are added and mixed. The test tube is inclined and 2 mL of concentrated sulfuric acid is added through the sides of the test tube. Reddish purple or violet ring is seen at the junction of the two liquids.

Principle: Disaccharides and polysaccharides are hydrolyzed by concentrated sulfuric acid into monosaccharides, which are further dehydrated, by the acid to form hydroxy methyl furfural or furfural respectively, which condense with alpha naphthol to form the violet colored complex.

Procedure: To 2 mL of Seliwanoff's reagent, 3 drops of fructose solution are added and boiled for 30 seconds (Prolonged heating may convert aldohexoses to ketohexoses and give a false positive test). Then the test tube is allowed to cool in the rack. Cherry red colored complex is formed, which shows the presence of fructose.

Principle: Fructose is dehydrated by concentrated hydrochloric acid (HCl) to form furfural derivative, which condenses with resorcinol to give the cherry red complex.

Procedure: To 3 mL of Foulger's reagent, 5 drops of fructose are added and boiled vigorously for one minute. Test tube is left in the rack for cooling. A blue color is obtained which shows the presence of fructose.

Principle: This test is based on the furfural formation, which condenses with urea in the presence of stannous chloride to give a blue color.

Procedure: To 5 mL of Bial's reagent in a test tube 0.5 mL of pentose solution is added and mixed, and heated until it begins to boil. Green colored solution is obtained.

Principle: By the action of concentrated HCl upon pentose, furfural is formed. This condenses with orcinol to give green colored compound.

Carbohydrates having a free aldehyde or keto group exhibit reducing property and they are called as reducing sugars.

Procedure: To 5 mL of Benedict's solution, 8 drops of reducing sugar are added and boiled for 2 minutes. It is then allowed to cool spontaneously. Depending on the amount of sugar present in the solution, green, yellow or red precipitate will be formed. The precipitate is cuprous oxide.

Principle: Under alkaline condition, sugar is reduced to enediol forms. Cupric ions of cupric hydroxide are then reduced by enediol forms of sugar to form cuprous hydroxide. Simultaneously, the enediol form of sugar is oxidized to sugar acid. The cuprous hydroxide during heating is converted to cuprous oxide, which may be green, yellow, or red precipitate.

Practical application: Benedict's test is the most widely used test for detection of glucose in urine. It is used commonly for screening diabetes mellitus. It answers for all reducing sugars. Depending upon the color produced, approximate concentration of sugar in urine may be ascertained.

4Procedure: Each 1 mL of Fehling's reagent A, B and C are taken in a test tube, mixed and boiled to check auto reduction. 1 mL of reducing sugar is added and boiled for 2 minutes and the test tube is allowed to cool. Green, yellow or red precipitate will appear depending upon the concentration of sugar present.

Principle: Same as Benedict's test.

Procedure: To 2 mL of Barfoed's reagent, 1 mL of sugar solution is added, mixed and boiled for 30 seconds only (Prolonged boiling should be avoided as it gives false positive result for disaccharides which get hydrolyzed). Test tube is allowed to cool and a red precipitate is formed at the bottom of the test tube, which is cuprous oxide.

Principle: This test differs from other 2 reduction tests as this test is carried out in mild acidic medium which will be answered only by strong reducing—monosaccharides.

5Procedure: A boiling water bath is set up. 10 mL of sugar solution (monosaccharides and reducing disaccharides) is taken in a test tube and one spatula full of phenylhydrazine hydrochloride and two spatulas full of sodium acetate and 1 mL of glacial acetic acid are added, and mixed well and filtered in a test tube. Filtrate is kept in boiling water bath. Yellow crystals of osazones are formed within few minutes in the case of monosaccharides. In the case of disaccharides, the osazone crystals are formed only after cooling. Few crystals are transferred to a glass slide with the help of a glass rod and covered with cover slip and observed under low power microscope. The following findings will be seen in different sugars.

Principle: When reducing carbohydrates are heated with phenylhydrazine hydrochloride at 100°C and at pH 4.3, they form the corresponding osazone, aniline and ammonia with the removal of one molecule of water. As glucose and fructose differ only with respect to first and second carbon atoms they form the same osazone. Sucrose cannot form osazone, as it is not a reducing sugar.

Procedure: Sucrose solution is first hydrolyzed by adding 3 drops of concentrated hydrochloric acid and boiled. After cooling the test tube under tap water, 20% sodium carbonate is added dropwise until there is no effervescence, to neutralize the acid. Then Benedict's reagent is added and boiled for 2 minutes and the test tube is allowed to cool spontaneously. A precipitate, which may be green, yellow or red in color is obtained depending upon the amount of glucose and fructose present.

6Principle: Sucrose will not answer for Benedict's test, as it is a non-reducing sugar. After hydrolysis by concentrated HCl sucrose is converted into glucose and fructose which are reducing monosaccharides, which answer this. This is a confirmatory test for sucrose.

Osazone test: Maltosazone will be formed within 30 minutes. These crystals are soluble in hot solution and so they separate out on cooling. The crystals are sunflower shaped or having the shape of sunflower petals, on viewing through the microscope.

It is a test for polysaccharides. Starch, dextrin and glycogen can be differentiated by this test.

Procedure: 2 mL of polysaccharide (starch, glycogen and dextrin) solution, 5 drops of glacial acetic acid are added. Then 3 drops of N/50 iodine solution are added drop by drop and mixed. Starch gives blue color which disappears on heating and reappears on cooling. Dextrin gives a reddish-purple color which disappears on heating but does not reappear on cooling. Glycogen gives a reddish-brown color which disappears on heating and appears on cooling.

Principle: This test depends upon the adsorption property of the large polysaccharide molecule, which adsorbs the smaller iodine molecules on their surface to form an ill-defined colored complex. On cooling, the complex is reformed and the color reappears except in dextrin.

These are also tests for polysaccharides to differentiate starch from dextrin.

Procedure: To 5 mL of polysaccharide solution, 5 mL of saturated ammonium sulfate solution is added and shaken vigorously and kept in the rack for 5 minutes. A white precipitate is formed. This is filtered to another test tube and 2 drops of glacial acetic acid is added to the filtrate and then 3 drops of N/50 iodine solution is added. Starch will not give any blue color whereas dextrin gives a red color.

Full saturation test: 5 mL of dextrin solution is taken in a test tube and solid ammonium sulfate is added with constant shaking until it begins to settle at the bottom of the test tube. This is filtered in another test tube and 2 drops of glacial acetic acid and 3 drops of N/50 iodine solution are added. Dextrin gives red color.

Principle: As starch has small surface area, it is precipitated by half saturation with ammonium sulfate. Dextrin is a small molecule with greater surface area and so it is not precipitated even by full saturation with ammonium sulfate.

Three mL of starch solution is taken in a test tube and 2 drops of concentrated HCl are added and boiled for 2 minutes and then cooled. It is then neutralized with 40% sodium hydroxide (or 20% sodium carbonate) till the effervescence is stopped. With the neutralized hydrolyzate Benedict's test is performed to get a positive result.

Principle: Starch is a non-reducing sugar. Acid hydrolysis breaks starch into glucose molecules. As glucose is a reducing sugar Benedict's test gives a positive result.

The most commonly available polysaccharide is starch, which is a mixture of amylose and amylopectin. The individual glucose unit in amylose are linked by 1,4-glycosidic linkages. Amylopectins have branching points contributed by alpha-1,6-glycosidic linkages. Starch is insoluble in cold water, but forms a colloidal solution in hot water. Starch has no detectable reducing property.

They are formed as intermediate products during the course of hydrolysis of starch by dilute mineral acid and also by the reaction of amylases. These heterogeneous compounds are grouped into amylodextrin, erythrodextrin and achrodextrin based on the color produced by N/50 iodine. These three groups, which are in the order of decreasing molecular weight gives violet, red and no color in iodine reaction. Dextrins are partially soluble in water. They have more reducing activity.

Proteins occupy the major portions of our body. It is a nitrogenous substance made up of carbon, hydrogen, oxygen and nitrogen. Some proteins contain sulfur and phosphorus.

All proteins are polymers of amino acids. There are 20 amino acids, which are genetically coded. According to their structure, they are classified as follows:

Phenylalanine—Tyrosine (Hydroxyphenylalanine).

Proline.

Peptides are formed when two or more amino acids are linked.

Proteins are made up of peptide chains. They are classified as follows:

Primary proteoses: They are not coagulated by heat, but precipitated by half saturation with ammonium sulfate.

Secondary proteoses: They are not coagulated by heat but they can be precipitated only by full saturation with ammonium sulfate.

Peptones: They are not coagulated by heat. They cannot be precipitated by half and full saturation with ammonium sulfate.

Globular proteins form colloidal solutions, in which the particles have the diameter between 1 millimicron to about 200 millimicrons.

The stability of suspensoids are due to the electrical charges over the surface of the molecule which prevent the formation of precipitation.

There are two factors for the stability of an emulsoid:

Lead acetate, Mercuric chloride, Mercuric nitrate

Picric acid, Trichloroacetic acid, Phosphotungstic acid, Sulfosalicylic acid, Tannic acid

Ammonium sulfate, Sodium sulfate

Isoelectric point or isoelectric pH is the pH of a protein at which level it carries equal number of positive and negative charges and the net charge is zero. At this pH, the protein does not move either to the anode or to cathode in an electric field. Isoelectric point varies from protein to protein.

Reagent: 10% lead acetate solution.

Procedure: To 2 mL of protein solution, 5–10 drops of lead acetate solution are added and mixed. A white precipitate is formed.

Reagent: Mercuric chloride or mercuric sulfate.

31Procedure: To 2 mL of protein solution, mercuric chloride or sulfate solution is added drop by drop. A white precipitate is formed.

Principle: Metal ions (lead, mercury) combine with the negatively charged groups on the protein to form the precipitate — metal proteinate. This principle is used in treating heavy metal poisoning by giving egg white (protein - albumin) orally which will precipitate the heavy metals.

Procedure: To 2 mL of protein solution 2–3 drops of sulfosalicylic acid are added and mixed. A white precipitate is formed.

Procedure: To 2 mL of protein solution few drops of freshly prepared solution of tannic acid are added. A light brown or yellow precipitate is obtained.

Procedure: To 2 mL of protein solution, 1 mL of picric acid is added and mixed. A yellow precipitate is formed.

Principle for test no 1 and 3: The negatively charged ions of the alkaloids—Sulphosalicylic acid, tannic acid and picric acid neutralize the positive charges on the protein causing denaturation which will end in precipitation.

Procedure: To 2 mL protein solution, 3 drops of glacial acetic acid are added and mixed. Few drops of potassium ferricyanide are then added drop by drop to get a yellow precipitate.

Principle: Glacial acetic acid confers a positive charge on protein. When the negatively charged ferricyanide ions are added, the proteins get precipitated by neutralizing the charges.

Half Saturation Test: To 3 mL of protein solution, 3 mL of saturated solution of ammonium sulfate is added and mixed well.

Full Saturation Test: To 3 mL of protein solution, solid ammonium sulfate is added and mixed well till few undissolved ammonium sulfate crystals get settled at the bottom (saturated solution).

Principle: The colloidal protein solution has electric charge and the shell of hydration to keep it stable.

Reagents: Acetic acid, bromocresol green

Procedure: To 3 mL of casein solution, 3 drops of bromocresol green are added and mixed. A blue color is seen on the alkaline side. Then 2% of acetic acid is added drop by drop till the color changes to light green (pH 4.6). A white curdy precipitate is formed.

Principle: At isoelectric pH, the solubility of proteins is minimum as the molecules become electrically neutral at this pH.

Reagent: 1% acetic acid.

Procedure: Protein solution is taken up to three-fourth level of a test tube. The tube is inclined slightly and by holding it at the bottom, the upper portion of the solution is heated over a flame. An opalescent coagulum is formed at the heated portion only and there is no change at the nonheated bottom portion. 3–5 drops of 1% of acetic acid is added to get maximum white precipitate.

Principle: Proteins undergo denaturation by denaturing agents such as heat. Denaturation is a process in which the physical, chemical and biological properties of proteins are altered due to the breaking up of peptide bonds. There will be change in the conformation of the molecule. Denaturation is a reversible process and the denatured protein can be converted to its original form when the denaturing agents are removed. Coagulation, on the other hand, is an irreversible process.

Color reactions of proteins are produced by a particular amino acid or by a particular group of amino acids. These reactions are helpful in identifying the protein in a given solution and also to identify the particular amino acid.

Reagents: 5% sodium hydroxide and 1% copper sulfate (Biuret reagent).

Procedure: To 2 mL of protein solution, 2 mL of 5% NaOH is added and mixed well. Then 2–3 drops of 1% copper sulphate solution is added. Protein gives a violet or purple colored complex.

Principle: This is a general test for proteins. Compounds which contain 2 or more peptide bonds will answer this. Dipeptide and free amino acids do not give biuret positive. The violet or purple color is due to the formation of the coordination complex between cupric ions and nitrogen atoms of the peptide bonds.

Reagent: To 2 mL of protein solution 2–3 drops of ninhydrin are added and boiled. After cooling a bluish-purple colored complex is formed.

33Principle: This test is answered by proteins containing free alpha amino groups only. Alpha amino groups of the protein, react with ninhydrin to give a blue-violet colored complex called Ruhemann's purple. Proline and hydroxyproline will not answer this test as they do not have alpha amino group. Instead they will give yellow color only.

Reagents: Concentrated nitric acid, 40% sodium hydroxide.

Procedure: To 3 mL of protein solution, 1 mL of concentrated nitric acid is added and heated for one minute. Due to denaturation, a yellow precipitate is formed. After cooling it under tap water, 2 mL of 40% sodium hydroxide solution is added which makes the precipitate to become orange in color.

Principle: Aromatic amino acids, which contain benzene ring, will answer this (phenylalanine, tyrosine and tryptophan). Nitration of benzene ring forms a yellow derivative which turns orange in alkaline medium.

Reagents: 10% mercuric sulfate, 10% sulfuric acid and 1% sodium nitrite.

Procedure: To 1 mL of protein solution, 1 mL of 10% mercuric sulfate in 10% sulfuric acid is added and boiled for half a minute. A yellow precipitate is formed. 3 drops of sodium nitrite are added to this and the precipitate is changed to red color.

Principle: This test is specific for hydroxyl group containing aromatic amino acid—tyrosine. The red color is due to mercury complex of nitrophenol derivative.

Reagents: Dilute formalin (1 in 500), 10% mercuric sulfate in 10% sulfuric acid, concentrated sulfuric acid.

Procedure: To 3 mL of protein solution, 2 drops of 1/500 formalin and one drop of 10% mercuric sulfate in 10% sulfuric acid are added and mixed well. Then 3 mL of concentrated sulfuric acid is added through the sides of the tube. A purple or violet ring is formed at the junction of the 2 liquids.

Principle: Indole group of tryptophan combines with formaldehyde in the presence of sulphuric acid (Oxidizing Agent) and mercuric sulfate to give the violet or purple colored complex.

Note: Collagen and gelatin will not answer this due to the absence of tryptophan.

Reagents: Alpha naphthol, 20% sodium hydroxide and bromine water.

Procedure: To 3 mL of protein solution, 5 drops of 20% sodium hydroxide and 4 drops of 1% alpha naphthol in alcohol are added and mixed. To this 10 drops of bromine water are added. A bright red color is formed.

Principle: This test is specific for guanidine group of arginine. In alkaline medium alpha naphthol combines with the guanidine group of arginine to form a complex which is oxidized by sodium hypobromite to produce a red color.

Reagents: 40% sodium hydroxide, 2% lead acetate.

Procedure: To 2 mL of protein solution, 2 mL of 40% sodium hydroxide is added and mixed and boiled for one minute. Then 5 drops of lead acetate solution are added. A black or brown precipitate is formed.

Principle: Sulfur containing amino acids — Cysteine and Cystine answer this but not Methionine. Sulfur present in the protein reacts with sodium hydroxide while boiling, and it is converted to inorganic form of sodium sulfide. This reacts with lead acetate to form a black precipitate of lead sulfide. Methionine does not answer this test as the sulfur is not released by alkaline digestion.

Note: Gelatin and casein do not answer this test due to the absence of cysteine.

Reagents: 0.5% sulfanilic acid, 1% sodium nitrite and 10% sodium carbonate.

Procedure: To 1 mL of 0.5% sulfanilic acid and 1 mL of sodium nitrite solution, 2 mL of protein solution is added and mixed and cooled under tap water. Then 1 mL of 10% sodium carbonate is added to make the solution alkaline. An orange red complex is formed.

Principle: This test is specific for imidazole group of histidine. Diazotized sulfanilic acid reacts with the imidazole group of histidine to form this cherry red complex in alkaline medium.

Procedure: To 2 mL of protein solution, 2 drops of 1% alpha naphthol in alcohol is added and then through the sides of the test tube, 2–3 mL of concentrated sulfuric acid is added to get a violet or purple ring at the junction of two liquids.

Principle: This test is a general test for carbohydrates, answered by carbohydrate containing glycoproteins such as mucin. Casein, gelatin albumin and peptones will not answer this as they are not glycoproteins.

It is a simple heat coagulable protein. It is soluble in water. It is precipitated by full saturation with ammonium sulfate. It contains all amino acids.

Gelatin is formed when collagen is hydrolyzed. It is not heat coagulable. It can be precipitated by half saturation.

Peptone is a secondary derived protein formed by the cleavage of peptide bonds. It is soluble in water. It is not heat coagulable. It is not precipitated by even full saturation with ammonium sulfate.

Casein is the chief protein of milk. It is a conjugated protein (phosphoprotein). In milk, it is present as calcium caseinate. It is insoluble in water but soluble in dilute acids and alkali. It is not coagulable by heat. Its isoelectric pH is 4.6. It can be precipitated by half saturation with ammonium sulfate.

Non-protein nitrogenous substances include all substances containing nitrogen other than proteins. They are: urea, uric acid, creatine, creatinine and ammonia.

It is the end product of amino acid metabolism. It is synthesized in liver. The normal blood urea level is 15 to 40 mg per 100 mL and normal urinary urea is 15–30 g per day.

Procedure: To 3 mL of urea solution, 3–5 drops of freshly prepared sodium hypobromite solution is added. A brisk effervescence is formed.

Principle: Sodium hypobromite acts on urea and decomposes it to give nitrogen, carbondioxide and water. Liberation of nitrogen gas produces brisk effervescence.

Procedure: 3 mL of urease suspension is added to 3 mL of urea solution and then a drop of phenolphthalein indicator is added. Warm the tube with the palm of the hands. Wait for 5–10 minutes. A pink color is produced.

Principle: Horse gram contains the enzyme urease which acts on urea to form ammonium carbonate which changes the solution to pink color in the presence of the indicator.

It is the product of catabolism of purines, in human body. It is synthesized in liver and excreted through urine.

Normal value in blood:

Male: 3–7 mg/100 mL

Female: 2.5–6.5 mg/100 mL

Normal level in urine

250–750 mg/day.

Procedure: To 3 mL of uric acid solution, 1 mL of Benedict's uric acid reagent and 1 mL of 20% sodium carbonate solution are added to get a deep blue color.

Principle: As uric acid is a reducing agent, it reduces phosphotungstic acid to tungsten blue in strong alkaline condition.

Reagents: Ammoniacal silver nitrate.

Procedure: A filter paper is moistened with ammoniacal silver nitrate. Then 2–3 drops of uric acid solution is added to that. A black color is produced.

Principle: Salt of silver is reduced by uric acid to metallic silver.

Procedure: In a China dish, 1 mL of uric acid is taken and few drops of concentrated nitric acid is added and heated over the flame gently. A reddish-yellow residue is formed. To this, 1–2 drops of dilute ammonia is added to get a purplish-red color.

Principle: Uric acid is oxidized by nitric acid to give the reddish-yellow purpuric acid which combines with ammonia to form ammonium purpurate or murexide, which is purple red in color.

Creatinine is the anhydride form of creatine phosphate. Creatine is synthesized from 3 amino acids—glycine, arginine and methionine.

Procedure: 3 mL of creatinine solution is taken in one test tube and in another test tube 3 mL of distilled water is taken as control. To both the test tubes 1 mL of saturated picric acid solution and 5210 drops of 10% sodium hydroxide are added. A deep orange color is produced in the first tube containing creatinine and in the second tube containing water, yellow color is produced.

Principle: Creatinine reacts with picric acid to form creatinine picrate, which turns to deep orange color in alkaline medium.

Scheme I: NPN—Urea, uric acid and creatinine

Scheme II: Proteins—Albumin, casein, gelatin and peptones

Scheme III: Carbohydrates—Glucose, fructose, maltose, sucrose, lactose, starch and dextrin.

Lipids are heterogeneous group of substances, which are insoluble in water and soluble in organic solvents. Lipids are classified into:

Principle: When unsaturated fatty acids are treated with halogens like bromine, double bonds are saturated. Bromine adds across the double bonds, resulting in the disappearance of the yellow color.

Procedure: To 1 mL of palmitic acid (saturated acid) solution, bromine water is added drop by drop and mixed, to get yellow color.

Cholesterol has the cyclopentanoperhydrophenanthrene nucleus with 27 carbon atoms. It is present only in animal tissues. The derivatives of cholesterol are sex hormones, steroid hormones, bile acids and salts, and vitamin D.

Principle: Cholesterol is dehydrated by sulfuric acid and acetic anhydride gradually and the color gets changed to red, blue and green.

Procedure: 2 mL of cholesterol in chloroform solution, is taken in a clean dry test tube and 10 drops of acetic anhydride and 3 drops of concentrated sulfuric acid are added and mixed. The solution turns to reddish-violet, blue and then emerald green. This reaction is used in the colorimetric estimation of cholesterol in blood.

Principle: Same as Liebermann-Burchard reaction.

Procedure: 2 mL of cholesterol in chloroform solution is taken in a clean dry test tube, and 2 mL of concentrated sulfuric acid is added along the sides of the test tube and mixed. A reddish-violet color is seen in the upper chloroform layer and a yellow color with green fluorescence is seen in the lower acid layer.

Milk is the secretion of lactating mammary gland. It is an emulsion of lipids in a solution of lactose, inorganic salts and proteins such as casein, lactalbumin and lactoglobulin. Milk is rich in vitamin A and riboflavin but poor in vitamin D. Minerals in milk include high calcium, phosphate, sodium, potassium and chloride. Milk is deficient in iron and copper. pH of fresh milk is 6.6 to 6.8. Specific gravity of milk is 1.030. Human milk has more lactose but less protein than cow's milk.

Gastric juice is secreted from the stomach by three types of cells, namely the chief cells, parietal cells, and mucous cells. It is highly acidic in nature. It is clear, odorless, pale yellow fluid having pH of 1–2. The important constituents of gastric juice are:

It is the chief secretion of liver, which is the largest gland in the body.

Hemoglobin is a conjugated protein consisting of globin, which is the protein part and heme, the prosthetic group. Heme is an iron porphyrin component.

68Derivatives of hemoglobin:

Spectroscope is a simple device that resolves white light into its seven component colors. It consists of a narrow slit through which light enters. A set of prisms resolves the light that can be viewed through an eyepiece. The wavelength region of light which the human eye can perceive, ranges from 400 to 700 nm. The wavelength of violet light is 400 nm and that of red light is 700 nm. When day light is viewed through the spectroscope, a few dark lines are seen. The two prominent lines are at 589 nm and 518 nm. They arise due to the absorption of light of particular wavelength by sodium and magnesium, respectively, present in the solar atmosphere. They are known as Fraunhofer's lines. When a solution of hemoglobin is viewed through a spectroscope, similar dark lines or bands are seen at definite wavelengths. They arise due to absorption of light by hemoglobin. The absorption maxima of these lines differ from one hemoglobin derivative to another, which is successfully used in differential identification of these compounds.

Urine is an excretory product of the body produced by the kidneys. Examination of urine may lead to the diagnosis of many metabolic and systemic diseases.

The ratio of day urine to night urine is 2:1 or 3:1. The ratio may be altered in renal diseases.

Normal odor—Fresh urine aromatic odor.

Abnormal odor

Normal urine: 1.015–1.025.

The pH of normal urine is 4.6–8 (average 6). Freshly voided urine is acidic. On standing, it may become alkaline due to the formation of ammonia from bacterial decomposition. pH of urine is influenced by the nature of diet.

High protein diet or low carbohydrate diet: Acidic urine.

Vegetables and fruits: Alkaline urine.

Normal value: 0.8 to 1.3 g/day.

^*(Presenting as inorganic as well as organic phosphates.)

(Normal excretion 0.7 g/day). Excreted as ammonium salts.

Increased in acidosis. Decreased in alkalosis.

Three forms of sulfates:

They are derived from the metabolism of sulfur containing amino acids such as cysteine, cystine and methionine.

Normal value of total inorganic sulfates: 0.7 to 1 g/day.

Increased in: High protein diet.

Decreased in: Renal dysfunction.

Normal value: 0.06 to 0.12 g/day. It consists of Na and K salts of sulfuric acid, esters of phenols such as indoxyl, etc. They are formed during putrifaction of amino acids in the intestines.

Major nitrogenous constituent of urine. It is produced from the breakdown of proteins.

It is the end product of purine catabolism in humans.

It is the anhydride of creatine. Urinary creatinine is derived from muscle creatine. It is not influenced by the protein intake. Normal value (1–2 g/24 h).

Increased in—Fever, myopathy.

Normal urine contains traces of urobilinogen, which is derived from bilirubin by the action of bacterial flora in the intestine, enters into circulation and then excreted by the kidneys.